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Mobility of cytochrome P 450 in the endoplasmic reticulum membrane

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signalyanchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retentionyretrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signalyanchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of f luorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 3 10210 cm2ys. A coefficient only slightly larger (7.1 3 10210 cm2ys) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes. The general rules governing mechanisms of subcelluar localization of membrane proteins remain poorly understood. Proteins in the membranes of the endoplasmic reticulum (ER), Golgi apparatus, plasma membrane, and transport and storage vesicles in the transport pathway are initially inserted into the membrane of the ER. The existence of positive or negative (retention) targeting signals and the formation of large complexes or networks that immobilize the protein and are incompatible with inclusion in transport vesicles have been proposed as mechanisms for targeting the proteins to their ultimate location (reviewed in ref. 1). Several lines of evidence suggest that a bulk-f low mechanism is a default pathway of transport from the ER, but the transport of secreted proteins at rates considerably higher than that of the bulk flow indicates that positive signals for efficient transport exist. Many ER resident proteins are transported from the ER and then retrieved by a retention signal-mediated process. The sequences KDEL and KKXX have been identified as retentiony retrieval signals for the soluble and membrane-bound ER proteins, respectively, and deletion of these sequences results in transport out of the ER, supporting bulk flow as a default pathway (1–3). Similar signals have not been detected for membrane proteins that are restricted to the ER without retrieval. Likewise, discrete signals specifying retention in the Golgi have not been identified; relatively long hydrophobic transmembrane segments and flanking regions of the proteins are important for retention (4, 5). Subsequently, it was suggested that retention of Golgi membrane proteins is mediated by ‘‘kin’’ recognition and formation of large oligomeric structures, the immobility of which prevents further transport (6, 7). Similarily, it also has been proposed that oligomerization and network formation immobilize andyor prevent transport of resident ER membrane proteins that do not have defined protein retention signal motifs (8, 9). Cytochromes P450 (P450) are resident ER membrane proteins that are retained directly in the ER and do not undergo recycling through the pre-Golgi compartment (10, 11). P450 is targeted to the ER by an N-terminal signal sequence, which is not cleaved; it also serves to anchor the protein to the membrane (12, 13). Redundant signals for retention are present in P450 because both the N-terminal signalyanchor and the catalytic domain cause retention of heterologous proteins in the ER (11, 14, 15). Specific sequence motifs have not been identified as retention signals, and multiple regions of the catalytic domain appear to contribute to the retention function (11, 14). These negative results are consistent with the hypothesis that the mechanism of P450 retention in the ER may be related to formation of a network or aggregate, rather than requiring specific sequence motifs as signals. Furthermore, P450 is known to form oligomers (16) and, additionally, it forms molecular complexes with its two partners, cytochrome P450 reductase and cytochrome b5, both ER membrane proteins (17), which potentially could result in a large network or an aggregation, thus immobilizing P450. The mobilities of membrane proteins have been estimated by measuring the recovery of fluorescence in a photobleached area that results from the movement of unbleached molecules into the bleached area (18, 19). Until recently, such studies have been largely restricted to cell-surface proteins, which can interact directly with fluorescent antibodies, whereas similar studies on intracellular proteins required microinjection of fluorescent antibodies. Introduction of the green fluorescent protein (GFP) as a fluorescent tag for subcellular localization studies (20) permits measurements of the mobilities of intracellular proteins. This technique has been applied to the Golgi-specific proteins, which were found to have mobilities greater than those of plasma-membrane proteins, arguing strongly against their retention in the Golgi via immobile network formation (21). Mobilities of ER resident proteins The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9514793-6$2.00y0 PNAS is available online at www.pnas.org. Abbreviations: P450, cytochrome P450; ER, endoplasmic reticulum; GFP, green fluorescent protein; EGFR, epidermal growth factor receptor; D, diffusion coefficient. †To whom reprint requests should be addressed at: Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, IL 61801. e-mail: byronkem@uiuc.edu.