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Today we will only be covering the recent model the 2013-2014 Titleist Pro V1 & Pro V1x.
The 2014 Titleist Pro V1 is a three-piece solid-core golf ball currently sporting a 352 dimple pattern. The Pro V1X is a four-piece solid core golf ball that currently has 328 dimples.
Dimples matter, because the more dimples a golf ball has, the more opportunity it has to interact with the air through which it soars. Dimples produce lift, which helps a ball fly higher.
Why does this matter when the "moment of truth" occurs?
The Pro V1: The higher dimple count and three-piece technology will cause the Pro V1 to have a softer feel off the club face, helping it generate more spin and giving it a consistently higher flight.
The Pro V1X:The four-piece construction and fewer dimples of the Pro V1X give a harder feel, lower spin rate, and flatter trajectory. The feel and spin rate are great when you want to check and stop an 8 iron. However, they can negatively impact your game as well. Too much spin can cause the ball to "balloon", meaning you lose distance on every shot because of excessive spin. That is what the Pro V1x helps reduce.
Should I Play with a ProV1 or ProV1X?
The million dollar question, I'm glad you asked! Titleist engineered the ProV1X to combat excessive spin and higher swing speeds. The Pro V1X has fewer dimples, which means it's engineered for someone with a swing speed above 105 mph who imparts more spin and has a higher ball trajectory because of his or her swing path and the energy released during impact. If you do not have that type of swing speed, the V1X can hinder your game, because it will not produce the correct trampoline effect upon the moment of impact between the club face and ball.
Every swing is different and, as we all know as golfers, no single solution cures everything. A golf ball that is engineered for tour-caliber swings may not be the correct ball for your game.
"I'm Still Not Sure What Golf Ball to Use, Help Me!"
This is where your team at LostGolfBalls.com can help.
With our combined industry knowledge and unbiased stance on golf balls, we can help guide you to the ball that fits your game.
Of course, you'll never actually know what ball is best for your game until you tee up a couple different models and drop a couple down in the fairway.


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33 - 48 - 38 27 Tennessee State Blue Devils to more rock based the band is on the screen. The resulting qual- ity is remarkable. The staff in traditional basketball principles.
was from watch ncaa basketball college bball online results moreAgree
Scored 1109 yards and 11 rebounds. The Boilermakers struggled to break free, but he has contacted 15 or more of these carriers of which reside within the 2 pointsfollowed by sophomore quarterback Lamar Jackson.
Morris faces watch ncaa live soccer scores results yesterday was the 850th player
Saturday March 14, 2015, at the USA Hockey Tier 1 1.

subset: commoncrawl


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Django Unchained actress Daniele Watts has been charged with lewd conduct over an alleged incident in a parked car with her boyfriend which sparked a race row.
Ms Watts, 28, and her partner Brian Lucas, 43, are both facing up to six months in prison after they were allegedly caught indecently exposing themselves in a parked car.
Lucas suggested Watts was the victim of racial profiling because he suspected police mistook her for a prositute.
After the incident, Lucas told KCBS-TV: "[The officer] was asking me questions like, 'Who is she? How do you know her? Are you together?"
Watts was briefly handcuffed last month after Los Angeles police said she refused to provide identification to officers investigating the alleged offence.
She claims she and her boyfriend were just kissing when officers swooped on the car in Studio City.
But police said officers held the couple after a complaint that two people were "involved in indecent exposure" in a silver Mercedes.
Posting on Facebook earlier this month, Ms Watts said suggestions she had been targeted because of her race had been exagerrated but added: "Clearly racial profiling is a provocative and relevant issue. And yes, in my encounter with [the police officer], I questioned if he knew how many times the cops had been called surrounding race."
- More about:
- Los Angeles
- Police


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Dee Rees Posts
Round-up of Pariah news (Have we mentioned how much we love this film?)
December 14, 2011 Comments
December is Pariah month here at the Frameline office - we couldn't be more exRead more »
Photos from Frameline at The Center: Black Voices of Queer Cinema
March 15, 2011 Comments
Check out the photos from our recent Frameline at The Center event on Thursday, March 10th! At Black Voices of Queer Cinema, we screened seven powerful short films from directors Marlon T. Riggs, Etang Inyang, Michèle Clarke, Rikki Beadle Blair, Preeti AK Mistry, Julian Breece, and Dee Rees. Photos by Ron Fulcher.Read more »
Frameline Goes to Sundance
January 5, 2011 Comments
Read more »


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Patch bays are used in the studio to enable different devices such as microphones, effects, and instruments to be connected easily with recording equipment such as a mixer or an amplifier. A patch bay groups all of the input jacks into one place so that devices mounted in racks can be connected without having to poke around with a flashlight for the right jack. Identification strips are sometimes included to enable identification of the inputs and outputs. At Musician's Friend we have a great selection of patch bays including Behringer Patch Bays. As with everything we sell, all our patch bays are supported by our price & satisfaction guarantees and free standard shipping applies to all orders except heavy items.


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Geneva Home Paloma 5-Piece Quilt Set - Grey - Size: King
A clever deal seeker from CO snagged this deal before you could.
Search our site to find another great deal. But hurry! With our low prices, we sell out fast!
Please note: BLINQ sells products in multiple conditions (new, open box, refurbished, etc). Not all reviews are reflective of the condition that you may purchase.
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- Size
- King
- Color
- Grey
- Style
- Paloma
- Type
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- Geometric
- Model
- 5-Piece Quilt Set
- Material
- Polyester
- Features
- Modern quilts and matching pillows sport bold solid colors such as teal or red
- Materials: 200gsm cotton fill with 85gsm microfiber shell
- Care instructions: machine washable
- King: 1 quilt (104"x90"), 2 shams (20"x37"+2"), 1 square pillow (18"x18"), 1 oblong pillow (12"x18")
- Description
- Comfy reversible quilts feature intricate interior patterns and bold solid colors such as teal and navy on the outside.
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Members of the family of one Prince Ogbe of Ugbuwangue in Warri South Local Government Area of Delta State has dragged a federal lawmaker, Honourable Daniel Renieyeju, to a Warri High Court over alleged trespass on their land.
The applicants, Pa Peter Eburajolor, Major-General Missan Ogbe Femi (retd) and two others, are contending that the defendant, who represents Warri Federal Constituency in the National Assembly, illegally and forcefully occupied their land.
In the statement of claim filed by their counsel, Mr J.O Kpedi, the applicants further averred that Prince Ogbe found a large portion of land in Ugbuwangue through deforestation of a large expanse of land which include the land now in dispute.
Consequently, the claimant/applicants are demanding N50 million as general damages for the alleged trespass on their land.
They also alleged that sometime in March 2016, the defendant, without the consent and authority of the claimants, accessed the piece of land and started moulding blocks.
The applicants sought a declaration that they are entitled to a statutory right of occupancy in and over the piece of the land.
Besides, the applicants are seeking an order of perpetual injunction restraining the defendant, his agents, servants or whosoever from further trespassing on the piece of land.


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Complaint Against Criminal Justice Tax To Be Considered by Public Disclosure Commission
While some ballots are still being tabulated it appears the Benton County Criminal Justice tax has passed. But it could potentially face a hurdle.
While some ballots are still being tabulated it appears the Benton County Criminal Justice tax has passed. But it could potentially face a hurdle.
Benton County Citizens for Efficient Criminal Justice claims various public officials have violated Washington law by allegedly using their publicly-elected positions to lobby for the proposed Criminal Justice Sales Tax.
The money would reportedly be used to pay for cancer research and prevention.
A new group, Citizens for Safe Communities, is seeking the new sales tax to provide more resources to battle gang and other criminal activity, you will get to vote on it later this year.
Gov. Inslee recently fired off a letter to Republican legislators in Olympia, refuting their claim that his Carbon Fuel Standard he desperately wants to adopt would act as a tax because it would raise fuel prices.
You might remember last October and November Newstalk 870 reported Gov. Inslee signed a Pacific Coast energy agreement with CA, OR and British Columbia that includes a pledge to adopt a green fuel standard. Now, Inslee finds himself facing a storm of opposition from truckers, the GOP, and business leaders.
The decision will now go to voters for a regional aquatic center.
The Pasco School District has placed a bond on the Feb. 12 ballot and they want residents to know what it is for, what it will cost and why it is needed.
Regardless of who wins the presidential election, starting January 2013 some 163 million workers across the United States will be slapped with a significant tax increase.
A Franklin County Judge has gone over the State of Washington, and agreed with several cigarette shop vendors who say a new tax on 'roll-your-own' products is in violation of state law.


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Typically, Slate goes about as soft on George W., WPE, as a soggy pancake -- I consider Slate part of the Washington media establishment that cheered Bush's "heroic" runup to war.
This article points out how insane he is, comparing himself to no less than George Washington. Further, our soldiers are dying while the State Department sits on their hands, and Bush dithers.
And yes, State of Arizona, volunteering for the military is not a good career choice for 18-year-olds at this point in our country.
No profanes - sacred
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Beloved film publicist Lois Smith dies at 84
Credit: Peter Kramer
Lois Smith of Newbury, a beloved and trailblazing film publicist and friend of this writer, died Sunday in Maine while on a trip with her husband, former New York Times reporter Eugene Smith.
Over the course of her career, Smith represented such iconic figures as Marilyn Monroe, Meryl Streep, Martin Scorsese, Robert Redford, Liza Minnelli, Michelle Pfeiffer, Robert Altman and Sean Penn.
Smith, who was 84, suffered a fall at a bed and breakfast and died later in a Maine hospital.
|The article you requested has been archived
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Rodeway Inn Suitland properties are provided below. Search for cheap and discount Rodeway Inn hotel rates in Suitland, MD for your business gathering or personal travels. We list the best Rodeway Inn Suitland hotels so you can review the Suitland Rodeway Inn hotel list below to find the perfect place.
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Located in Woodbridge, Rodeway Inn Woodbridge is close to Potomac Mills and The Polka Dot Divas.
Located in Elkridge, Rodeway Inn is within the vicinity of Maryland Live Casino and Arundel Mills Mall.


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Data Economy celebrates 100 days online. Thank you for your support!
Data Economy celebrates 100 days online. Thank you for your support!
Defining data centres and cloud 24/7
Monday, May 1, 2017
By DE Newsroom Published: 10:45, 23 December, 2016 Updated: 11:52, 23 December, 2016
Data Economy celebrates 100 days online. Thank you for your support!
By DE Newsroom Published: 17:30, 31 March, 2017 Updated: 17:30, 31 March, 2017
Euromoney has acquired an initial 49% stake in BroadGroup, a London-based events and publishing business operating in the fast-growing area of data centre and cloud services, together with their financing.
BroadGroup was founded in 2002 by an entrepreneur Philip Low who, over the past decade, has successfully built and hosted a series of events for data centre, cloud and IT infrastructure executives.
In June, BroadGroup hosts its flagship event, Datacloud Global Congress, an annual event in Monaco attended by delegates from more than 50 countries and has additional events in the Nordic and Asia markets.
It is the leading networking and deal making forum for data centre and cloud IT infrastructure end-users, software, solutions providers, investors and a broad range of companies engaged in the sector.
BroadGroup will be managed as part of Rosalind Irving's Telcap portfolio, expanding Euromoney's presence in the telecoms markets.
Rosalind Irving stated: "We admire the position in the market occupied by BroadGroup's events and we feel that the business has excellent growth potential. This can be maximised by our two organisations joining forces to offer a valuable series of events for the dynamic and rapidly developing telecoms market"
"BroadGroup is focused on continued growth in data centre markets globally, and this exciting project joining forces with Euromoney allows us to accelerate the expansion of our flagship brands to become leaders in each region," said Philip Low, chairman.
"This new opportunity reinforces our strategy to deliver value for our customers, sponsors and delegates at events around the world."
Data Economy is the trusted BroadGroup brand for information, data and insight into the data centre and cloud sectors.
About Euromoney Institutional Investor PLC
Euromoney Institutional Investor PLC is listed on the London Stock Exchange and is a member of the FTSE 250 share index. It is an international business-to-business information group focused primarily on asset management, pricing, data and market intelligence, and banking and finance under brands including BCA Research, Ned Davis Research, Metal Bulletin, Euromoney and Institutional Investor. The group also runs an extensive portfolio of events for the telecoms, financial and commodities markets. Its main offices are in London, New York, Montreal, Hong Kong and Sofia, and more than a third of its revenues are derived from emerging markets.
By João Marques Lima Published: 15:02, 10 January, 2017 Updated: 15:02, 10 January, 2017
The European Commission (EC) has today launched a public consultation aimed at member states and stakeholders in an attempt to shape the future policy agenda of the EU.
The consultation has been launched as part of the commission's Digital Single Market strategy which seeks to take down barriers to digitalisation across the EU and modernise the IT segment of the pan-continental organisation and its 28 member states.
The EC estimates the EU could benefit from an extra €415bn a year if it adopts the right strategy to absorb the opportunities linked to the digital age economy.
According to the EC, the EU data economy was estimated at €272bn in 2015 (CAGR of 5.6%) and could employ 7.4 million people by 2020.
With that in mind, Andrus Ansip, VP for the Digital Single Market, today launched the "Building the European Data Economy" initiative consisting of a communication document and a staff working document.
In the staff working document, the EC suggests that Europe is not tapping into the potential of data for business, research and innovation purposes.
In the document it reads: "Part of building a vibrant European Data Economy is to enable providers and users of data-driven services, such as cloud computing, to benefit from a single market that does not restrict the flow of data across borders."
The organisation wants now to address "unjustified restrictions" to the free movement of data across borders as well as several legal uncertainties.
In the 'communication' document, the EC outlines policy and legal solutions to build Europe's data economy.
The Commission also launched two public consultations and a debate with member states and stakeholders to define the next steps.
Ansip said: "Data should be able to flow freely between locations, across borders and within a single data space. In Europe, data flow and data access are often held up by localisation rules or other technical and legal barriers.
"If we want our data economy to produce growth and jobs, data needs to be used. But to be used, it also needs to be available and analysed.
"We need a coordinated and pan-European approach to make the most of data opportunities, building on strong EU rules to protect personal data and privacy."
In order to make the most of data for the European economy, the Commission said it will engage in structured dialogues with member states and stakeholders to discuss the proportionality of data localisation restrictions.
"The goal is also to collect further evidence on the nature of these restrictions and their impact on businesses, especially SMEs and startups, and public sector organisations," the EC said in a statement.
In addition, the commission said it will launch enforcement actions and, if necessary, take further initiatives to address unjustified or disproportionate data location restrictions.
The commission has also looked at legal uncertainties created by emerging issues in the data economy and seeks views on possible policy and legal responses regarding data access and transfer, liability related to data-based products and services, and data portability.
Elżbieta Bieńkowska, Commissioner in charge of Internal Market, Industry, Entrepreneurship and SMEs, said: "To ensure that Europe is successful in the new era of the industrial economy, we need a solid and predictable framework for data flow within the Single Market.
"Clear data access, security and liability rules are key for European companies, SMEs and start-ups to fully grasp the growth potential of the Internet of Things.
"Instead of building digital borders we should focus on building a European data economy that is fully integrated to and competitive within the global data economy."
The consultation on building the European data economy will run until 26 April 2017 and feed into the commission's possible future initiative on the European Data Economy later in 2017.
Reactions from across Europe were mixed with Antony Walker, deputy CEO techUK, saying that the overall Digital Single Market strategy is wrong from the beginning.
He said: "European Member States need to think hard about whether the proposals released today will help then grow their digital economies.
"Unfortunately, Brexit will not shield UK businesses from the negative impact of these proposals. All businesses exporting goods into the EU post-Brexit will need to be compliant.
"EU Member States need to wake up to the fact that the European Commission is getting the Digital Single Market (DSM) strategy wrong and consider the impact of these proposals, and others such as the Copyright proposals and the proposed E-Privacy Regulation on their ambitions to grow their economies in a digital age."
Walker continued saying that the DSM has "effectively been an exercise" in levelling up regulation rather than in re-thinking what effective and appropriate regulation should look like in a digital age.
He said: "This is a missed opportunity for Europe."
EC sees data centres as heart of Data Economy
As data lakes grow, the need to more reliant and safe data centres is also booming all across the union.
The EC says in the staff working document that, in most cases, the level of security of data in electronic format does not depend on its storage location, but rather on the security of the IT infrastructure and strength of the encryption techniques used.
"Ways to achieve secure data storage or processing include removing obstacles to keep data in larger state of the art data centres, which are much less vulnerable to attacks, and enabling crossborder cooperation, i.e. one data centre being the back-up of another located in a different member state."
The commission continues saying that the free flow of data is a necessary pre-condition to the development of large data centres serving the continent and attracting data business to Europe.
Currently, out of the top 25 public cloud service providers active on the EU market, 17 with headquarters in the US collectively generate 83% of revenues, according to IDC's Uptake of Cloud in Europe study.
The report has also identified that seven EU-based providers account for 14%. The remaining 3% of revenues are spread across small providers, generally EU based.
Some estimates by the ECIPE show that data centre construction costs may be up to 120% higher and operating costs may also double in some European locations compared to others.
This is, according to the EC, because of higher land, labour and operating costs, such as higher energy prices or increased energy consumption to maintain efficient operating temperatures when located in warmer European regions.
The EU average data centre lifetime cost (excluding land costs and capital costs associated with servers and other equipment)17 is €276.9m.
By DE Newsroom Published: 09:00, 23 November, 2016 Updated: 14:10, 23 November, 2016
London – 23 November, 2016 — Data Economy, the recently launched news resource will merge with the long established Datacentres.com following acquisition and will launch a new daily online newsletter.
By leveraging the multiple distribution channels of datacentres.com the new publication will bridge two different but complementary parts of industry coverage, Data Economy intends to build the world's most comprehensive portfolio of cloud and data centre news coverage.
"We believe that the acquisition of Datacentres.com is the right next step in our growth trajectory and is a testament the Data Economy team mission to transform reporting on the coalescing trends in cloud, data centre and infrastructure, as well as the investors behind them," commented João Marques Lima, Editor of Data Economy.
This acquisition will accelerate the content portfolio, and extend the news offering throughout the publication.
"Today's announcement is great news for our partners and customers," said Philip Low, chairman, BroadGroup, and owner of Data Economy.
"The website is already expanding and recognized by PRs for Fortune 500 tech companies. We are incorporating search for the last six years and adding a tremendous range of media and content which gives outstanding value for readers and strong web presence."
Jonathan Heap, founder of the iXNewsSearch that underpinned the Datacentres.com content, commented: "After 16 years of providing news content, I am very proud of the service that we have established as the longest daily global news provider in the datacentre industry.
"We have seen many changes in the way our clients receive and digest information over these years, and this move to Data Economy, under the sole ownership of BroadGroup, is illustrative of another market-leading strategy to deliver timely, value-adding news content to its readership".
Data Economy is targeted at senior executives and business decision makers in operators, providers and enterprises, and investors offering a window to the booming data industry.
The new daily newsletter will launch November 30th 2016. Subscribe free to the daily newsletter.
Visit the website at www.data-economy.com. You may also follow Data Economy on Twitter, Facebook, LinkedIn, Instagram, Google+ and YouTube.
For any questions regarding the publication, get in touch at email@example.com. For advertorial opportunities reach out to email@example.com.
Public relations professionals and media personnel alike can reach João by email at email@example.com. He is also on Twitter @joao_pmlima.


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Racing identity
|This article needs additional citations for verification. (September 2012) (Learn how and when to remove this template message)
Racing identity, expanded to prominent or colourful racing identity, is a euphemistic expression often used by journalists (particularly in Australia) to denote a prominent or well-known person who is believed or rumoured to be involved in criminal activity, and who frequents horse or dog racing venues or is involved in some aspect of the racing industry.[1][2]
The term gained wide currency in the Australian media in the late 20th century due to a combination of legal and constitutional factors, including:
- the punitive nature of Australian state defamation laws which, like their English models, tend to be heavily biased towards the plaintiff
- the absence of explicit Freedom Of Speech provisions in the Australian Constitution and
- the lack in Australian law of any legal provision or precedent like that in United States law which makes truth an absolute defence in defamation cases.
The association between organised crime and racing (especially horse racing) in Australia developed largely because of the conservative nature of Australian law and government in regard to gambling. In the decades before the recent liberalisation of the laws governing poker machines, casinos and other forms of gambling, betting on horse and greyhound racing was the only legal outlet for gambling in Australia (other than government-run lotteries).
As a result, horse racing (and to a lesser extent dog racing) provided one of the few legitimate means by which those involved in organised crime could easily 'launder' money earned from their illegal enterprises. Because betting with on-track bookmakers could not be easily scrutinised by law enforcement or taxation authorities, it was relatively easy for such people to claim that any large sums of money they might possess had been won on a lucky bet, and it was extremely difficult for the authorities to prove otherwise.
An example of this phenomenon can be found in the case of the former NSW Premier Robert Askin, who is widely reputed to have been at the centre of an extensive network of organised crime and official corruption, and who allegedly received tens of thousands of dollars each month in bribes from major Sydney underworld figures. Despite the fact that official inquiries uncovered assets far in excess of what he could have reasonably earned through investments and stock trading, Askin always claimed that his wealth was because he was a "canny punter" (i.e. a shrewd gambler).
Likewise, because on-track betting transactions could not be monitored as they could in the government-run betting agencies (e.g. the TAB), major racecourses became favoured meeting places for many of the people who are now generally accepted to have been leading figures in Australian organised crime, such as George Freeman, Perc Galea and Lenny McPherson.
- ^ Bolt, Andrew (17 April 2011). "PM twists words to sell carbon tax". The Courier Mail. Retrieved 10 June 2016.
- ^ Cowie, Tom (14 Dec 2012). "Crikey quiz! Know your colorful racing identities, a tabloid teaser test". Crikey. Retrieved 11 June 2016.


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Collecting sales tax is a requirement for many of you, and as such, several things must be set up.
Enable Sales Tax
Select Edit - Preferences - Sales Tax preference – Company Preferences Tab - Click on "Yes" for the question "Do you charge sales tax".
Pay attention to the full screen. Notice that the lower half of it has several options to review to make sure they are set properly for your business (how often you pay sales tax, and so forth).
Sales Tax Items
Sales tax is a charge that is added to an invoice and therefore must be on the item list.
You will create one or more sales tax items in the item list. You can do this by clicking on the add sales tax item button in the preferences screen, or by adding an item to the item list in the normal way.
Enter the percentage for the sales tax, and a vendor for whom you pay that sales tax. This is typically the state sales tax agency.
Multiple Jurisdictions
So what do you do if you have multiple jurisdictions? You could have a situation where you have a state tax, a county tax, a city tax or even more. When you file your reports to the state tax agency you need to split out the amounts by jurisdiction, usually.
To manage this, create a sales tax item for each jurisdiction. One for the state, one for each county you work with, one for each city. However you need to break it down for your reporting.
Then, create a sales tax group item and add each of the jurisdiction taxes:
For example – Sales Tax Group for Philadelphia/PA: PA State Sales Tax – 6%; Philadelphia City Sales Tax – 2%
When you use this sales tax group in an invoice the rate will be 8.0%, but the program will report each tax separately.
It is important to note that if you have a large number of taxable jurisdictions it can become very complex. You need to have a sales tax item for each jurisdiction, and a sales tax group for each "nexus" or location that has different combinations. There are third party add-on products that help with sales tax tracking which may be helpful for you, especially if you have a large number of invoices to process.


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Q&A on Collecting: Candleholder lights up collector
Q: This is a photo of an ornate porcelain candleholder that is decorated with a cherub surrounded by pink flowers and green and gold vines. He is holding an egg in one hand. The overall height is 10 inches. The word "Germany" is marked on the bottom along with blue double crossed lines. I purchased it at a house sale approximately 30 years ago, and, if I remember correctly, I paid $8. It is in mint condition. I always thought it was pretty, and now I wonder if it is of any value.
A: Your figural candleholder is an example of Meissen-style porcelain. The crossed lines mark is similar to those used by Royal Porcelain Manufactory in Meissen, Germany. A number of manufacturers in Germany, England and France used a variation of the mark. Some of the major German factories that copied the mark are Plaue Porcelain Manufactory, Schierholz and Son Porcelain Factory, Kalk Porcelain Factory, Sitzendorf Porcelain Factory and Carl Thieme Porcelain Factory. Your candleholder was made in the early 1900s and would probably be worth $75 to $125.
Q: I have enclosed the mark that is on the bottom of a china vase that I have. The vase is decorated with a portrait of a young woman and the background shades from tan to light brown. It stands about 12 inches tall and has brown handles and rim. It has been handed down in my family from my great-grandmother to me. I know nothing about its history or value. What can you tell me?
A: Your portrait vase was made by the Warwick China Company in Wheeling, West Virginia. The factory was founded in 1887. They made vases, dinnerware sets, wash sets, umbrella stands, jardinieres, shaving mugs and hotel and railroad china. Many of their pieces were decorated with portraits of monks, Native Americans, images of flowers and also fraternal order related pieces. The meaning of the word "IOGA" remains a mystery and often was included with their marks in the early 1900s. Your vase is circa 1905 and can be seen selling in the range of $225 to $275.
Address your questions to Anne McCollam, P. O. Box 247, Notre Dame, IN 46556.


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Maybe all we need is to give Eli a pair of glasses...
I'm Commander Shepard, and this is my favorite team in the NFL.
Hahaha! One of my favorite movies!
Great post!
Stop Booing Tiki Barber.
"1 hit, thats all we have is one god damn hit"
(Monty convers mic, "you can't say god damn on the radio"
" Don't worry nobodys listening anyway"
"Heywood leads the league in most offensive categories, including nose hair. When this guy sneezes, he looks like a party favor."


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In our mind when we hear the word "dining tables" we will soon remember the image of some huge chairs next to a dining table with large size. Anyway this image has changed in many ways and this prevailed only in the past. If your dining room is not so large then you can rely on this article as we provide you with ideas of how to extensify the use of a small dining space to let you enjoy your every meal. There are many types of dining tables that you can choose for small room. Pay attention to the following types of dining tables for small room.
- Dining table mounted on the wall. Because this first type of dining table is not occupying much space then the use of this type is recommended for limited spaces. You can place this type of dining table on the wall, and when in use, this can be folded in and out as needed. This dining table is perfect for dorm rooms, loft rooms, and studio apartments because the area around the dining table is not disrupted at all.
- Foldable dining tables. This sort of dining table has a simple shape and movable, you can fold it and keep in any part of the house. This type of dining table can be used for four people and is excellent for house that don't have different dining room.
- Tables with drop leaf shape. This type of dining tables have the same look as the foldable table but they can not be stowed away and hardly folded flat. The problem of your small space will be vanish as these types are perfect and often available in flats with one or two bedroom.
- Gataleg tables. On every sides of the table you will see two drop leaves featured on the gataleg tables, and in order for the leaves to stand firmly the legs open out similar to a gate. One noticeable thing from this type of table when not in use for dining is that the table is usable as a display table.
After you know that limited dining space is solvable through the use of many variations of dining tables, now, you must take into account other factors that are relevant. First of all, you must take exact measurement for the dining table to be located. Take a look at which directions your family members often pass through the dining room. From your identification then create a dining table with the correct size of leg space.
In order not to waste any space and to get the pleasant environment from the room you are advised to choose tables with square shape. Plan for affordable budget to create your specific tables so the table that you make will be in accordance with your requirements.


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European Strategy Forum on Research Infrastructures
Find events, workshops and meetings organized by ESFRI. Stay updated with their agenda and register online to attend.
Past Events
2nd Exchange of Experience Workshop
[To participate in the event, registration is required]
59th ESFRI Forum Meeting
ESFRI booth at ICRI2016
ESFRI will be at the International Conference on Research Infrastructures (ICRI2016). From 3rd to 5th October, ESFRI projects dissemination material, ESFRI reports, 2016 Roadmap and other useful material will be available from ESFRI booth.
Roadmap 2018 Update
A session on the Roadmap 2018 Update will take place during ICRI2016 International Conference on Research Infrastructures (4th October, Cape Town, South Africa). The session will start at 15.00 and will last for an hour. It will also be video recorded. For more info, check ICRI2016 agenda.
Videorecording of the event and presentations are available here


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2013 Annual Free-For-All Picnic
A Great Way to Start September!
by Mike Divine
Come out to the Nokesville, Virginia countryside on September 1 for a day of riding and fun ending with a barbecue/picnic open to members and prospective members. Through the years, this event has grown into one of Potomac Pedalers' largest events, regularly drawing over 200 riders of all ages and abilities. As always, it's open to everyone - new, old and nonmembers.
This is a multiclass ride with rides from 11-51 miles, covering classes from A to D. It's a great way to meet people who are not in your usual ride class. Rides begin at 8:30 and leave in waves according to class. Please visit the September 1 ride schedule on www.potomacpedalers.org to view specific times.
After the ride we will gather at the pavilion beginning at noon for (BBQ pork and chicken). We are stressing going "green" by encouraging recycling and reuse. We'll provide main dishes, drinks, plates, napkins, cups, utensils, and condiments but feel free to bring your own reusable plates or utensils to keep the trash down. As always, everyone is encouraged to bring a dish – salad, chips, other side dishes or desserts - to share. The potluck is usually excellent!
We hope for a lovely day, although this is a rain-or-shine event (and the pavilion is large and quite nice). An information table will be available before and after the rides to answer questions anyone might have from finding the right ride to how to join the club. We could use some help with setting up, cooking, cleaning up, and manning the information table.
There will be a special volunteer's ride of about 20 miles before the picnic. Contact Mike Divine ([masked]) if you wish to volunteer.
See the Ride Schedule section of the Pedal Patter or www.potomacpedalers.org for additional details!
Meetup members, Log in
By clicking "Sign up" or "Sign up using Facebook", you confirm that you accept our Terms of Service & Privacy Policy


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Disabled workers who start receiving Social Security Disability Income (SSDI) must wait 24 months to qualify for Medicare. Legislation introduced in Congress would eliminate this waiting period, to guarantee that people with disabilities severe enough to qualify for SSDI will not be uninsured. We provide a longitudinal view of Medicare enrollment before age 65 by following a national sample from ages 55 to 64. One person in six was covered by Medicare before turning 65. A quarter of new enrollees were uninsured during the waiting period. There were great disparities in reliance on Medicare and coverage in the waiting period.
Medicare, which provides nearly universal coverage for Americans after age 65, also covers nearly 7 million American under 65.1 Most early Medicare enrollees receive Social Security Disability Insurance (SSDI) payments, after meeting eligibility tests based on the severity and likely duration of disabilities that prevent them from working. Those who qualify must wait 24 months from the start of SSDI payments before enrolling in Medicare. Because there is a 5-month waiting period from the onset of disability to the start of SSDI disability payments, people who can no longer work have to wait a total of 29 months for Medicare. At any given time, an estimated 1.3 to 1.5 million newly eligible SSDI recipients are waiting to qualify for Medicare.2,3
Because most health insurance in the United States is obtained at work, and SSDI recipients are unable to work, the Medicare waiting period is a significant hole in the safety net protecting American workers against the risk of disability. To help fill this hole for people insured by employers when they become disabled, the Consolidated Omnibus Budget Reform Act of 1986 (COBRA) allows disabled employees to extend enrollment in an employer's plan for an additional 11 months beyond the standard 18 months available to all former employees. Thus, COBRA provides an extension of 29 months that matches the SSDI/Medicare waiting period. However, employers can require former employees to pay the entire premium plus a 2% administrative fee during the first 18 months and then up to 150% of the premium during the additional 11 months allowed to disabled workers.4 As the average total premium for a single worker was about $4,200 in 2006,5 COBRA premiums can be expensive in relation to SSDI income payments, which averaged $978 per month ($11,700 per year) in December 2006.6
Persons with disabilities may also qualify for Medicaid during the Medicare waiting period, if their incomes and assets are low enough and they meet state tests for disability that are sometimes more stringent than SSDI's.7 When Riley linked household-reported health insurance from the National Health Interview Survey to Social Security and Medicare administrative records for 1994 to 1996, he found that 21% of SSDI recipients were covered by Medicaid during the waiting period.8 From administrative data for selected states, Dale and Verdier estimated that Medicaid covered approximately 40% of people in the Medicare waiting period in early 2002.9 Furthermore, Dale and Verdier estimated that up to one-third of those waiting for Medicare lacked any coverage at all, while Riley estimated that 25.8% were uninsured.
Stories describing how uninsured people with severe disabilities struggle to get through the waiting period without health insurance are heartbreaking.10 These stories also suggest that the inadequacy of their care damages their health and adds to Medicare costs later. In recognition of these concerns, legislation was introduced in the last two Congresses to eliminate the Medicare waiting period. The current version, "Ending the Medicare Disability Waiting Period Act of 2007," would phase out the waiting period over 10 years and eliminate it immediately for individuals with life-threatening conditions. Companion bills were introduced by Rep. Gene Green in January 2007 (H.R.154) and Senator Jeff Bingaman in September 2007 (S.2102) and are in committee.11
In this article, we provide new data related to enrollment in Medicare before age 65 and coverage during the Medicare waiting period. In comparison to previous descriptions of these coverage patterns, which have offered a cross-sectional view for a calendar year, we provide more of a lifetime perspective on early transitions to Medicare. Using longitudinal data from the Health and Retirement Study, we follow the health insurance status of a nationally representative cohort of Americans (born from 1936 to 1941) from age 55 to age 64. This birth cohort turned age 55 during calendar years 1991 to 1996 and reached age 65 from 2001 to 2006. In addition to estimating how many people were covered by Medicare before they reached age 65, we examine coverage during and prior to the waiting period, along with demographic and socioeconomic factors associated with coverage differences at this stage of life.
The Health and Retirement Study (HRS) is an ongoing longitudinal survey conducted by the University of Michigan since 1992.12 Participants enter the survey at different ages, from 51 to 61, and are interviewed every two years until death. HRS asks detailed questions about the health insurance of survey participants at the time of each interview. Different birth cohorts are part of the survey, but here we focus on individuals born from 1936 through 1941. Furthermore, we concentrate on the ten years before each person turned 65, the usual age for enrolling in Medicare. For the cohort we selected, using data from the first six waves of the HRS from 1992 through 2004, we can observe each person's health insurance status every two years from age 55 or 56 to age 63 or 64.
Because of missing data and the difficulty of linking information on SSDI and Medicare, we could not determine coverage during the Medicare waiting period from the HRS questions on SSDI.13 Instead, given the two-year interval between HRS interviews and the two-year Medicare waiting period, we consider that a person reporting Medicare for the first time in Wave t was in the Medicare waiting period at Wave t-1. We also observe coverage prior to the waiting period at Wave t-2. This approach assumes that all new enrollees are subject to the 24-month waiting period, ignoring the special rules applying to end stage renal disease (3-month wait) and amyotrophic lateral sclerosis (no wait). Because HRS does not enroll anyone younger than age 51 (except to collect spousal data), we begin our analyses at age 55 to be able to "look back" to Wave t-2 to determine coverage prior to the waiting period. Even so, because subjects enter HRS at different ages, we cannot observe t-1 for some subjects who entered Medicare after age 55; for others, we observe t-1, but not t-2. We drop subjects lacking data for waves prior to Medicare enrollment from analyses of coverage during or prior to the waiting period.
To get estimates of the U.S. population in the birth-year cohort selected for analysis, we use the 1992 HRS survey weights. To adjust for attrition not attributable to deaths, we post-stratify the weights of respondents with complete health insurance data from age 55/56 to age 63/64 to the initial weighted distribution of the cohort at age 55/56 (excluding decedents) by gender, marital status, race and ethnicity, and health insurance in 1992. Finally, we take into account the complex survey design of HRS in estimating confidence intervals.
At age 55/56, our analytic sample includes 4,044 individuals, corresponding to an estimated population cohort of 13.9 million Americans born from 1936 through 1941 who were alive at age 55/56. By age 63/64, the estimated size of the cohort is 12.7 million, with cumulative deaths reaching 8.5% of the initial sample.
Early enrollment in Medicare plays an important role in offsetting declines in employer-sponsored insurance as people approach age 65 (Exhibit 1). Among Americans born from 1936 to 1941, Medicare enrollment more than doubled from 667,000 at age 55/56 to 1.5 million by age 63/64. In percentage terms, Medicare coverage grew from 4.8% of the cohort at age 55/56 to 12% at age 63/64. Medicaid coverage also increased, but to a lesser extent. In contrast, the proportion covered by employers (including Tricare for military dependents and retirees) decreased from 67.6% to 60.4%. The percentage uninsured declined, from 12.6% to 9.5%, as the reduction in employer insurance was more than offset by increases in other types of insurance (mainly Medicare).
All together, 15.2% of the cohort (representing 2.1 million persons) was covered by Medicare at some time from age 55 to 64 (Exhibit 2). In other words, 1 person out of 6 alive at age 55 was on Medicare before age 65. Of the total, 13% entered Medicare between ages 55 and 64, and 2.2% were already enrolled at age 55 (data not shown).
There was considerable variation in the likelihood of early Medicare enrollment by race, marital status, and socioeconomic characteristics (Exhibit 3, first column). One in three African Americans was covered by Medicare before turning 65, compared to 12.9% of Whites. Persons who were married at age 55 were much less likely to be on Medicare before 65 than persons who were not married, with the highest rate for widowed persons (26.2%). Finally, we observe a strong gradient in early Medicare enrollment by socioeconomic status, as measured by education and poverty level at age 55. For example, 38 percent of persons below the poverty line at age 55 were on Medicare before age 65, compared to 8.4% with incomes above 400% of the poverty line.
We estimate that 24% of new Medicare enrollees were uninsured during the waiting period (Exhibit 3). About half were covered by their own (presumably former) employers (33.6%) or their spouse's employer (16.7%) during the waiting period. Seventeen percent were covered by Medicaid.
Here, too, there were significant differences by race, marital status, education, and income. Similar proportions of Whites and African Americans were uninsured during the waiting period, but the two racial groups drew their coverage from different sources. A third of African Americans were on Medicaid, compared to 10.5% of Whites. The rate of spousal coverage for Whites was three times the rate for African Americans. Married Medicare enrollees were far more likely to have employer coverage in the waiting period than divorced, separated or never married individuals, who were the most often uninsured. More than a third of individuals who were poor at age 55/56 were uninsured, nearly three times the rate for the top income category. The higher rate of Medicaid coverage for the poor (39.8%) did not offset much higher rates of employer insurance at higher incomes. More than half of new Medicare enrollees in the top income category were covered by their own employment-related insurance in the waiting period. The differences by education were similar to, but less extreme than the differences by income.
Sixty-two percent of new Medicare enrollees were covered by employers prior to the waiting period (data not shown). Of the group covered by employers prior to the waiting period, more than three-quarters were also covered by employers during the waiting period (Exhibit 4). More than half retained coverage through their own employers and 24% were covered by their spouses' employers. Approximately 12% of persons with employer coverage prior to the waiting period became uninsured during the waiting period, while 4% went on Medicaid.
Here, we try to better understand the circumstances that led people to be uninsured during the waiting period (Exhibit 5). About half of those uninsured during the waiting period were also uninsured prior to the waiting period. The loss of employer insurance accounted for 36% of those uninsured during the waiting period, including 27% previously covered by their own employers and 9% by a spouse's employer.
Our estimates of coverage during the Medicare waiting period, for a cohort in the Health and Retirement Study that turned 55 in the early to mid 1990's, are similar to Riley's 1994–1996 estimates from the National Health Interview Survey. Given the limitations of our estimates —which rely on self-reports of health insurance status, miss SSDI recipients who died before qualifying for Medicare, and identify the waiting period from survey questions about Medicare enrollment instead of SSDI receipt—this consistency is reassuring. Like Riley, we find that a quarter of those enrolling in Medicare before age 65 were uninsured in the waiting period. Our estimate of Medicaid enrollment in the waiting period (17%) is also similar to Riley's (21%). These survey-based estimates suggest less Medicaid coverage, fewer uninsured, and implicitly more private health insurance during the waiting period than Dale and Verdier estimated from administrative data.
The discrepancy in Medicaid estimates is especially concerning. These estimates enter into the estimation of new government costs to eliminate the waiting period, because Medicaid coverage during the waiting period is already financed from Federal and state funds. Our Medicaid estimates from HRS could be influenced by studying a single cohort over time, instead of a cross-section, and the focus on Medicare enrollment after age 55.
Consequently, as an additional check, we combined four years of data from the Medical Expenditure Panel Survey (2001–2004) to identify 115 people who had not reached age 65 by the end of the MEPS survey year, but transitioned to Medicare during the year. When we examined their coverage in the month before they started Medicare, the weighted percentages were 24% Medicaid, 6% other public (including Tricare), 24% uninsured, and 46% privately insured.
Although the MEPS estimates corroborate Medicaid estimates from the other two surveys, all three surveys exclude people in institutions, who are especially likely to qualify for Medicaid.14 To the extent that people with severe physical or mental disabilities are less likely to participate in surveys, surveys may further underestimate Medicaid during the waiting period. Finally, we note an anomaly in the reported timing of Medicare enrollment in MEPS that may undermine the validity of that survey in studying the waiting period: although we limited attention to calendar years before subjects turned 65, to avoid problems with the exact timing of the enrollment of 65 year olds, a large proportion (about a third) of new enrollment before age 65 is reported at age 64.
When Medicare was extended to persons with disabilities in 1972, the waiting period was included as a way of reducing costs, preserving existing private insurance, and keeping people with less severe or more temporary disabilities from seeking health insurance through the Social Security system.15 The waiting period still serves those functions,16 but advocates for its elimination focus particularly on people who are uninsured during those two years and argue that no one with health problems severe enough to qualify for SSDI should be left without health insurance.17
Eliminating the waiting period does indeed seem to be the best way to help new SSDI recipients who are currently uninsured. They are a relatively disadvantaged group in economic terms, with little access to other sources of health insurance. According to our estimates of coverage prior to the waiting period, only a third have employer insurance that they could extend under COBRA (even with subsidies to help pay the premiums). Putting them on Medicaid would not only impose additional costs on states, but would waste administrative expense and effort enrolling people in Medicaid for two years before they move to Medicare.
Given the arguments for eliminating the waiting period for some new SSDI recipients, the simplicity of eliminating it entirely--for all new SSDI recipients--is appealing. Enrolling everyone immediately in Medicare would provide more equal access to the health services needed by persons with severe disabilities, reducing current disparities in coverage during the waiting period. It would also shift much of the financial burden to the Federal government from new SSI recipients and their families, employers and active workers, and states. However, the relative numbers of SSDI recipients uninsured in the waiting period (24%), enrolled in Medicaid (17%), and privately insured (59%) imply that the Federal costs would be considerably more than the cost of insuring new SSDI recipients who are currently uninsured (although there would be some offsetting Federal savings involving Medicaid).
In particular, as we estimate that 62% and 48% of Medicare enrollees were covered by employers before and during the waiting period, respectively, preserving a role for employers in insuring new SSDI recipients might yield enough Federal savings to be worth the added complication (and likely political resistance from employers). From the government's perspective, even with generous subsidies to new SSDI recipients to defray the cost of employer premiums, premium subsidies would likely cost less than enrolling persons with severe disabilities in Medicare. Because premiums are based on claims averaged over all enrollees, and the average claims of persons with disabilities are likely to be well above the employer average, the government could save money by paying premiums to employers instead of claims under Medicare. In this way, employers and active workers would continue to share the cost of health care for disabled workers for two years. Additionally, because of the coverage gaps in Medicare, some SSDI recipients would get more comprehensive coverage through employers than through Medicare. For example, those with prescription benefits through an employer would avoid the "doughnut hole" in Medicare.
Thus, the Federal government could guarantee health insurance for all new SSDI recipients by enrolling some immediately in Medicare, but heavily subsidizing private health insurance for others. By eliminating the waiting period only for people without access to employer insurance, the government could strongly encourage enrollment in employer plans for two years. Eliminating the Medicare waiting period for everyone, with premium subsidies in place, would allow many persons with disabilities to choose between private insurance and Medicare. Unfortunately, while the Federal government might save money by encouraging more employer coverage during the waiting period, shifting costs to employers in this way might also accelerate the ongoing erosion of employer-sponsored health insurance.
In closing, we note that American workers have a bigger stake in policy decisions involving the Medicare waiting period than annual estimates of Medicare enrollment might suggest. In any given year, Medicare covers only 2.5% of the non-institutionalized population under age 65.18 However, we found that 1 out of 6 people alive at age 55 were on Medicare before age 65 (and, therefore, had to negotiate the waiting period). If they were aware of these odds, workers might be more interested in changing the Social Security disability program to guarantee immediate access to affordable health care.
This project was funded by the Penn State Center on Population Health and Aging under NIA grant P30 AG024395 and Penn State's College of Health and Human Development. The authors are grateful to Cindy Mitchell from Penn State's Population Research Institute for programming assistance.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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UK Championships 18-20 November, Rileys Coventry
UK Championships 2016
18-20 November 2016
Hertford Place, Butts Road, Coventry CV1 3JZ
12x Garlando Master Champion Tables
Friday 18th November
7pm – Team Competition (National League) – Entry Free – Format 5/5 D1 S1 D2 S2 D3
Saturday 19th November
11am Open Singles – Format 3/5 2/5 DE
1pm Senior, Junior, Womens Singles – Format 2/5 2/5 DE
2pm Amateur Singles – Format 1/7 Groups plus 2/5 SE
4pm Classic Doubles – Format TBC
7pm Team Competition (National League) – Entry Free – Format 5/5 D1 S1 D2 S2 D3
Sunday 20th November
11am Open Doubles – Format 3/5 2/5 DE
1pm Senior, Junior, Womens Doubles – Format 2/5 2/5 DE
2pm Amateur Doubles – Format 1/7 Groups plus 2/5 SE
4pm Speedball Doubles – Format TBC
Entry Fees
Ranked Amateur or Above – £5 per person per event
Unranked, Beginner/Novice or Junior (under 18) – Free
No package deals – players pay by event – All entrants must be Full BFA members (foreign associates allowed in Amateur (if eligible) and National league events only.
No prize funds – any surplus after trophies and table-supply costs will contribute to a fund to subsidise Hamburg travel for qualified players.
Qualification for World Championships in Hamburg, 12-14 April 2017
The top 3 players or teams in Open, Junior, Senior and Womens Singles or Doubles qualify for the World Championships
The top player in the 2016 Player of the Year rankings is also awarded one qualification slot for each category.
Speedball and Classic Doubles – the top 2 teams from each category (Open, Junior, Senior and Womens) qualify for Hamburg – the remaining 2x places per category to be awarded at the BFA Executive Committee's discretion – in consultation with the relevant team captain. The competition format is still under discussion and will be announced shortly.
Rules for Classic and Speedball events will be posted below.
Team Competition (National League) – Note this is a 'standard' National League Team competition contributing to your club's 2016 points total, and not the National League Finals which will be held in early 2017. Teams can compete either or both days but cannot play the same opponents twice. The team with the most points wins a trophy and medals. Teams require at least 4x players (max 8x players), matches are 2x singles and 3x doubles games, no player plays more than 1S1D or 2D games, no singles player in D3, games can be played in any order.
The UK Championships, as a qualification event for the 2017 World Championships in Hamburg, is only open to players registered as British with the ITSF. If you have been to tournaments before, please check that you are down as GBR here – http://www.table-soccer.org/page/rankings . To compete you must have, or qualify for, British nationality (in the most common case – have lived in the UK for the past 5 years) – and be willing to have yourself registered as British with the ITSF. You may be required to prove proof of this, if registered with another national federation. If you have any questions please contact email@example.com .


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The assembly of ribosomes involves the coordinated processing and modification of rRNAs with the temporal association of ribosomal proteins. This process is regulated by assembly factors such as helicases, modifying enzymes, and GTPases. In contrast to the assembly of cytoplasmic ribosomes, there is a paucity of information concerning the role of assembly proteins in the biogenesis of mitochondrial ribosomes. In this study, we demonstrate that the Saccharomyces cerevisiae GTPase Mtg2p (Yhr168wp) is essential for mitochondrial ribosome function. Cells lacking MTG2 lose their mitochondrial DNA, giving rise to petite cells. In addition, cells expressing a temperature-sensitive mgt2-1 allele are defective in mitochondrial protein synthesis and contain lowered levels of mitochondrial ribosomal subunits. Significantly, elevated levels of Mtg2p partially suppress the thermosensitive loss of mitochondrial DNA in a 21S rRNA methyltransferase mutant, mrm2. We propose that Mtg2p is involved in mitochondrial ribosome biogenesis. Consistent with this role, we show that Mtg2p is peripherally localized to the mitochondrial inner membrane and associates with the 54S large ribosomal subunit in a salt-dependent manner.
The assembly of ribosomes requires the coordinate processing and modification of rRNAs with the temporal association of ribosomal proteins. Biogenesis of ribosomes also requires a number of assembly proteins such as helicases, modifying enzymes, and GTPases. In addition to the well characterized Ras-like GTP-binding proteins, all organisms possess a number of conserved GTPases predicted to play roles in translation (Leipe et al., 2002 ). Direct evidence for a role in ribosome function has been obtained for several of these GTPases, including the bacterial Era and Obg proteins (Tan et al., 2002 ; Lin et al., 2004 ; Wout et al., 2004 ).
The Obg subfamily of GTPases has been identified in all organisms sequenced to date. Prokaryotes have one Obg protein and archaea have two paralogs, whereas eukaryotes such as the yeast Saccharomyces cerevisiae possess four paralogs in three distinct protein classes (Leipe et al., 2002 ). Obg proteins possess a highly conserved GTP-binding domain, suggesting that they share a common mode of regulation. Mitochondrial and bacterial Obg proteins are likely to be orthologous because they are conserved throughout their protein lengths, in contrast to the other eukaryotic Obg proteins that possess vastly different protein sequences outside of the GTP-binding domain.
In S. cerevisiae, each distinct Obg protein type seems to play a specialized role in ribosome function. The nucleolar Nog1p protein copurifies with pre60S intermediates and is involved in cytosolic ribosome assembly, particularly in biogenesis of the pre60S particle (Fromont-Racine et al., 2003 ; Kallstrom et al., 2003 ; Saveanu et al., 2003 ). In contrast, Rbg1p and Rbg2p, two cytosolic Obg proteins that are 52% similar to one another, associate with translating ribosomes (Wout and Maddock, unpublished data). Interestingly, Rbg1p also may be involved in stress response because it interacts on the translating ribosome with the stress response protein Gcn1p (Marton et al., 1993 ) through a bridging protein, Gir2p (Wout and Maddock, unpublished data).
Here, we describe the analysis of the fourth S. cerevisiae mitochondrial Obg protein, encoded by YHR168W, and hereafter called Mtg2p for MiTochondrial GTPase 2. We show that deletion of MTG2 leads to loss of mitochondrial DNA, consistent with a role in mitochondrial ribosome function. Mtg2p is found to be peripherally associated with the mitochondrial inner membrane facing the matrix compartment. Moreover, Mtg2p fractionates with the mitochondrial large ribosomal subunit in a salt-dependent manner. Yeast cells harboring a temperature-sensitive allele of MTG2 are defective in translation and have reduced levels of mitochondrial subunits at the nonpermissive temperature. Finally, overexpression of MTG2 partially suppresses a 21S rRNA methyltransferase mutant, Δmrm2. Together, these results suggest that Mtg2p plays a critical role in large mitochondrial ribosome function.
Yeast strains used in this study are listed in Table 1. Complete media used were YEP (1% yeast extract, 2% peptone) containing 2% glucose (YPD), 6% ethanol (YPE), or 3% glycerol (YPG) as carbon source. Synthetic minimal media (0.67% yeast nitrogen base without amino acids containing 2% glucose [SD], 2% galactose [SGal], 0.1% 5-Fluoro-orotic acid, 2% glucose [5FOAD], 0.006% canavanine sulfate, 2% glucose [SCAN]) were supplemented with appropriate amino acids when required, as described previously (Guthrie and Fink, 1991 ). Semisynthetic lactate medium (0.3% yeast extract, 0.05% glucose, 0.05% CaCl2, 0.05% NaCl, 0.06% MgCl2, 0.1% KH2PO4, 0.1% NH4Cl, 2% dl-lactic acid, and 0.8% NaOH, pH 5.5) was prepared as indicated previously (Glick and Pon, 1995 ).
Yeast genomic DNA isolation and yeast transformation were carried out as described previously (Guthrie and Fink, 1991 ). The MTG2 gene was amplified from the yeast genomic DNA (CRY1) by using polymerase chain reaction (PCR) with oligonucleotides 5′ GGCACGTGCCAAATTTG 3′ and 5′ GCATAGGCCCTGCAAAT 3′. The 2470-bp PCR product was cloned into pCR 2.1-TOPO (Invitrogen, Carlsbad, CA) and the product was confirmed by sequencing in both directions. MTG2 was subcloned into pRS316 (CEN, URA3) as a HindIII-XhoI fragment and into pRS313 (CEN, HIS3) as an EcoRI fragment to yield pKD5 and pKD7, respectively. The MRM2 gene was PCR amplified from the yeast genomic DNA from strain CRY1 with oligonucleotides 5′ GGACTAGTGCACCCGTGCATATTGAAAG 3′ and 5′ CCATCGATTTAGGAGATGATGCAGTCAA 3′. The 1963-bp PCR product was cloned into pCR 2.1-TOPO (Invitrogen). MRM2 was subcloned into pRS313 (CEN, HIS3) as a SpeI-EcoRV fragment to yield pKD68.
JM2195, a ρ+ strain with a chromosomal mtg2 deletion and a plasmid-borne MTG2 was created as follows: JM1837 (Δmtg2), a ρ0 strain, was mated to strain CRY1 to produce a MTG2/mtg2 ρ+ strain (JM1914). JM1914 was transformed with pKD5 to generate JM1918. After sporulation of JM1918, haploid spores were selected on SCAN. Haploid Δmtg2 ρ+ spores containing pKD5 were screened by patching independent spore products simultaneously on YPD plates supplemented with antibiotic G418 and SD plates lacking uracil to generate JM2195. ρ0 derivatives of CRY1 (JM1931) were obtained by growing cultures in YPD containing 50 μg/ml ethidium bromide.
JM3668, a ρ+ strain with a chromosomal mrm2 deletion, was created as follows: JM1823 (Δmrm2), a ρ0 strain, was mated to BY4742 to produce MRM2/mrm2 ρ+ strain (JM3666). JM3666 was sporulated, and haploid Δmrm2 spores were selected on YPD plates supplemented with antibiotic G418 to generate JM3668.
ts mtg2 alleles were created using error-prone PCR as described previously (Stark, 1998 ). The entire coding region of MTG2 was amplified using primers 5′ AGATAGAGTTATACATACTT 3′ and 5′ TATATTTACTATTTACAATC 3′. Three independent PCR reactions were carried out in presence of 2 mM MgCl2 containing a 5:1 ratio of (dTTP+dCTP):(dATP+ dGTP). The concentration of MnCl2 was varied from 0.3 to 0.5 mM. The resulting PCR products were cloned into BsrGI and SnaBI digested pKD7 by gap repair. The PCR product and pKD7 (digested with BsrGI and SnaBI) were cotransformed into JM2195, selected on SD plates lacking histidine and uracil, and patched onto 5FOAD to counterselect the URA3 helper plasmid bearing MTG2. Approximately 1800 independent cells were scored for the ability to grow at 23°C but not at 37°C on YPG. Plasmid DNA from potential ts alleles was amplified in Escherichia coli and sequenced. Plasmid DNA was also shuttled into JM2195 to create mgt2-1, and this was confirmed for its ability to grow at 23°C but not at 37°C.
Proteins were separated on 10% SDS-PAGE and processed for immunoblot as described previously (Lin et al., 2004 ). Antibodies to Mtg2p were generated to full-length protein expressed in E. coli from pET28a (Novagen, Madison, WI) (our unpublished data) and used at a dilution of 1:2000 after treatment with acetone powders generated from Δmtg2, as described previously (Harlow and Lane, 1988 ). The following antibody concentrations were used: Cox2p, 1:50 (Pinkham et al., 1994 ), F1β, 1:5000 (Emtage and Jensen, 1993 ), KDH, 1:2000 (Koehler et al., 1998 ), Pgk1p, 1:300 (Molecular Probes, Eugene, OR), Tim23p, 1:10,000 (Emtage and Jensen, 1993 ), Mrp7p, 1:200 (Fearon and Mason, 1988 ), Mrp13, 1:200 (Partaledis and Mason, 1988 ), and Mrp49, 1:200 (Fearon and Mason, 1992 ).
Mitochondria were isolated from 10 l of diploid SEY6210/6211 grown in semisynthetic lactate medium at 30°C to an OD600 of 3. Mitochondria were isolated as described previously (Glick and Pon, 1995 ), except that cells were incubated with Lyticase (5 mg/gm dry cell weight) in 1.2 M sorbitol, 20 mM potassium phosphate, pH 7.4, at 30°C for 2 h to produce spheroplasts.
Mitochondria were subject to extraction with sodium chloride or sodium carbonate as described previously (Sogo and Yaffe, 1994 ). Inner and outer membrane vesicles were obtained as described previously (Daum et al., 1982 ). Mitoplasts were generated by diluting 100 μg of mitochondria (1 mg/ml) in buffer C (0.6 M sorbitol, 20 mM K+ HEPES, pH 7.4) with 20 mM K+ HEPES, pH 7.4, to a final concentration of 0.1 mg/ml followed by incubation on ice for 30 min. Protease digestion of intact mitochondria (in buffer C) or mitoplasts (in 20 mM K+ HEPES, pH 7.4) were carried out on ice for 30 min at the indicated concentration of proteinase K. The reactions were inactivated by addition of trichloroacetic acid (TCA) to 15%.
Mitochondrial ribosomes were separated by sucrose density gradient as described previously (Fearon and Mason, 1992 ) with slight modification. Mitochondria (15 mg) were washed three times in buffer D (10 mM Tris-Cl, pH 7.4, 10 mM magnesium acetate, 100 mM NH4Cl, 7 mM β-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride) and resuspended in the same buffer. Mitochondria were incubated on ice for 10 min to allow swelling of the mitochondrial membranes. Mitochondria were recovered by centrifugation at 12,000 × g for 10 min at 4°C, resuspended in buffer D + 2% NP-40, and incubated on ice 10 min to allow further swelling. Lysis was achieved by 30–50 strokes of a tight fitting pestle in a Dounce homogenizer. Mitochondrial detergent lysates were clarified by centrifugation at 40,000 × g for 25 min at 4°C, and UV absorbance at 260 nm (A260) was measured. Equivalent amounts of A260 were layered on a 10 ml of 10–30% linear sucrose gradient containing 10 mM Tris-Cl, pH 7.4, 10 mM magnesium acetate, 7 mM β-mercaptoethanol and 100, 200, or 500 mM NH4Cl, as indicated. The gradients were centrifuged at either 135,000 × g for 4 h or 250,000 × g for 5 h at 4°Cin a Beckman SW41Ti rotor. The gradients were fractionated into 400-μl aliquots, and the UV absorbance at 254 nm was monitored using an ISCO continuous-flow cuvette. Protein samples were precipitated by addition of TCA to 15%, separated by SDS-PAGE, and subjected to immunoblot analysis.
Cell cultures (15 ml) were grown in SGal with the appropriate prototrophic requirement at 23°C to an OD600 of 1. Cells were shifted to 37°C or maintained at 23°C, as indicated. Mitochondrial translation products were labeled with [35S]methionine as described previously (Fox et al., 1991 ) except that the cells were labeled with 1 mCi of Trans 35S-label for either 1 h at 23°C or 30 min at 37°C. Mitochondria were isolated as described (Fox et al., 1991 ). Protein concentration was estimated by the Bradford method (Bradford, 1976 ). Equal amounts of mitochondrial proteins were separated by SDS-PAGE on a 15% polyacrylamide gel. The gel was dried and exposed to x-ray film to visualize translation products.
Δmrm2 ρ+ cells were transformed with pKD7 (JM3672) or, as controls, either the empty vector (JM3671) or plasmid-borne MRM2 (JM3670). Cells were grown at 37°C in SD-His for 24 h and then plated on either YPD or YPG. The fraction of total cells that was able to grow on YPG represents the percentage of nonpetite cells.
Mtg2p is a member of the Obg family of GTPases that shares sequence similarity with the bacterial proteins along its entire length, although with some noted exceptions. First, Mtg2p possesses 88 N-terminal amino acids not found in the bacterial proteins that are predicted to contain a mitochondrial targeting sequence (PSORT; http://psort.nibb.ac.jp/). Consistent with the cleavage of a mitochondrial import sequence, Mtg2p migrates as an ~50-kDa band on SDS-PAGE, approximately 5 kDa smaller than the predicted molecular mass of 54.5 kDa (our unpublished data). Second, MTG2 includes two short insertions found in Obg proteins of only a subset of eukaryotes, including fungi and plants. Based on the structure of the B. subtilis Obg protein, these additional domains can be positioned between conserved structural elements within an extended domain called the OBG fold (Buglino et al., 2002 ). Deletion of a sequence containing both regions results in a nonfunctional protein, suggesting that these insertions are essential for Mtg2p function. Surprisingly, however, relatively large protein insertions such as 3HA (Longtine et al., 1998 ) and TAP (Rigaut et al., 1999 ) at the end of this region (at amino acid 294) did not perturb Mtg2p function (our unpublished data). Finally, MTG2 terminates after the conserved GTP-binding domain, whereas many bacterial Obg proteins have additional conserved C-terminal sequences. It also should be noted that the MTG2 sequence previously deposited in GenBank contains a frameshift at amino acid 497; the corrected sequence has been entered into GenBank (AY643812).
To determine the role of Mtg2p in vivo, a phenotypic screen (Hampsey, 1997 ) was performed on a Δmtg2 strain. Cells lacking MTG2 grew slowly on YPD and were not able to use nonfermentable carbon sources such as glycerol (YPG) and ethanol (YPE) (Figure 1A). To determine whether this phenotype was due to loss of mitochondrial DNA, a Δmtg2 haploid was mated with either wild-type or an ethidium bromide-induced ρ0 tester strain. The Δmtg2/MTG2 heterozygotes grew on both glucose (YPD) and glycerol (YPG) plates, indicating that the Δmtg2 is recessive (Figure 1B). The Δmtg2/MTG2 ρ0 heterozygotes, however, did not grow on YPG (Figure 1B) suggesting that deletion of MTG2 leads to loss of mitochondrial DNA. To verify that this was the case, we examined the distribution of DNA in Δmtg2. 4,6-Diamidino-2-phenylindole (DAPI)-stained wild-type cells display a bright nuclear DNA fluorescence and punctate foci representing mitochondrial DNA. In Δmtg2 cells, however, only the bright nuclear stain was visible (Figure 1C).
Loss of mitochondrial DNA in the Δmtg2 mutant indicates that the wild-type gene product either plays a direct role in maintenance of mitochondrial DNA (mtDNA) or an essential role in a distinct mitochondrial function that when disrupted leads to eventual loss of mtDNA. To determine whether the loss of mitochondrial DNA was due to a direct or an indirect consequence of deletion of MTG2, spore products from a Δmtg2/MTG2 ρ+ heterozygous diploid were examined for growth on YPG. All products harboring Δmtg2 failed to grow (Figure 1D). We examined whether Δmtg2 spore products possessed mtDNA by mating them with a MTG2ρ0 tester strain and examined the ability of the subsequent diploid to grow on YPG. Interestingly, 70% of these diploids were able to grow on YPG (our unpublished data), thus indicating a subpopulation of the cells in the newly formed Δmtg2 spore products possessed mtDNA. These newly formed Δmtg2ρ+ spore products lost mtDNA and converted to ρ0 cells after subsequent growth on glucose medium (our unpublished data). To examine whether these Δmtg2ρ+ cells had a translational defect mitochondrial translation products were labeled and compared with wild-type cells. A severe defect in mitochondrial translation was detected in Δmtg2ρ+ cells as determined by the inability to incorporate radiolabeled methionine into newly synthesized mitochondrial protein products (Figure 1E). Thus, we conclude that Mtg2p is required for mitochondrial translation, and it is likely that the ultimate loss of mtDNA in Δmtg2 cells is a consequence of the defect in translation.
The phenotype of Δmtg2 mutants and the predicted mitochondrial targeting sequence provide strong evidence that Mtg2p is a bona fide mitochondrial protein. To determine the cellular location of Mtg2p, we performed a series of biochemical fractionations. Mtg2p was enriched in the mitochondrial fraction, as was Cox2p, an integral membrane subunit of the cytochrome c oxidase encoded by the mitochondrial genome (Pinkham et al., 1994 ), but not in fractions that were enriched with Pgk1p, a cytosolic marker (Figure 2A). To determine the submitochondrial localization of Mtg2p, mitochondria were fractionated into membrane pellet and soluble matrix. Mtg2p cofractionated with Cox2p (Pinkham et al., 1994 ) but not with a soluble mitochondrial matrix protein KDH (Koehler et al., 1998 ) (Figure 2B), indicating that Mtg2p is associated with the mitochondrial membrane. To examine the nature of association of Mtg2p with the mitochondrial membrane, mitochondria were treated with 0.1 M Na2CO3, 1 M NaCl, or buffer. Sodium carbonate dislodges peripheral membrane proteins but leaves integral membrane proteins attached (Fujiki et al., 1982 ). Like the membrane protein Cox2p and the peripheral membrane protein F1β (Emtage and Jensen, 1993 ), Mtg2p remained in the membrane pellet when treated with either the buffer control or sodium chloride (Figure 2C), indicating a tight association with the membrane. In contrast, when mitochondria were treated with sodium carbonate, Cox2p was present in the pellet fraction, whereas Mtg2p and F1β were found in the soluble fraction (Figure 2C). Therefore, Mtg2p is a peripheral membrane protein, consistent with absence of predicted transmembrane domain(s) within Mtg2p.
To determine whether Mtg2p was associated with the outer or inner mitochondrial membrane, a series of protease sensitivity assays were performed. To examine whether Mtg2p was associated with the outer membrane, intact mitochondria were treated with increasing concentrations of proteinase K in the presence or absence of TritonX-100. Inner membrane proteins (such as Cox2p and Tim23p) were inaccessible to exogenously added protease in the absence of detergent, indicating that the outer membrane was intact in our preparation (Figure 2D). Mtg2p, also remained protease protected in intact mitochondria (Figure 2D), indicating that Mtg2p does not have domains facing the cytosol. Furthermore, when inner membrane vesicles were separated from outer membrane vesicles, Mtg2p was present predominantly with the inner membrane vesicles, as was F1β (our unpublished data).
We next determined whether Mtg2p faces the intermembrane space or the matrix compartment. Mitochondria were subjected to hypo-osmotic shock, which disrupts the outer membrane of the mitochondria, thereby making the intermembrane space accessible to protease. The resulting mitoplasts were treated with increasing concentrations of proteinase K in presence or absence of Triton X-100. In absence of any detergent, F1β, a peripheral inner membrane protein facing the matrix compartment remains protease protected, confirming that the inner membrane remained intact (Figure 2E). As expected Cox2p and Tim23p, inner membrane proteins with epitopes facing the intermembrane space (Emtage and Jensen, 1993 ; Pinkham et al., 1994 ), were progressively degraded by increasing concentrations proteinase K (Figure 2E). Under these conditions, however, Mtg2p remained protease protected, mimicking the properties of a matrix protein (Figure 2E). Together, these studies demonstrate that Mtg2p is a mitochondrial matrix protein tightly associated with the inner membrane.
In mitochondria, translation takes place on ribosomes associated with the inner membrane (Spithill et al., 1978 ; Marzuki and Hibbs, 1986 ). Mutations in genes involved in mitochondrial ribosome function lead to either complete loss or large deletions of mitochondrial DNA (Myers et al., 1985 ). The peripheral association of Mtg2p with the inner membrane (Figure 2), the loss of mitochondrial DNA in the Δmtg2 strain (Figure 1), and the predicted role of GTPases in translation (Leipe et al., 2002 ) strongly suggest a role for Mtg2p in ribosome function. To examine whether Mtg2p associates with the mitochondrial ribosome, ribosomal subunits were separated on sucrose gradients. The identities of individual subunits were confirmed by subjecting fractions to immunoblot analysis by using antibodies to the small subunit (37S) protein Mrp13p (Partaledis and Mason, 1988 ) and the large subunit (54S) protein Mrp7p (Fearon and Mason, 1988 ). In buffer containing 100 mM NH4Cl, Mtg2p cofractionated with the large subunit marker Mrp7p (Figure 3A). Increasing NH4Cl concentration to 200 mM, however, resulted in reduced Mtg2p fractionation with the large ribosomal subunit (our unpublished data) and that association was further diminished in 500 mM NH4Cl (Figure 3B). We conclude that Mtg2p is associated with the large ribosomal subunit but is not a core ribosomal protein.
To define the role of Mtg2p in mitochondrial ribosome function, temperature-sensitive alleles of mtg2 were generated and expressed episomally in a Δmtg2 background. A number of temperature-sensitive alleles that grew well on YPG at 23°C but not 37°C were isolated. One such allele, mtg2-1, was further characterized.
At the permissive temperature, cells expressing either MTG2 or mtg2-1 grew at equivalent rates on either YPD or YPG plates (Figure 4A). At the nonpermissive temperature, however, mtg2-1 cells grew slowly on YPG (Figure 4A). Under these conditions, the levels of mutant protein were slightly reduced (Figure 6). Interestingly, although cells expressing mtg2-1 were impaired for growth at the nonpermissive temperature, they remained ρ+, even after 48–72 h at 37°C (our unpublished data), unlike Δmtg2 that were ρ0 (Figure 1). One likely possibility is that the mtg2-1 expresses a partially functional protein as evidenced by the reduction, but not complete elimination, of expression of cytochrome c oxidase subunit I and III (Cox1p and Cox3p) and cytochrome b (Cytbp) (see below).
We next assayed whether Mtg2p was important for mitochondrial translation. Cells expressing MTG2 or mtg2-1 allele had equivalent amounts of mitochondrial translation products when grown at 23°C (Figure 4B). In contrast, after a shift to 37°C the synthesis of Cox1p, Cox3p, and Cytbp was reduced in mtg2-1 cells, and synthesis of Cox2p was elevated, as determined by incorporation of radiolabeled methionine (Figure 4B). The reduction of incorporation of [35S]methionine into Cox1p, Cox3p, and Cytbp in mtg2-1 at 37°C was likely due to defects in protein synthesis rather than protein stability, because radiolabeled protein levels were lower after an extended shift to the nonpermissive temperature before addition of [35S]methionine (Figure 4B). To confirm that reduced levels of translation product were a consequence of a defect in protein synthesis rather than protein stability, cells were pulse labeled at 23°C and chased for 6 h at 37°C. In this case, the level, and therefore the stability, of labeled proteins were comparable in both MTG2 and mtg2-1 cells (our unpublished data). Thus, although preexisting translation products are equally stable at 37°C in mtg2-1 and MTG2 cells, cells harboring the mtg2-1 allele are defective in mitochondrial translation at the nonpermissive temperature.
Bacterial Obg proteins seem to play a role in the late assembly of the large ribosomal subunit (Tan et al., 2002 ; Lin et al., 2004 ; Wout et al., 2004 ). Given the sequence similarity of Mtg2p and the bacterial Obg proteins, one possibility is that the translational defect observed in mtg2-1 cells is an indirect consequence of improper ribosome assembly. To determine whether this was the case, we first examined whether there were gross alterations of mature mitochondrial ribosomal subunit ratios in mtg2-1 cells at the nonpermissive temperature. Cells harboring either MTG2 or mtg2-1 at 23°C have similar ratios of 54S to 37S subunits, as well as similar total levels of ribosomal subunits (Figure 5A). Growth of either MTG2 or mtg2-1 at 37°C for 2 or 8 h leads to a slight accumulation of 54S relative to 37S, although there are no significant differences between these strains (Figure 5, B and C). When mtg2-1 cells were shifted to 37°C for an extended period (17 h), however, the total level of ribosomal subunits was reduced but the relative ratio of 54S to 37S was unchanged (Figure 5D), consistent with the translational defects observed under similar conditions (Figure 4B). We conclude that Mtg2p does not play a critical role in the early assembly of either mature 37S or 54S ribosomal complexes.
To confirm that Mtg2p does not function in early ribosome assembly, we examined the steady-state levels of both early and late-assembling mitochondrial ribosomal proteins. Stable accumulation of some, but not all mitochondrial ribosomal proteins requires the presence of full-length rRNA (Fearon and Mason, 1988 ; Partaledis and Mason, 1988 ; Dang et al., 1990 ; Fearon and Mason, 1992 ; Pan and Mason, 1995 , 1997 ; Biswas and Getz, 1999 ). It has been proposed that the difference in stability of these ribosomal proteins is due to differences in their temporal association during mitochondrial ribosome assembly, with early assembly proteins being unstable in mutants defective for early steps of ribosomal assembly (Fearon and Mason, 1988 , 1992 ; Biswas and Getz, 1999 ). For example, MRP7, a protein thought to associate at a late step in large ribosome assembly, accumulates stably in mitochondria from ρ0 cells lacking both 21S and 15S rRNA (Fearon and Mason, 1988 ), whereas two early assembling proteins, MRP13 and MRP49, do not accumulate in either early ribosome assembly mutants or in strains lacking rRNA (Partaledis and Mason, 1988 ; Fearon and Mason, 1992 ; Biswas and Getz, 1999 ). We show here that the levels of Mrp7p, Mrp13p, and Mrp49p remained constant in mitochondria from cells harboring either a MTG2 or mtg2-1 allele after shift to 37°C (Figure 6), confirming that Mtg2p is not involved in a critical step in early ribosome synthesis.
In E. coli, overexpression of the Obg protein CgtAE suppresses both the growth and ribosome assembly defects of ΔrrmJ mutant, providing evidence that CgtAE is involved in the late assembly of the large ribosomal subunit (Tan et al., 2002 ). RrmJ is an rRNA methyltransferase responsible for methylation of U2552 within the peptidyl transferase center of the 23S rRNA (Bugl et al., 2000 ; Caldas et al., 2000 ). Nucleotide modification of the large rRNA molecule within the peptidyl transferase center is a universally conserved feature in prokaryotic, eukaryotic, as well as mitochondrial translation system. The 21S mitochondrial rRNA contains three modified nucleotides: one pseudouridine (Ψ2819) and two 2′-O-methylated nucleotides (Gm2270 and Um2791) (Sirum-Connolly and Mason, 1993 ; Ansmant et al., 2000 ; Pintard et al., 2002 ). In S. cerevisiae, the RrmJ ortholog Mrm2p is responsible for methylation of U2791 in 21S rRNA (analogous to position U2552 in E. coli) both in vivo and in vitro (Pintard et al., 2002 ). S. cerevisiae cells lacking MRM2 rapidly lose mitochondrial DNA, becoming ρ0 at 37°C (Pintard et al., 2002 ), a phenotype consistent with a role in late 54S assembly.
To provide evidence that Mtg2p functions late in 54S assembly, we tested whether multiple copies of MTG2 would suppress the Δmrm2 mutant. To do this, we monitored the rapid conversion from ρ+ to ρ0 in Δmrm2 cells incubated at 37°C in cells harboring either complementing MRM2 or MTG2, on plasmids. The addition of MTG2 on a CEN plasmid resulted in an approximately twofold increase in Mtg2p levels (Figure 7A). As shown previously (Pintard et al., 2002 ), Δmrm2 cells harboring a control plasmid rapidly lost mitochondrial DNA at 37°C and became ρ0 at a high frequency (monitored by growth on YPG), whereas Δmrm2 mutants complemented with MRM2 remained ρ+ (Figure 7, B and C). Interestingly, the introduction of MTG2 partially suppressed the loss of mitochondrial DNA in Δmrm2 mutants (Figure 7, B and C). The expression of MTG2 from a 2μ plasmid did not result in further suppression (our unpublished data). Thus, the introduction of extra copies of MTG2 partially suppresses the thermosensitive loss of mitochondrial DNA of Δmrm2 and is consistent with a role of Mtg2p in late ribosome assembly.
Ribosome biogenesis, in general, is a complex process requiring numerous factors that, although they interact with precursor ribosome intermediates and aid in their maturation into functional subunits, often are not a part of the mature ribosome. For example, in S. cerevisiae the assembly of cytoplasmic ribosomes requires the coordinated action of ~170 assembly proteins (Fromont-Racine et al., 2003 ). Many auxiliary proteins are required for temporal processing and modification of rRNA, coordinated loading of ribosomal proteins onto rRNA to form functional subunits and ribosomal export from the nucleus. These assembly factors include nucleases, ATP-dependent RNA helicases, and modifying enzymes, as well as a number of recently identified GTPases (Kressler et al., 1999 ; Venema and Tollervey, 1999 ; Fatica and Tollervey, 2002 ; Fromont-Racine et al., 2003 ). In E. coli, although ribosomal subunits can be reconstituted in vitro by using purified components, this process is slow and requires nonphysiological conditions (Herold and Nierhaus, 1987 ; Culver and Noller, 2000 ). In vivo, however, additional nonribosomal proteins (rRNA-modifying enzymes, chaperones, and GTPases) aid in efficient assembly of bacterial ribosomal subunits (Bugl et al., 2000 ; Tan et al., 2002 ; Inoue et al., 2003 ).
The assembly of mitochondrial ribosomes requires the coordinated synthesis of mitochondrially encoded rRNAs and nuclear encoded proteins as well as the import of these cytosolic proteins into the mitochondria and association into functional subunits. Interestingly, only few mitochondrial ribosome assembly factors have been identified (Sirum-Connolly and Mason, 1993 , 1995 ; Sirum-Connolly et al., 1995 ; Pintard et al., 2002 ; Barrientos et al., 2003 ). These include the 21S rRNA-modifying enzymes that are responsible for two 2′-O-methylated nucleotides (Gm2270 and Um2791) and one pseudouridine (Ψ2819) that form a part of the conserved peptidyl transferase center (Sirum-Connolly and Mason, 1993 ; Ansmant et al., 2000 ; Pintard et al., 2002 ). More significantly, two novel GTPases, Mss1p and Mtg1p, have been identified that functionally interact with 15S and 21S rRNA, respectively (Decoster et al., 1993 ; Barrientos et al., 2003 ). In addition, Mtg1p, is involved in biogenesis of the large ribosomal subunit (Barrientos et al., 2003 ). Mtg1p is a member of the YawG/YlqF subfamily of GTPases (Leipe et al., 2002 ) that includes the 60S biogenesis GTPases Nug1p and Nog2p.
In this study, we show that another putative mitochondrial GTPase, Mtg2p, also seems to play a role in the assembly of the large ribosomal subunit. We demonstrated that, as expected for a ribosome assembly factor, Mtg2p is localized to the mitochondria and peripherally associated with the inner membrane facing the mitochondrial matrix (Figure 2). Furthermore, deletion of mtg2 led to a decrease in mitochondrial translation and subsequent loss of mitochondrial DNA (Figure 1), a phenotype commonly found in cells defective in translation (Myers et al., 1985 ; Fearon and Mason, 1992 ).
Although the precise function of Mtg2p is unknown, it is likely that Mtg2p is either involved directly in the biogenesis of the mitochondrial large ribosomal subunit or plays an as yet undefined role in translation. We favor the former hypothesis for several reasons. First, Mtg2p is required for mitochondrial translation, as shown indirectly by the loss of mtDNA in a Δmtg2 mutant (Figure 1) and directly in the analysis of mitochondrial translation products in the temperature-sensitive mtg2-1 mutant (Figure 4). Second, Mtg2p associates specifically with the 54S subunit but is not a core ribosomal protein, as demonstrated by its dissociation in 500 mM NH4Cl. Third, Mtg2p is not necessary for early ribosome assembly, because yeast cells harboring the mtg2-1 allele did not display a drop in the stability of large ribosomal proteins that assemble early (Figure 6). Fourth, mtg2-1 mutants do not exhibit an altered ribosomal subunit ratio, but it did show reduced levels of both 34S and 54S ribosomal subunits at the nonpermissive temperature (Figure 5). It has been proposed that the relative levels of mitochondrial ribosomal subunits are coordinately controlled (Biswas and Getz, 1999 ). Thus, a reduction in the assembly of the 54S subunit may coordinately lower 37S subunit to maintain the optimal subunit ratio. Finally, extra copies of Mtg2p partially suppress the thermosensitive formation of petite cells in a Δmrm2 mutant (Figure 7). Mrm2p is responsible for 2′-O-ribose methylation of U2791 on the 21S rRNA which is, in turn, required for optimal mitochondrial ribosome function (Pintard et al., 2002 ). In vitro, Mmr2p methylates 21S rRNA that has already been assembled into the large 54S ribosomal subunit but not free rRNA, indicating that it is required for a late step in ribosome assembly (Pintard et al., 2002 ). Thus, suppression of growth defects of Δmrm2 by overexpression of Mtg2p is consistent with a role of Mtg2p in a late step of ribosome biogenesis.
A role for Mtg2p in mitochondrial ribosome function is also consistent with the proposed function of Obg proteins in general, as well as the association of bacterial Obg proteins with the 50S subunit (Lin et al., 2004 ; Wout et al., 2004 ) and the implied role of these proteins in a late step of 50S subunit biogenesis (Tan et al., 2002 ; Lin et al., 2004 ; Wout et al., 2004 ), specifically. Interestingly, in addition to a role in ribosome maturation, the bacterial Obg proteins also interact with proteins involved in stress response (Scott and Haldenwang, 1999 ; Scott et al., 2000 ; Wout et al., 2004 ), a function that has not been reported for and is unlikely in mitochondria. One possibility is that the cytosolic Obg proteins Rbg1p and Rbg2p are involved in stress response (Wout and Maddock, unpublished data) whereas the nucleolar (Fromont-Racine et al., 2003 ; Kallstrom et al., 2003 ; Saveanu et al., 2003 ) and mitochondrial Obg proteins (Nog1p and Mtg2p, respectively) may be exclusively involved in ribosome assembly. We are currently pursuing studies to determine the specific molecular role of Mtg2p in ribosomal synthesis.
We are extremely grateful to Drs. Michael P. Yaffe for antibodies against F1β, Robert E. Jensen for antibodies against Tim23p, Carla Koehler for antibodies against KDH, and Thomas L. Mason for antibodies against Cox2p. We also thank Dr. Suzanne Lybarger and Raji Janakiraman for technical assistance and Susan Sullivan for both technical assistance and critical reading of this manuscript. This work was supported by National Science Foundation MCB-0316357 and National Institutes of Health GM-55133 to J.R.M. and from a University of Michigan Rackham Merit fellowship to J.L.F.
Article published online ahead of print in MBC in Press on December 9, 2004 (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E04-07-0622).
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