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Mannosyl-Diinositolphospho-Ceramide, the Major Yeast Plasma Membrane Sphingolipid, Governs Toxicity of Kluyveromyces lactis Zymocin

ABSTRACT Kluyveromyces lactis zymocin, a trimeric (αβγ) protein toxin complex, inhibits proliferation of Saccharomyces cerevisiae cells. Here we present an analysis of kti6 mutants, which resist exogenous zymocin but are sensitive to intracellular expression of its inhibitory γ-toxin subunit, suggesting that KTI6 encodes a factor needed for toxin entry into the cell. Consistent with altered cell surface properties, kti6 cells resist hygromycin B, syringomycin E, and nystatin, antibiotics that require intact membrane potentials or provoke membrane disruption. KTI6 is allelic to IPT1, coding for mannosyl-diinositolphospho-ceramide [M(IP)2C] synthase, which produces M(IP)2C, the major plasma membrane sphingolipid. kti6 membranes lack M(IP)2C and sphingolipid mutants that have reduced levels of M(IP)2C precursors, including the sphingolipid building block ceramide survive zymocin. In addition, kti6/ipt1 cells allow zymocin docking but prevent import of its toxic γ-subunit. Genetic analysis indicates that Kti6 is likely to act upstream of lipid raft proton pump Kti10/Pma1, a previously identified zymocin sensitivity factor. In sum, M(IP)2C operates in a plasma membrane step that follows recognition of cell wall chitin by zymocin but precedes the involvement of elongator, the potential toxin target.

As judged from recent data that kti14 casein kinase I mutants express Elongator-like defects and that hyperphosphorylation of Elongator in sit4 phosphatase mutants coincide with zymocin survival, TOT function seems to be kept in check by Elongator phosphorylation (32,34,42). In support of Elongator's role as a key zymocin effector, its TOT function is further suppressed by Elongator proteolysis and by lack of protein urmylation (18,21). Notably, without Elongator, protein urmylation is affected and removal of TOT partner Kti11 enhances Elongator instability and defeats diphtheria toxicity (18,23,40).
Despite these advances towards identifying components of the toxin target process, the early steps of the zymocin response are poorly understood (53). Consistent with an impact on early events, class I mutations in KTI2/CHS3 and KTI10/ PMA1 protect solely against exozymocin and affect chitin synthesis and plasma membrane H ϩ -ATPase function, respectively (11,33,43). In line with this, holozymocin binds chitin in vitro and has exochitinase activity (10,33). As judged from the finding that hygromycin B, whose antibiotic action requires an intact membrane potential established by H ϩ -ATPase Pma1/ Kti10, is inert against kti10 cells, plasma membrane energization is likely to be required for zymocin action (43,50). Given the strongly hydrophobic nature of the ␤-subunit, to which the active ␥-subunit is linked via a disulfide bond, a tentative picture that emerges for early zymocin-induced events includes chitin docking followed by delivery of the ␥-toxin subunit to the membrane and subsequent subcellular import (33,43,56).
In support of class I zymocin protection, we present in this report evidence that kti6 cells express a membrane defect that allows them to survive antifungal drugs whose actions require an intact plasma membrane potential or that work by disrupting membranes. Consistently, KTI6 is allelic to IPT1, coding for mannosyl-diinositolphospho-ceramide [M(IP) 2 C] synthase, the terminal sphingolipid biosynthetic enzyme in yeast. In line with this, membranes of kti6 cells lack M(IP) 2 C and, in contrast to what occurs in Elongator tot-mutants, ␥-toxin import is blocked in kti6 cells. Presumably, early during the zymocin response, M(IP) 2 C acts in a step at the cell membrane that follows zymocin docking but precedes both the engagement of plasma membrane H ϩ -ATPase and Elongator. This suggests a possible function of M(IP) 2 C as a secondary membrane receptor required for ␥-toxin uptake.
Identification of KTI6/IPT1 and sphingolipid measurements. Zymocin was concentrated from 1.3 liters of YPD culture broth of K. lactis killer strain IFO1267 (Table 1) to 13 ml by hydroxyapatite column chromatography as described previously (59). We screened the EUROSCARF deletion set of 4,898 nonessential S. cerevisiae gene disruptions in the BY4741 background (Table 1) for altered sensitivity against K. lactis zymocin by transfer onto 96-well micro- EUKARYOT. CELL plates using an 8 by 6 array stainless steel replicator. Approximately 10 3 mutant cells were grown in 60 l of 1 M sorbitol-YPD liquid medium with 1:300 or 1:1,500 dilution zymocin in round-bottomed 96-well microplates at 30°C. Observation for 3 days revealed that the wild-type strain failed to grow in the presence of a 1:1,500 zymocin dilution. Two hundred six resistant mutants which grew on 1:300-diluted zymocin were retested to confirm their phenotype. KTI6 was isolated as follows. Zymocinresistant mutants isolated from the BY4741 deletion set (MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0 kanMX4 [G418 r ]) were grown for 18 h at 30°C and mixed with ARB27 (kti6-2), a killer-resistant mutant strain derived from LL20 (MAT␣ leu2 his3 URA3) (11) ( Table 1). After overnight incubation at 30°C, the mixed cells were suspended in 100 l sterilized water in a 96-well microplate and transferred onto YPD/G418 plates. Cells were resuspended in water and then transferred into SD medium without uracil. Upon replica plating twice on selective medium, diploids were concentrated by single colony isolation on SD medium without uracil and reselected on YPD/G418 medium.
Reverse transcription-PCR and PCR-based gene manipulations. For IPT1/ KTI6 expression studies, total RNA from yeast was isolated with RNAeasy columns (QIAGEN, Hilden, Germany). Following DNase I treatment, 4 g RNA was subjected to first-strand cDNA synthesis at 42°C for 1 h using 20 l reverse transcription-PCR mixtures (RevertAid kit; MBI Fermentas, Lithuania). Next, 1/10 was used in 25 PCR cycles using Taq  Immunological techniques. Protein extraction for use in Western blot studies was as described previously (19). Protein loadings were checked using an antibody against the yeast Pfk1 ␣ and ␤ subunits (a gift of J. J. Heinisch, University of Osnabrück, Germany) and Cdc19, pyruvate kinase (kindly provided by J. Thorner, University of California at Berkeley). Cell fractionation was done by sucrose ultracentrifugation (38). Separation of yeast cytoplasmic, soluble fractions from insoluble cell wall material followed a previous protocol (49). Rather than quantifying protein content from the insoluble and clotty pellet fractions, protein quantity was checked in the parallel supernatants by immuoprobing for phosphofructokinase content (see above). Zymocin treatment of S. cerevisiae cells prior to protein extraction or cell fractionation involved growth in 100 ml YPD to an optical density at 600 nm of Ϸ1.0 followed by addition of 50% (vol/vol) zymocin (see above) and further incubation for 6 h.

RESULTS
Phenotypic analysis of the kti6 mutant. Like kti2 and kti10 cells, the kti6 mutant was found to resist holozymocin (zym R ) in killer eclipse assays ( Fig. 1A) (11,33,43). Similar to the toxin-sensitive (tox S ) response of wild-type and class I kti10 cells, GAL1-promoter driven expression of zymocin's ␥-toxin caused kti6-2 cells to cease growth, while class II Elongator tot3⌬ cells resisted ␥-toxin (tox R ) (Fig. 1B). This typical class I response predicts kti6 cells to be affected early in the zymocin response rather than being altered in Elongator's toxin target (TOT) capacity. Based on further phenotypic studies, kti6-2 cells performed like the wild type, with viability not detectably being affected (Fig. 1C and D).
In contrast to a class II Tot Ϫ mutant (tot3⌬), kti6-2 cells expressed wild-type tolerance to caffeine and Calcofluor White (Fig. 1D). The latter contrasted with the pronounced resistance of class I chitin-deficient (kti2⌬/chs3⌬) cells to the chitin indicator drug Calcofluor White (Fig. 1D), suggesting that kti6-2 cells have normal chitin levels and do not resist zymocin by preventing chitin docking. In contrast to the ethanol sensitivities of chitin and Elongator defective strains, kti6-2 cells expressed ethanol tolerance like the wild type (Fig. 1D). Unlike the latter, however, kti6-2 cells recovered less well from heat shock although significantly better than kti10 cells (Fig.  1E). Together with strong resistance of kti6-2 cells to hygromycin B (Fig. 1F), an antibiotic that requires membrane energization through the H ϩ -ATPase Pma1/Kti10 (50), these phenotypes suggest that KTI6 is relevant for cell surface integrity. Consistently, kti6-2 cells resisted the membrane disruptor nystatin ( Fig. 1F) (35). As for the zymocin response, kti6-2 cells are therefore expected to express a plasma membrane defect that affects a step following chitin docking but that does not change Elongator's TOT capacity.
KTI6 is epistatic to KTI10, the plasma membrane H ؉ -ATPase gene. Since underassembly of the H ϩ -pump Kti10/ Pma1 causes class I zym R (43), we investigated the relationship between the KTI10 and KTI6 genes. In common with class I zymocin resistance, both kti10 and kti6-2 cells survived the antibiotic hygromycin B, with protection being more pronounced in kti6-2 cells (Fig. 1F). However, while the kti10 H ϩ -pump defect caused cell death at pH 3 (43), kti6-2 cells were pH insensitive (not shown). Studying pH effects on the zymocin response, kti6-2 cells remained zym R at pH 7 ( Fig. 2A) and were fairly resistant to zymocin at pH 5 ( Fig. 2A). In contrast, the zym R phenotype of kti10 cells was prominent at pH 7 but suppressed at pH 5 ( Fig. 2A). At pH 4, a condition inactivating zymocin, all strains tested were insensitive, indicating that the pH effect on kti10 was not due to impaired viability (not shown).
In support of this, the kti6-2 defect was complemented by a plasmid-coupled IPT1 gene on pITP1 (Fig. 3B) (58). Similarly, a zym R ipt1 W93STOP allele isolated in a screen for plant defensin DmAMP1 tolerance and shown to truncate the IPT1 product (60) was complemented by pIPT1, too (Fig. 3B).

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SPHINGOLIPID M(IP) 2 C IMPACTS TOXIN UPTAKE 883 M(IP) 2 C in governing a plasma membrane event required for zymocin toxicity and whose block in kti6 cells abrogates zymocin action. kti6 cells lack M(IP) 2 C and IPT1/KTI6 repression protects against zymocin. The amount of M(IP) 2 C in kti6-2 cells was determined by long term labeling with [ 3 H]myo-inositol. Following lipid extraction and separation by thin-layer chromatography, radiolabeled sphingolipids were detected and compared with sphingolipids in wild-type KTI6 cells and kti6-2 mutant cells transformed with pIPT1. kti6-2 cells (Fig. 6A, bottom panel) completely lacked the radioactive fraction representing alkali-stable M(IP) 2 C species typical of wild-type cells (Fig. 6A, top panel). Consistent with previous reports on M(IP) 2 C-minus strains (14), mutant kti6-2 cells apparetly com-pensated for the lack of M(IP) 2 C by up-regulating the level of MIPC (Fig. 6A, bottom panel).

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ZINK ET AL. EUKARYOT. CELL related with the zymocin response. Again, this shows the importance of Ipt1-mediated formation of M(IP) 2 C for zymocin action. kti6 cells allow zymocin to bind but prevent import of ␥-toxin. Taking into account that kti6-2 cells resist exozymocin without protecting against endogenous ␥-toxin, we probed for a kti6-2 defect in the primary contact with holozymocin. Together with other class I (chs3⌬ and kti10) mutants, protein extracts of zymocin-treated kti6-2 and wild-type cells were subdivided into soluble cytosolic and insoluble cell wall fractions. Following immunoprobing with anti-␥-toxin antibody, ␥-toxin was absent from cell wall fractions of chitin-deficient chs3⌬ cells (Fig. 7A), consistent with the known binding of holozymocin to chitin in vitro (33). Equivalent cell wall fractions from wild-type, kti10 and kti6-2 cells, however, produced reactive anti-␥-toxin signals that corresponded to a ␥-toxin control from culture filtrates of K. lactis killer cells (Fig. 7A). Hence, holozymocin is able to dock onto the cell wall of kti6 cells in a manner similar to sensitive wild-type or resistant Tot Ϫ and kti10/pma1 mutant cells. Moreover, indistinguishably from what occurs with wild-type sensitive or resistant kti10/pma1 cell walls, holozymocin is able to dock onto the surface of kti6-2 cells. This strongly suggests that the sphingolipid M(IP) 2 C is required after zymocin has attached to its chitin receptor.
To ask whether M(IP) 2 C-minus kti6-2 cells survive zymocin as a result of reduced ␥-toxin uptake, equal amounts of protein extracts from zymocin-treated kti6-2 and reference cells were subjected to cell fractionation and Western blots using anti-␥toxin and anti-Cdc19 antibodies. As expected, we found that in the absence of zymocin, ␥-toxin was not detectable in wild-type cells (Fig. 7B), while zymocin-treated cells significantly accumulated the toxic ␥-subunit intracellularly (Fig. 7B). In contrast, however, ␥-toxin import by M(IP) 2 C-minus kti6-2 cells was completely absent and copied the behavior of chitin-deficient chs3⌬ cells (Fig. 7A), which are unable to establish the primary contact with holozymocin (33, 43) (Fig. 7A). Given that kti6-2 cells allow zymocin contact, while endogenous ␥-toxin induces kti6-2 cell death (Fig. 1B), nondetectable ␥-toxin levels suggest an import or uptake defect of kti6-2 cells. Whether this reflects a role for sphingolipid M(IP) 2 C in ␥-toxin binding to or translocation across the membrane is currently being investigated.

DISCUSSION
Zymocin resistance of kti6 cells. Based on chitin affinity purification of zymocin and the refractory nature of chitindeficient chs3⌬ cells, zymocin's primary cell contact requires chitin interaction (33). Searching for nonchitin factors required for zymocin binding and/or uptake, kti6 cells are shown here to survive exozymocin but to be killed by a chitin poison (Calcofluor White) or intracellular ␥-toxin expression. These data indicate a response defect distinct from Elongator or chitin mutants and are consistent with our findings (Fig. 7) that ␥-toxin binds kti6 cells with normal efficiency, but does not enter their cytosol. Based on heat shock sensitivity and nystatin resistance, phenotypes shared by kti6 and kti10 plasma membrane H ϩ -ATPase mutants (50), KTI6 is possibly involved in membrane function. In support of this idea, resistance to zymocin and antifungals (hygromycin B, syringomycin E, and defensin DmAMP1) that require and alter membrane function and permeability suggests that kti6 cells deny the docking or uptake of antimycotics (15,58,60). This reinforces a plasma membrane role of KTI6 and its function for antizymosis.
Consistently, kti6 cells are complemented by the plasma membrane sphingolipid M(IP) 2 C synthase gene IPT1, fail to form M(IP) 2 C, and accumulate precursor sphingolipid MIPC. Together with our data that removal, truncation, or underproduction of Ipt1 phenocopies zymocin protection, while IPT1 overexpression enhances zymocin toxicity, it is clear that M(IP) 2 C is an integral zymocin sensitivity determinant. Yeast sphingolipids primarily populate the plasma membrane, with M(IP) 2 C representing the major fraction (27). They associate in lipid rafts (30) with proteins such as major H ϩ -pump Pma1/ Kti10 (2,3). Based on reports that sphingolipids influence lipid raft protein content, M(IP) 2 C-minus kti6 cells may impact the H ϩ -pump Pma1/Kti10 and thus evoke zymocin resistance similar to kti10/pma1 mutants (43).
Indeed, our finding that zym R of kti10 cells, in contrast to kti6 single and kti6kti10 double mutants, is suppressed by H ϩ suggests that KTI6 functions before KTI10. Also, the fact that kti6-2 cells suppress synthetic sickness associated with cooverexpression of Pma1/Kti10 and Ast1, a plasma membrane protein and suppressor of defective Pma1 H ϩ -pumps (3,12), shows that M(IP) 2 C sphingolipids are required to mediate membrane associated communication between Ast1 and Pma1/Kti10 that impairs cell performance and viability. However, green fluorescent protein studies indicated normal Pma1/ Kti10 distribution in ipt1/kti6 membranes (4), ruling out the option that Kti6 acts solely by Kti10 localization.
Role of M(IP) 2 C in zymocin action. Consistent with a role for sphingolipid biosynthesis in the zymocin response pathway, ceramide synthase lag1⌬lac1⌬ double knockouts and csh1⌬ csg1⌬csg2⌬ mutants with reduced MIPC and M(IP) 2 C levels are shown here to be zymocin resistant (54,63). As a major constituent of lipid rafts, M(IP) 2 C may influence raft-embedded proteins such as End6, Pma1/Kti10, or Ggp1 (2, 4). Indeed, cell surface defects of a ggp1⌬ mutant elicited hypersensitivity to zymocin, while kti10/pma1 cells were shown to resist the toxic complex in a class I fashion (33,43). Based on the tolerance of kti6 and kti10 cells to antifungals that require membrane energization by the H ϩ pump Pma1/Kti10, there may be raft communication between M(IP) 2 C and Pma1/ Kti10. Consistently, synthesis of sphingoid base and ceramide is required for Pma1/Kti10 function and exogenously applied phytosphingosine does rescue Pma1/Kti10 defects (39,64).
Based on our findings that kti6 protection against zymocin is barely affected by low pH in contrast to the pH-suppressible resistance of kti10, H ϩ -ATPase is likely to follow M(IP) 2 C function in promoting zymocin sensitivity. In spite of intact zymocin docking, lack of M(IP) 2 C in kti6 cells may prevent zymocin from binding to a secondary receptor in the plasma membrane required for ␥-toxin uptake. Whether M(IP) 2 C allows contact between ␥-toxin and such a receptor or represents this receptor, as speculated for toxicity of syringomycin E (58) and demonstrated for several plant defensins (60,61), remains open. However, the zymocin-relevant function of M(IP) 2 C cannot be replaced by its precursor sphingolipids MIPC or IPC, and this specificity may relate to the terminal myo-inositol phosphate moiety that distinguishes M(IP) 2 C from MIPC and IPC. A M(IP) 2 C-dependent membrane protein may serve as a second receptor, and although recently ruled out for lethality of plant defensin DmAMP1 (61), this scenario applies to yeast killer toxin K1. This ionophore hijacks, in a two-step-docking process, ␤-1,6-D-glucan and the glycosylphosphatidylinositol (GPI)-anchored protein Kre1 as primary cell surface and sec- , KTI6 wild-type cells (LL20) (upper panel) and thin-layer chromatography were as described previously (14). Peak sphingolipid frac-FIG. 7. Zymocin docking onto kti6 cells is unaffected while ␥-toxin import is denied. A. Zymocin docking studies. Protein extracts from zymocin-treated (ϩ) or nontreated (Ϫ) cells of the wild-type (wt; LL20) and resistant kti10 (ARB68), chs3⌬ (DJY3) or kti6-2 (ARB27) strains were fractionated into soluble (supernatant) and insoluble cell wall/debris (pellet) material. Following SDS-PAGE, supernatants were immunoprobed by anti-Pfk1 antibody (arrows indicate ␣ and ␤ subunits of Pfk1), while pelleted cell wall material was immunoprobed by an anti-␥-toxin antibody. Concentrated culture filtrate (cf) of the zymocin producer K. lactis strain AWJ137 served as a positive ␥-toxin control. B. ␥-Toxin import studies. Following sucrose gradient ultracentrifugation, cell fractions obtained from wild-type (LL20) cells or kti6-2 (ARB27) and chs3⌬ (DJY3) mutants that were treated (ϩ) or not treated (Ϫ) with zymocin were separated by SDS-PAGE and subjected to Western analysis using anti-␥-toxin and anti-Cdc19 antibodies. Concentrated killer culture filtrate (cf) served as a positive ␥-toxin reference (see A). Arrows indicate the positions of ␥-toxin and pyruvate kinase Cdc19.

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SPHINGOLIPID M(IP) 2 C IMPACTS TOXIN UPTAKE 887 ondary membrane receptors, respectively (7,29). Similarly, aerolysin, an Aeromonas hydrophilia toxin, binds GPI-anchored membrane proteins prior to multimerization (20). Finally, as membrane traffic and endocytosis are impacted by lipid rafts and sphingoid bases, M(IP) 2 C may rather be involved in internalization of ␥-toxin (30,66). Intriguingly, the phenotypes associated with loss of END6 are suppressed by mutations in IPT1, suggesting that M(IP) 2 C relates to the performance of End6, a lipid raft protein with roles in endocytosis and actin polarization (4,44). Although green fluorescent protein studies failed to demonstrate mislocalization of End6 in ipt1⌬ membranes, biogenesis and sorting of lipid raft proteins do require sphingoid base synthesis (4). M(IP) 2 Cminus kti6/ipt1 cells may nonetheless impair compartmentalization of membrane proteins to lipid rafts. Indeed, cell surface clustering of lipid rafts at the tips of mating projections has been shown to correlate with a 50% increase of raft-associated M(IP) 2 C content in response to pheromones (1). Whether kti6/ipt1 cells have mating defects comparable to those of other lipid biosynthetic mutants (1) will be important to know; also important will be studying whether sphingolipid mutants interfere with endocytosis of proteins other than ␥-toxin and whether this involves membrane docking or internalization steps.