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J. Biol. Chem., Vol. 275, Issue 25, 19409-19415, June 23, 2000

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Endoplasmic Reticulum Retention Determinants in the Transmembrane and Linker Domains of Cytochrome P450 2C1^*

Elzbieta Szczesna-Skorupa and Byron Kemper

From the Departments of Molecular and Integrative Physiology and Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received for publication, March 21, 2000, and in revised form, April 18, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cytochrome P450 2C1 N-terminal signal anchor sequence mediates direct retention of the protein in the endoplasmic reticulum and consists of a hydrophobic transmembrane domain, residues 3-20, followed by a hydrophilic linker, residues 21-28. Fusions of the N-terminal 21 or 28 amino acids of P450 2C1 to green fluorescent protein resulted in endoplasmic reticulum localization of the chimera in transfected cells. Disruption of microtubules by nocodazole treatment resulted in redistribution into a punctate pattern for the 1-21, but not for the 1-28, chimera indicating that the linker was preventing transport from the endoplasmic reticulum but was not required for retrieval to the endoplasmic reticulum from the pre-Golgi compartment. In the 1-28 chimera, mutations of residues 21-23 (KQS) in the linker resulted in redistribution of the chimera after nocodazole treatment. Mutations in the transmembrane domain affected both direct retention in the endoplasmic reticulum and retrieval from the pre-Golgi compartment, and although structural requirements for each process are distinct, in both cases the arrangement of amino acids and distribution of hydrophobicity are critical. In contrast, the linker region exhibits a sequence-specific requirement for direct retention in the endoplasmic reticulum.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sorting of membrane proteins entering the secretory pathway starts with their insertion into the endoplasmic reticulum (ER)¹ membrane. Further targeting of the proteins results from either selection for transport by packaging into transport vesicles or by exclusion from transport resulting in the ER retention. Selection of a protein for inclusion in transport vesicles may depend on the specific interaction of vesicle coat subunits or adaptors with targeting signals present in the protein (for reviews, see Refs. 1-3). Retention in the ER can result from the lack of a positive sorting signal, the presence of a signal for retrieval of the protein from pre-Golgi compartments if a protein is compatible with packaging into the transport vesicles, or a combination of both. For proteins completely excluded from the transport vesicles, a negative signal or property must be present that either prevents the protein from being included in the vesicles or targets it to an area of the ER away from transport vesicle formation.

In the absence of positive transport signals, localization of a protein may result from the properties of the transmembrane domain (TMD) and its interaction with the membranes. It has been postulated that the main characteristic distinguishing proteins localized to the membrane of the Golgi apparatus or plasma membrane is the length of their hydrophobic TMDs, shorter ones being characteristic for the Golgi and longer ones for the plasma membrane (4). As a result of a higher content of cholesterol, the plasma membrane is usually thicker than the membranes of the Golgi, thus the longer TMDs would be selected for the transport out of the Golgi to the plasma membrane, whereas those with shorter TMDs would be retained in the Golgi (5). However, it has also been shown that specific amino acid sequences in the TMD of the protein and its flanking region and/or network formation may contribute to selective retention in the Golgi (6-8).

In contrast, there is no consistent difference in TMD length between ER membrane proteins and those in the Golgi. This suggests that other properties of these sequences are responsible for their function in selective localization of proteins in either organelle. Although several studies on transmembrane anchors of ER membrane-localized proteins have been described, the actual signal-mediating retention of these proteins has not been defined. Significant differences in the mechanism of these retention signals are likely, because some of them mediate direct retention (exclusion from the transport vesicles) (9-13), whereas others function via the retrieval pathway (14-16). In addition, studies on ER and Golgi membrane resident proteins show that their retention may be mediated not only by the TMD but also a cytoplasmic domain (7, 14, 17). Contributions of multiple domains to the final disposition of the protein have been shown for several proteins (18-22).

P450 2C1/2 are unusual type I proteins in which the N-terminal signal sequence spans the membrane with the N terminus on the luminal side (9). The N-terminal sequence of P450 2C1 fused to several reporter proteins mediates the localization to the ER of the chimeric proteins (23, 24). Either the first 21 amino acids, which includes the TMD of the protein and terminates with a charged residue, lysine, or the first 28 amino acids, which includes seven additional, mostly hydrophilic, residues, have an ER retention function. Amino acids 21-28 exhibit length, but relaxed sequence, requirements for efficient assembly of P450 2C2 suggesting that they function as a linker sequence (25). The amino acid sequence and/or structure responsible for ER retention is not known. It has been shown that microsomal P450 2C2 and M1 are directly retained in the ER and do not undergo recycling through a pre-Golgi compartment (9, 10). P450 2C2 has been also shown to contain a redundant ER retention signal in its cytoplasmic (catalytic) domain (17). In contrast, ER retention of P450 2E1 has been shown to involve recycling, and only the N-terminal signal anchor has an ER retention function (26). Thus, some discrete sequence or structural differences between these enzymes are responsible for their differing ability to escape the retention in the ER. Although most P450s are localized to the ER, some microsomal P450s have been detected in the Golgi or plasma membrane (27-29). This escape from the ER might be related to different mechanisms of retention, as for P450 2C2 and P450 2E1, or might occur only under certain physiological conditions.

To characterize the sequence or structural requirement of the N-terminal ER retention signal of microsomal P450 2C1, we analyzed the effect of mutations in the transmembrane domain and linker region on the subcellular localization of green fluorescent protein (GFP) fused to the mutated sequences. Our results unexpectedly indicate that exclusion of P450 2C1 from ER transport vesicles is dependent on three specific amino acids in the linker region. Changes in the linear gradient of hydrophobicity along the transmembrane domain or across the membrane helix (amphipathicity) also can result in transport out of the ER. Retrieval back to the ER of the mutant proteins that enter ER transport vesicles is not dependent on the linker sequence but mutations in the transmembrane domain affect retrieval. The sequence requirements of the transmembrane domain for exclusion from entry into ER transport vesicles are different from those for retrieval.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Tran³⁵S-label was from ICN Radiochemicals (Costa Mesa, CA), N-glycosidase F was from New England BioLabs, the antibody against GFP was from Roche (Indianapolis, IN), and protein A-Sepharose was from Amersham Pharmacia Biotech. Cell culture media and antibiotics were from Life Technologies, Inc. and calf serum and nocodazole were from Sigma.

Plasmid Constructions-- Chimera C1-(1-28)/GFP was constructed as described (24). All mutations of the P450 N-terminal signal anchor were prepared by polymerase chain reaction using C1-(1-28)/GFP DNA as a template and a set of designed primers. 5'-primers had BglII sites and 3'-primers HindIII sites introduced in them, so that all polymerase chain reaction-amplified DNA fragments were inserted into the same site (BglII and HindIII) of the GFP vector, pEGFP-N1 (CLONTECH). Construction of the N-terminal signal with a glycosylation tag in front of it, NC1, was described previously (9). The glycosylation tag sequence contains amino acids 150-178 of P450 2C2 with an N-glycosylation site at asparagine 160. To construct a plasmid encoding a glycosylation tag at the C terminus of GFP, the sequence encoding the tag was amplified with a set of oligonucleotide primers that introduced a NotI site at the 5'-end and a BsrG I site at the 3'-end. The amplified DNA was digested with these enzymes and inserted into the pEGFP-N1 vector digested with the same enzymes.

Expression in COS1 Cells-- COS1 cells were transfected with the expression plasmids using liposome-mediated DNA transfer as described (30). Subcellular localization of chimeric proteins by fluorescent microscopy was analyzed in cells transfected for 48 h and, when indicated, incubated with 10 µM nocodazole for 2 h before fixation with 4% paraformaldehyde (24). Biosynthetic radiolabeling of transfected cells, immunoprecipitation with anti-GFP antibody and N-glycosidase treatment were performed as described (9, 24).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Linker Mutations Affect Direct ER Retention and Induce Transport to the Post-ER Compartment-- The sequence of the 28-amino acid P450 2C1 N-terminal signal anchor sequence, which is sufficient to mediate ER retention of several reporter proteins (23, 24), is shown in Fig. 1. The signal anchor includes a hydrophobic core, flanked by Asp² and Lys²¹, which represents the TMD and a mostly hydrophilic linker peptide (amino acids 21-28). We have shown that the N-terminal 1-28 signal anchor mediates direct retention in the ER without recycling through the pre-Golgi compartment (26). The first 21 amino acids also target reporter proteins to the ER, but whether the proteins are directly retained in the ER by this sequence has not been studied. The 1-21 sequence fused to GFP-mediated ER retention as expected (Fig. 2C), although in some cells Golgi-like localized chimeras were also observed. To test whether ER retention mediated by the 1-21 segment prevents transport out of the ER, we analyzed the effect of nocodazole on the localization of the chimeras. If an ER-retained protein is maintained in the ER by retrieval from the pre-Golgi compartment, disruption of microtubules with nocodazole results in redistribution of the protein into a punctate pattern characteristic of fragmented Golgi (31). As Fig. 2D shows, nocodazole treatment of cells transfected with the chimera C1-21/GFP, results in a shift in the localization of the protein to a punctate pattern (in about half of the cells), which is consistent with its presence in the early Golgi compartment. A punctate pattern obtained by immunostaining these cells with antibodies to sec13, which is predominantly in the pre-Golgi compartment (26), was colocalized with the punctate fluorescence from C1-21/GFP (data not shown). In contrast, nocodazole did not affect the distribution of the chimera with the N-terminal 28 amino acids fused to GFP (Fig. 2, A and B). Thus, in the absence of the linker region, chimeras localized in the ER are not strictly excluded from ER transport vesicles as the 1-28 P450 2C1 chimeras are. To identify the structural determinants in the linker region required for static ER retention, we analyzed the effects of nocodazole treatment on the localization of chimeras containing a series of mutations introduced into the linker sequence (Fig. 1B). Representative photographs of cells expressing some of the GFP chimeras are shown in Fig. 2, and a summary of the results is in Fig. 1. A chimera in which the linker sequence was replaced with six Ala (C1-20/6A) was localized in the ER, but nocodazole treatment resulted in the redistribution of the protein into a punctate pattern (Fig. 2, E and F). To delineate which amino acids were important for the ER retention function, additional mutations were made in the linker region. Mutation of the three Gly (25-27) to Ala did not affect the distribution of the chimeric protein (not shown). However, substitution of three Ala for Lys²¹-Ser²²-Gln²³ resulted in the redistribution of the chimera after nocodazole treatment (Fig. 2, G and H). Substitution of Ala for both Gln²² and Ser²³ also resulted in a chimera that was redistributed by nocodazole treatment. Individual substitutions of Asn for Lys²¹ (C1-20/KN) or Val for Ser²³ (C1-20/SV) did not affect the distribution of the chimeric proteins in nocodazole-treated cells. However, when substitutions of Asn and Val for Lys²¹ and Ser²³, respectively, were made, the chimeric protein was redistributed after nocodazole treatment (Fig. 2, I and J). These data suggest that the specific sequence of Lys-Gln-Ser in the linker sequence is important for exclusion of the chimeric proteins from the ER transport vesicles.

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence of the native P450 2C1 N-terminal signal anchor and of mutations in the linker region (residues 21-28) (A), transmembrane domain 1-21 (B), and in both regions (C). The gap separates the transmembrane domain and the linker region. The mutated residues are in bold and are underlined. At the right, the cellular distributions of the chimeras in absence or presence of nocodazole (Noc.) are summarized. + indicates that the distribution was that expected for localization in the ER, and  indicates that the distribution was not consistent with ER localization in most cells. nd, not determined. In D, helical wheels representations of wild-type transmembrane domain 1-21, and selected mutants in an -helical form are shown. Polar and charged amino acids are shown in black.

View larger version (57K): [in this window] [in a new window]   Fig. 2.   Linker sequence mutations interfere with direct retention of C1-28-GFP chimera in the ER. 48 h after transfection with expression plasmids, COS1 cells were incubated for an additional 2 h without (A, C, E, G, and I) or with (B, D, F, H, and J) 10 µM nocodazole. The cells were fixed and photographed using a fluorescent microscope. The panels show representative images of cells transfected with GFP chimeras containing the following at the N terminus: C1-28 (A and B), C1-21 (C and D), C1-20/6A (E and F), C1-20/3A (G and H), and C1-20/NV (I and J).

Mutations of the TMD of P450 2C1 Affecting Direct ER Retention-- To test whether the hydrophobic TMD also plays a role in exclusion from the transport vesicles, we analyzed the effect of nocodazole on the behavior of chimeras with mutations in this sequence. Several possible properties of the TMD could contribute to its role in exclusion of the chimeric proteins from the ER transport vesicles. The length of the hydrophobic region of the TMD and the linear gradient of hydrophobicity along the TMD may play a role in the targeting of Golgi or ER membrane proteins (5, 14, 16). In addition, TMDs are usually predicted to adopt an -helical conformation in the membrane, and the amphipathicity of the helix could alter the function of the protein. Polar amino acids in the TMDs and their position in the -helix have been suggested to be important for retention in the ER and Golgi for some proteins (8, 14, 16, 32). To examine these possibilities, we have constructed mutations in the 1-28 fragment of P450 2C1 that replace polar with nonpolar amino acids, which alter the arrangement of amino acids in the TMD without changing the length or overall hydrophobicity of the TMD, and that insert amino acids to alter the length and/or hydrophobicity characteristics of the TMD (Fig. 1, B and D). Glu² was not altered in these mutants, because substitution of basic residues for this amino acid results in translocation of P450 across the membrane (33).

To examine the requirement for the nonhydrophobic amino acids, substitutions of Leu for Gly⁸, Cys¹⁰, Ser¹², and Cys¹³ in the middle of the TMD (mutation 4L) or for Ser¹⁸ and Trp²⁰ near the C-terminal end (mutation SWLL) were made. In addition to changing the specific amino acids, these substitutions increase the overall hydrophobicity of the TMD. The 4L mutation also alters the linear gradient of hydrophobicity by shifting its peak to the center of the TMD, and the increase in C-terminal hydrophobicity in SWLL also reduces the N to C terminus linear gradient. Nevertheless, neither of these mutants exhibited altered distribution of the chimeric proteins in the cells and no redistribution was observed after treatment with nocodazole (Fig. 3, A and B). However, when all the nonhydrophobic residues from Gly⁸ to Trp²⁰ were replaced by leucines (mutant 14L), the chimeric protein was not retained in the ER and was transported to the plasma membrane (Fig. 3G). This mutation does not change the overall length of the TMD; however, it increases the total hydrophobicity and reverses the polarity of its linear gradient, which is higher at the C-terminal side than at the N terminus.

View larger version (76K): [in this window] [in a new window]   Fig. 3.   Subcellular localization of chimeras with TMD mutations and effects of nocodazole treatment. COS1 cells were transfected, treated with nocodazole, and analyzed as described in the legend to Fig. 2. The cells were transfected with GFP chimeras containing the following at the N terminus: 4L (A and B), SLLS (C and D), C6L (E), N6L (F), 14L (G), and REV (H). Nocodazole-treated cells are shown in B, D, and F.

Two mutations were examined, which alter the arrangement of the amino acids in the TMD. Switching the position of Ser¹⁸ and Leu¹⁶ (mutant SLLS) is predicted to increase the amphipathicity of the -helix and creates a longer hydrophobic face on one side of the helix (Fig. 1D). This mutant was localized to the ER, but treatment with nocodazole resulted in a change of distribution characteristic of proteins that are transported from the ER and retrieved back to the ER from a pre-Golgi compartment (Fig. 3, C and D). A more extensive redistribution of residues was constructed by switching the amino acids in the N-terminal half of the TMD with those in the C-terminal half (Fig. 1B, mutant REV). The main effect of this change is to reverse the linear hydrophobic gradient along the helix. This mutant is not efficiently retained in the ER and has a heterogenous distribution pattern. In most cells, it is present in punctate/vesicular structures but also has a Golgi-like distribution and is detected on the surface of the cells (Fig. 3H). In the presence of brefeldin A, which disrupts the Golgi and prevents transport from the ER (34), this chimeric protein has an ER localization, indicating that it is initially inserted into the ER and is transported to post-ER compartments in the untreated cells (not shown). Because the overall length and hydrophobicity of the TMD were not changed in these mutants, these data indicate that the arrangement of the amino acids affecting the amphipathicity or the linear hydrophobic gradient of the TMD are important for exclusion of the chimeric proteins from ER transport vesicles.

The role of the three valines at positions 4-6 were examined by replacing these residues with either leucine or alanine. With the leucine substitutions, the overall hydrophobicity and the linear gradient are not greatly changed while the alanine substitutions decrease both. Both mutants were localized in the ER and nocodazole treatment had little effect on their distribution (not shown). The valines are, therefore, not specifically required for exclusion from ER transport vesicles, and altering the linear gradient substantially also does not affect the transport from the ER.

To examine the importance of the length of the TMD on ER retention, leucine or alanine residues were inserted at either the N-terminal end or the C-terminal end of the TMD. Insertion of two leucines before Ser¹⁸ (mutant C2L) did not affect the ER localization of the chimera, but nocodazole treatment caused a redistribution of the protein so that increasing the length allowed the protein to enter the ER transport vesicles (not shown). Insertion of two residues increases the length of the TMD but also rotates the -helix by about 240^o and in this case increases amphipathicity (Fig. 1D). Therefore, six leucines were inserted before Ser¹⁸ (Fig. 1B, mutant C6L), which more dramatically increases the length whereas maintaining the helix in approximately its normal rotational orientation relative to the rest of the protein. For C6L, a significant fraction of the protein molecules were present at the surface of most cells indicating that these chimera were no longer excluded from the ER transport vesicles (Fig. 3E). Insertion of six alanines before Ser¹⁸, like insertion of the leucines, resulted in transport of the chimera to the plasma membrane (not shown), so that the ability of the C6L protein to enter ER transport vesicles was not primarily the result of the increase in hydrophobicity but, more likely, the change in length of the TMD. In contrast, insertion of six leucines at the N-terminal side of the TMD, before Val⁴ (mutant N6L), did not affect the ER distribution of the chimera nor did nocodazole treatment affect distribution in most cells. In about 30% of cells, however, nocodazole induced a shift to punctate staining indicating that the protein was less efficiently excluded from the ER transport vesicles than wild type (Fig. 3F). These results indicate that the increased length of the TMD reduces the efficiency of exclusion of the chimeras from the ER transport vesicles.

Increasing the length of the hydrophobic core region can convert the TMD from a stop transfer signal to a translocation signal (35, 36), which might alter distribution of the protein. To examine this possibility in mutants with six leucines inserted, a 29-amino acid peptide encoding a signal for N-glycosylation was inserted at the N terminus of C6L and N6L. In both cases, a slower migrating glycosylated form was observed indicating that the N terminus was oriented into the lumen as expected (Fig. 4, lanes 2 and 4). However, the glycosylation level of C6L was lower than that of N6L, which may reflect less efficient glycosylation of C6L, but might also be because of a reversed orientation of a significant fraction of the C6L molecules. To examine this possibility, the glycosylation tag was added to the C terminus of GFP in chimera C6L. No glycosylation of the C-terminal tag was observed, which indicates that C6L has the expected membrane topology (Fig. 4, lanes 6 and 7).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Glycosylation of the GFP chimeras containing lengthened hydrophobic cores in the TMD. COS1 cells were transfected with the GFP chimeras containing a 29-amino acid glycosylation tag sequence attached to the N terminus of either mutant N6L (lanes 2 and 3) or C6L (lanes 4 and 5) or to the C terminus of GFP in mutant C6L (lanes 6 and 7). Lane 1 shows the position of the N6L chimera without the glycosylation tag. Radiolabeled proteins were immunoprecipitated with anti-GFP antibody and subjected to the digestion with N-glycosidase F (PF) (lanes 3, 5, and 7).

Mutations in the TMD Affecting Retrieval to the ER-- The results above show that deletion of the linker sequence or mutation of residues 21-23 in the linker results in most cases in the transport of the chimeric protein from the ER followed by retrieval from a pre-Golgi compartment back to the ER. This indicates that a signal for retrieval must be present in the TMD. Even with a wild type linker sequence, some TMD mutations resulted in transport of the proteins to the Golgi or plasma membrane indicating that signals to exclude the proteins from the ER transport vesicles and the retrieval signal were both defective. These mutants included C6L, C6A, and 14L so that mutations of all the polar residues in the TMD (14L) or lengthening the TMD at the C-terminal side (C6A and C6L) interfered with retrieval from the pre-Golgi compartment.

To better define the TMD sequence requirements for retrieval, TMD mutations were examined in the presence of the linker sequence containing substitutions of Asn for Lys²¹ and Val for Ser²³ (Fig. 1C). In these constructs, the effect of the TMD mutations on retrieval from the pre-Golgi compartment could be examined independently of effects on exclusion from transport vesicles. Similar results were also obtained when TMD mutations were examined in the absence of the linker or with a linker mutated to alanines at positions 21-23 (not shown). There were clearly different requirements for the retrieval of the protein from the pre-Golgi compartment compared with that for exclusion from ER transport vesicles (summarized in Fig. 1C). The mutation SWLL did not affect either process (Fig. 5A). Mutations SLLS and C2L, which interfere with exclusion from ER transport vesicles, did not affect retrieval (C2L/NV not shown, SLLS/NV shown in Fig. 5B). Conversely, two mutations that had little effect on exclusion of the proteins from ER transport had dramatic effects on retrieval. In the context of linker mutations, substitution of leucines for the four nonhydrophobic residues in the middle of the TMD (mutant 4L) resulted in localization of chimeric proteins on the surface of cells (4L/NV, Fig. 5C). Similarly, substitution of valines 4-6 to Ala (mutant 3V3A) resulted in Golgi and surface localization of a significant fraction of the protein in most cells (3V3A/NV, Fig. 5D). Interestingly, substitutions of leucines for these valines did not affect retrieval (not shown). These results indicate that the requirements for retrieval and exclusion from ER transport vesicles are distinct but overlapping. Extension of the length of the C-terminal region of the TMD affects both, whereas hydrophobicity at the N-terminal side and the nonhydrophobic residues in the center of the TMD are important for retrieval but not vesicle exclusion.

View larger version (149K): [in this window] [in a new window]   Fig. 5.   Effect of mutations in the TMD on retrieval to the ER from the pre-Golgi compartment. COS1 cells were transfected and analyzed as described in the legend to Fig. 2 with GFP chimeras containing the following at the N terminus: SLLS/NV (A), SWLL/NV (B), 4L/NV (C), and 3V3A/NV (D).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Retention of microsomal P450s in the endoplasmic reticulum is mediated primarily by the N-terminal signal anchor of the proteins. Although a redundant ER retention signal is present in the cytoplasmic catalytic domain of P450 2C1/2, only the N-terminal signal anchor mediates ER retention of another P450, 2E1 (26). These two proteins differ also in that P450 2C1/2 is strictly retained in the ER, whereas P450 2E1 is transported from the ER and is maintained as an ER protein by retrieval from a pre-Golgi compartment (26). Mutagenesis of the N-terminal sequence of P450 2C1, fused to a GFP reporter, indicates that both the TMD, which terminates at Lys²¹, and the linker region from 21-28 are important determinants for the exclusion of P450 2C1 from ER transport vesicles. The linker requirement is relatively sequence-specific so that mutation of two of the three residues from 21-23 permits transport of the chimeric protein from the ER as assessed by a redistribution of the protein after treatment with nocodazole. This sequence requirement suggests that relatively specific interactions with membrane lipids or proteins are required for this function. In contrast, the requirements in the TMD are relatively sequence-independent. Mutations that increase the amphipathicity and alignment of polar residue along one face of TMD in an -helical form (mutants SLLS and C2L) or that increase the length of the hydrophobic core and its overall hydrophobicity (mutants C2L, N6L, C6A, C6L, and 14L) result in transport of the proteins from the ER. Similar conclusions were reached for the resident ER proteins Ufe1p and UBC6 (32, 37). Lengthening the TMD of Ufe1p or UBC6 or rearranging the order of the amino acids of Ufe1p allowed transport from the ER. Interestingly, in P450 2E1, the hydrophobic core is longer than for P450 2C1, and the sequence of the linker region is different as well, including two differences in residues 21-23. These sequence differences probably underlie the inability of the N-terminal sequence of P450 2E1 to exclude the protein from the ER transport vesicles.

Changes in the P450 2C1 TMD, which affect exclusion of the chimeric proteins from the ER transport vesicles, are those that may potentially change the topology or the position of the peptide in the membrane. Lengthening the hydrophobic core may change the amino acids at the cytoplasm-membrane interface or result in tilting of the peptide in the membrane. Likewise, it has been suggested that amphipathic helices may insert obliquely in the membrane so that increasing amphipathicity might increase the tilt of the TMD (38). Such tilting may have a substantial functional significance, for example, a correlation has been found between the predicted orientation of some viral proteins in the membrane and membrane fusion (38) and between the predicted tilt of secretory signals and the efficiency in mediating secretion (39). In P450 2C1, the three amino acids immediately following the TMD in the linker region also are critical for exclusion of the proteins from the ER transport vesicles. This fact combined with the observations that mutations near the C-terminal portion of the TMD appear to be more disruptive that those at the N-terminal side (compare mutation of residues 4-6, 3V3A with the SLLS or CL2 mutations near the C-terminal side) suggests that the C-terminal region of the TMD extending three amino acids into the linker are most critical for vesicle exclusion. A plausible, but speculative, explanation then would be that the orientation or spatial relationship of this region relative to the membrane is critical for vesicle exclusion and that changes in the length, hydrophobicity, or amphipathicity of the TMD would alter this relationship. The sequence requirements in the linker region suggest that a relatively specific interaction of this region with lipids or other protein is important. These interactions could contribute further to maintaining the proper relationship of the TMD to the membrane or could be primary interactions mediating vesicle exclusion. In the latter case, changes in the TMD might affect the orientation of the linker relative to the membrane and interfere with these specific interactions.

An alternative explanation is based on a hypothesis that in the absence of positive sorting signals, only the physical properties of the TMD sequence determine the sorting of the proteins, which results in partitioning of the protein into membrane microdomains of differing lipid composition as has been suggested for Golgi (5) and ER (37) proteins. Recently, TMD-mediated interaction of viral hemagglutinin with lipid rafts has been shown to be involved in polarized sorting and possibly vesicular transport (40). Association with the raft lipids depended on the hydrophobic residues in the exoplasmic half of the TMD of the protein, and it was suggested that they could shape the TMD into a conformation compatible with a microdomain of high cholesterol. It is possible that budding vesicles represent such membrane microdomains of unique composition or thickness; the thickness of the ER membrane and the transport vesicle membrane differs (41). The length of the TMD or its tilt in the membrane, which would affect its cross-membrane length, could determine whether or not a protein is included in the transport vesicles. The effects of lengthening or altering amphipathicity of the P450 2C1 TMD are consistent with this hypothesis.

Deletion of the linker region or mutation of residues 21-23 resulted in transport from the ER, based on redistribution of the proteins after treatment with nocodazole, but did not affect the ER distribution in untreated cells. The linker sequence, therefore, is dispensable for retrieval of the protein back to the ER from the pre-Golgi compartment, and the retrieval signal must reside in the TMD. P450 lacks any of the known ER retrieval signals, KDEL, KKXX, or RRXX (1). Analysis of ER retention of TMD mutations in chimeras with deleted or mutated linker sequences indicated that the polar residues in the center of the TMD (4L) and high hydrophobicity in the N-terminal region (3V3A) were important for retrieval. These requirements differ from those for exclusion of the protein from the ER vesicles, because neither of these mutations resulted in the transport of the protein from the ER. Insertions of leucines or alanines at the C-terminal end, but not the N-terminal end, also interfered with retrieval again differing from effects on vesicle exclusion, which was affected by insertions at either end, so that the structural requirements for these two processes are clearly different. The only known receptor that may interact with TMDs in the retrieval of ER membrane proteins that do not contain known signal motifs is Rer1p (42-45). Similar to the observations with the P450 2C1 TMD, the presence of polar residues in the TMD of Gas 1p (16) and a gradient of hydrophobicity in the TMD of sec12 (14) were suggested to be important for Rer1p-mediated retrieval and, thus, Rer1p could be a candidate receptor for the retrieval of the P450 2C1 chimeric proteins. However, membrane proteins like Ufe1p are retrieved independently of Rer1p so that other receptors or adaptors for retrieval may exist.

Increasing the length of the TMD disrupts ER retention of cytochrome b₅ (46) and UBC6 (37), which has led to the proposal that the length of the TMD is the primary determinant for TMD-based sorting. This idea is analogous with the hypothesis that the length of the TMD is important for distinguishing targeting to the Golgi and plasma membrane (4, 5). Although the length of the TMD also affects transport from the ER and retrieval mediated by the P450 2C1 TMD, other properties are also important, because mutations that do not lengthen the TMD (substitution of alanines for valines 4-6) interfered with retrieval, and a mutation that increased the length of the TMD (insertion of six leucines before Val⁴) did not affect retrieval. Likewise, both changing the length of the TMD and the rearranging of amino acids in the TMD affected ER retention of Ufe1p (32). The importance of the length of the TMD may depend in part on the location of the TMD. In contrast to P450 2C1, the TMDs of cytochrome b₅ and UBC6 are at the C terminus of the proteins. The 1-28 N-terminal signal of P450 2C1 loses its ER retention property when located internally or at the C terminus of a chimeric protein (17) and shortening the TMD restores ER retention.² The relative role within the TMD of length, the linear gradient of hydrophobicity, and amphipathicity may, thus, depend on the location of the TMD and the topology of the protein. The importance of these properties rather than specific sequences in TMD-mediated sorting are consistent with the increasing evidence for the role of phospholipids in protein sorting in general and for ER transport mediated by coat proteins II vesicle budding in particular (47).

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM 35897.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-333-1146; Fax: 217-333-1133; E-mail: byronkem@uiuc.edu.

Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M002394200

² E. S. Skorupa and B. Kemper, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; TMD, transmembrane domain; P450, cytochrome P450; GFP, green fluorescent protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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