©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Determination of the Structural Requirements for Palmitoylation of p63 (*)

Anja Schweizer (§) , Jack Rohrer (¶) , Stuart Kornfeld

From the (1) Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Palmitoylation of p63, a type II membrane protein localized in the endoplasmic reticulum, is induced in a reversible manner by the drug brefeldin A. To study the requirements for palmitoylation, mutant forms of p63 were expressed in COS cells and analyzed by metabolic labeling with [H]palmitate, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis. By investigating deletion and point mutations, Cysin the 106-amino acid cytoplasmic tail of p63 has been identified as the site of acylation. Site-directed mutagenesis of residues 99-105 together with cytoplasmic tail truncation mutants showed that the amino acids surrounding Cysare not critical for palmitoylation of this residue. Analysis of a chimeric construct between p63 and the plasma membrane protein dipeptidylpeptidase IV further revealed that p63 palmitoylation is not dependent on its transmembrane domain. In contrast, the six-amino acid distance between the end of the predicted transmembrane domain and the palmitoylation site was found to be essential for proper acylation of p63.


INTRODUCTION

A wide variety of viral and cellular proteins are covalently modified by the post-translational addition of the 16-carbon saturated fatty acid palmitate. Palmitoylation can be either a stable or reversible modification. Irreversible palmitoylation has been documented for different viral (Schmidt and Schlesinger, 1979; Schmidt et al., 1979; Schmidt, 1982) and cellular proteins (Crise and Rose, 1992), while proteins such as the mammalian transferrin receptor (Omary and Trowbridge, 1981), the (proto)oncogene product p21(Magee et al., 1987), a neuronal growth cone protein (GAP-43) (Skene and Virag, 1989), erythrocyte ankyrin (Staufenbiel, 1987), as well as the BCH1 cell protein p63 (James and Olson, 1989), the CHO cell protein p62 (Mundy and Warren, 1992), and the protein tyrosine kinase p56 (Paige et al., 1993) show a reversible type of palmitoylation.

Various subcellular sites have been reported for the palmitoylation of different proteins. Palmitoylation of viral membrane glycoproteins takes place in a pre-/early Golgi compartment (Bonatti et al., 1989; Veit and Schmidt, 1993), whereas the attachment of fatty acids to many cellular proteins including the immunoglobulin E receptor (Kinet et al., 1985) and the transferrin receptor (Omary and Trowbridge, 1981) has been shown to occur at the plasma membrane. Gutierrez and Magee (1991), on the other hand, have identified a protein palmitoyltransferase activity that co-fractionated with Golgi membranes. These findings suggest that multiple palmitoyltransferases with different subcellular locations may exist.

In almost all of the proteins examined, palmitoylation occurs on internal cysteine residues through a thioester bond. An important aspect of protein palmitoylation concerns the specificity involved in selecting distinct cysteine residues on a particular protein for modification. Most of the cysteines identified as acylation sites in transmembrane proteins are found in the portion of the cytoplasmic domain of the polypeptide that is near the lipid bilayer or in the part of the transmembrane domain that is adjacent to the cytoplasmic sequence (Sefton and Buss, 1987; Schlesinger et al., 1994). However, the structural requirements necessary for palmitoylation have not been determined. The exact position of the cysteines varies considerably, and comparison of the amino acid sequences surrounding the acylation sites fails to reveal a general consensus sequence (Schlesinger et al., 1994).

As a step toward elucidating the structural requirements for palmitoylation of membrane proteins, we have studied the acylation of p63. p63 is a 63-kDa integral membrane protein that is localized in the endoplasmic reticulum (ER).()() Sequence analysis together with biochemical studies have demonstrated that it is a nonglycosylated type II transmembrane protein with a 106-amino acid NH-terminal cytosolic tail, a single transmembrane domain, and a large extracytoplasmic domain of 474 amino acids (Schweizer et al., 1993a, 1993b). p63 is reversibly palmitoylated in vivo in the presence of brefeldin A (BFA), but is only weakly acylated in the absence of the drug (Schweizer et al., 1993b). In the present paper, we have identified Cysas the site of palmitoylation in the p63 protein. The acylation of Cysappeared to be independent of the amino acid sequences surrounding this residue, whereas the spacing of the cysteine relative to the transmembrane domain was found to be critical for successful palmitoylation.


EXPERIMENTAL PROCEDURES

Materials

Enzymes used in molecular cloning were obtained from Boehringer Mannheim, New England Biolabs, or Promega. Dulbecco's modified Eagle's medium (4.5 g/liter glucose) and RPMI 1640 medium were from Life Technologies, Inc.; fetal calf serum from Hazleton Biologics (Lenexa, KS); Nusera from Collaborative Biomedical Products; DEAE-dextran, chloroquine, and protease inhibitors were from Sigma; [H]palmitate and Amplify from Amersham Corp.; EXPRESS protein labeling mixture from DuPont NEN; protein A-Sepharose beads from Repligen Corp. (Cambridge, MA); and cell culture dishes from Falcon (Becton Dickinson Co.).

Oligonucleotides were synthesized with a solid phase synthesizer (380A, Applied Biosystems) by the Protein Chemistry Facility of Washington University.

Recombinant DNA Procedures

All basic DNA procedures were as described (Sambrook et al., 1989).

The p63 wild-type (wt) cDNA has been described previously (Schweizer et al., 1993b) and consisted of the 5`-untranslated region, base pairs 1-84, the 1803-nucleotide coding region, and 1023 base pairs of the 3`-noncoding sequence. The full-length cDNA was inserted into the EcoRI site in the polylinker of the Bluescript SKor KSvector (Stratagene), respectively, with the initiator ATG 3` to the BamHI restriction site of the polylinker. The resulting constructs were designated pBSK-p63 or pBKS-p63, respectively. For transient expression in COS cells, the p63 insert was subcloned into the EcoRI site of the pECE vector (kindly provided by Dr. M. Spiess, Biozentrum, Basel, Switzerland) (Ellis et al., 1986) to give plasmid pECE-p63.

The construction of the 24-101 mutant (p63 with a deletion of amino acids 24-101) has been described in Schweizer et al. (1994). For transient expression in COS cells, plasmid pECE-24-101 (Schweizer et al., 1994) was used.

All new mutant forms of p63 were created by standard PCR protocols using the overlap extension technique (Ho et al., 1989). All mutants start at base pair 78 of the original wt p63 cDNA (Schweizer et al., 1993b). Base pairs 774-791 of the p63 sequence and base pairs 170-193 of the Bluescript KSvector were used as downstream and upstream flanking primers. The final PCR products were directly subcloned into the SmaI site of the pECE vector for transient expression in COS cells.

To create the 24-98 construct (p63 with deletion of amino acids 24-98) and the P-A100 construct (24-98 with alanine substitution of Cys), pBKS-24-101 was used as PCR template. pBSK-24-101 was obtained by digestion of plasmid pECE-24-101 with EcoRI and subsequent subcloning of the 24-101 insert into the EcoRI site in the polylinker of the Bluescript KSvector with the initiator ATG facing the BamHI restriction site of the polylinker. The internal PCR primers were TCG GAG AAG GGT GCC TCC TGC TCG CGC AGG CTC GGC AGG GCG TCC GCA TCG CGC AGG CTC GGC AGG GCG CTC AAC (24-98) or TCG GAG AAG GGT GCC TCC GCA TCG CGC AGG CTC GGC AGG GCG CTC AAC (P-A100) for the downstream reaction and CCT GCC GAG CCT GCG CGA GCA GGA GGC ACC CTT CTC CGA GGG GCT CGC GGC GCC (24-98) or CTT GCC GAG CCT GCG CGA TGC GGA GGC ACC CTT CTC CGA GGG GCT CGC GGC GCC (24-98A100) for the upstream reaction, respectively. The final plasmids were designated pECE-24-98 and pECE-P-A100.

For the construction of mutants P1-P12 (see Figs. 2, 4, 6, and 8), plasmid pBKS-24-98 was used as PCR template. pBSK-24-98 was generated by digesting plasmid pECE-24-98 with EcoRI and subsequent subcloning of the 24-98 insert into the EcoRI site of the polylinker. Appropriate pairs of partially complementary oligonucleotides which encoded the desired mutation were chosen as internal primers. The final plasmids were designated pECE-P1-pECE-P12.

The p63-DPPIV chimera 24-98PDP (24-98 cytoplasmic tail of p63, transmembrane domain of DPPIV, and lumenal domain of p63) was constructed based on the existence of a previously described chimera: 24-101PDP (Schweizer et al., 1994). The chimeric construct was precisely joined at the transition between two domains. To generate the 24-98PDP construct, pBKS-24-101PDP as template together with GAG AAG GGT GCC TCC TGC TCG CGC AGG CTC GGC AGG GTT CTT CTG GGA CTG GGA CTG CTG and CCT GCC GAG CCT GCG CGA GCA GGA GGC ACC CTT CTC CGA GGG GCT CGC GGC GCC as internal primers were used in the PCR reaction. Upstream and downstream flanking primers as well as further treatment of the final PCR product were as described above.

All mutants were verified by sequencing at the level of the final plasmid.

Cell Culture and Transfection

COS cells (African green monkey kidney cells, CRL1650; American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and fungizone in a humidified 5% COatmosphere. Transient transfection of COS cells (grown in 60-mm plates) was performed as described (Schweizer et al., 1994).

Antibodies

For the detection of p63, monoclonal antibody G1/296 (Schweizer et al., 1993a) was used. Metabolic Labeling with [S]Methionine and [H]Palmitate-COS cells were grown in 60-mm dishes. For labeling with [S]methionine, the cells were rinsed 43 h post-transfection with phosphate-buffered saline, preincubated in 3 ml of phosphate-buffered saline, 1% nonessential amino acids, 10% dialyzed fetal calf serum at 37 °C for 15 min, and pulsed for 90 min with 150 µCi of EXPRESS protein labeling mixture in 2 ml of preincubation medium. For labeling with [H]palmitate, the cells were washed twice with serum-free Dulbecco's modified Eagle's growth medium and labeled in 2 ml of Dulbecco's modified Eagle's medium containing 5% fetal calf serum and 700 µCi of [H]palmitate for 90 min at 37 °C in the presence or absence of 10 µg/ml brefeldin A (kindly provided by Sandoz AG, Basel, Switzerland).

Immunoprecipitation, SDS-PAGE, and Fluorography

Antigens were immunoprecipitated from Triton X-100-solubilized cells as described (Schweizer et al., 1988). The immunocomplexes were released from the beads by boiling for 3 min in electrophoresis sample buffer containing 62.5 m M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1 M dithiothreitol, and 0.001% bromphenol blue. Proteins were separated on 8% SDS-polyacrylamide minigels (Bio-Rad Laboratories) using the Laemmli (1970) system and visualized by fluorography using Amplify and Kodak X-Omat AR films.

Quantitation of fluorograms was carried out by means of a Molecular Dynamics Personal Densitometer. The amount of [H]palmitate incorporated was calculated relative to the expression found with [S]methionine.


RESULTS

Palmitoylation of p63 in COS Cells

In a previous study, it was demonstrated that brefeldin A (BFA) treatment of Vero cells markedly stimulated the palmitoylation of p63 (Schweizer et al., 1993b). To investigate whether the same phenomenon occurs in COS cells, the cells were transfected with p63wt cDNA, and, after 43 h, the cells were labeled with [H]palmitate for 90 min in the absence or presence of the fungal metabolite BFA. The expressed p63wt was then immunoprecipitated and subjected to SDS-PAGE. As seen in Fig. 1, p63wt showed weak labeling in the absence of BFA, whereas, in the presence of the drug, palmitoylation was strongly induced.


Figure 1: Palmitoylation of p63wt in COS cells. COS cells transfected with p63wt were labeled for 90 min with [H]palmitate in the absence or presence of 10 µg/ml brefeldin A. p63 was immunoprecipitated and subjected to SDS-PAGE (8% gel). The numbers at the left margin of the gel indicate known molecular mass in kilodaltons.



CysIs the Site of Palmitoylation in p63

The entire p63 sequence (Schweizer et al., 1993b) contains only two cysteine residues. Cysis located in the 106-amino acid cytoplasmic tail close to the membrane spanning segment (Fig. 2), while Cysis found within the transmembrane domain adjacent to the lumenal part of the protein. Since cysteines close to the membrane bilayer have often been found to undergo palmitoylation via a thioester linkage (Sefton and Buss, 1987), these two residues were the logical candidates for being the site(s) of palmitoylation in p63. To pursue this, we initially analyzed two constructs which altered the region of the protein that contains Cys. The first construct was a truncated form of p63 in which residues 24-101 of the p63 tail were deleted (24-101, Fig. 2and Schweizer et al. (1994)). We also prepared a construct (24-98) that has amino acids 99-101 added back to the 24-101 deletion mutant (Fig. 2). These two constructs along with p63wt were then expressed in COS cells. The cells were metabolically labeled with either [S]methionine or [H]palmitate in the presence of BFA, subjected to immunoprecipitation with anti-p63 monoclonal antibodies, and analyzed by SDS-PAGE. As shown in Fig. 3, p63wt and 24-98 were labeled with [H]palmitate ( lanes 1 and 3), whereas no [H]palmitate incorporation was detected for 24-101 ( lane 2). In contrast, all three proteins were equally labeled with [S]methionine ( lanes 5-7).


Figure 2: Schematic illustration of deletion and point mutants within the cytoplasmic tail of p63. Selected amino acids of the p63 cytoplasmic tail are shown ( single-letter code). Boxes represent the single transmembrane domain of p63. Straight lines indicate deleted regions of the p63 sequence. The mutants are named by the position of their deletions and point mutation as shown at the left margin of the figure.




Figure 3: Cys is the site of palmitoylation in p63. COS cells transfected with wt or mutant p63 were labeled for 90 min with [S]methionine and [H]palmitate in the presence of 10 µg/ml brefeldin A. p63 was immunoprecipitated and subjected to SDS-PAGE (8% gels). The numbers at the left margin of the gels indicate known molecular mass in kilodaltons.



These results are consistent with amino acids 99-101 containing the p63 palmitoylation site and implicate Cysas the residue that is actually palmitoylated. To test this directly, site-directed mutagenesis of 24-98 was used to replace Cyswith alanine (P-A100; Fig. 2). The alanine amino acid lacks the thiol group needed for acylation, but is similar in size to cysteine. When P-A100 was analyzed by labeling with [S]methionine and [H]palmitate in the presence of BFA after transfection, the resultant protein behaved like 24-101. The mutant showed normal incorporation of [S]methionine (Fig. 3, lane 8), while no band was visible with [H]palmitate (Fig. 3, lane 4). Thus, the specific replacement of Cysby alanine completely abolished palmitoylation.

The correct subcellular localization of P-A100 and 24-98 was confirmed by immunofluorescence analysis of transfected cells (data not shown).

Taken together, these data demonstrated that p63 is palmitoylated at Cyswhile Cysis not modified by this fatty acid.

As expected for a thioester linkage (Schlesinger et al., 1980; Kaufman et al., 1984), the H label bound to p63 was susceptible to cleavage with hydroxylamine at neutral pH (not shown). In addition, about 60% of the fatty acid label was removed from immunoprecipitated p63 upon boiling for 3 min in sample buffer containing 0.1 M dithiothreitol as compared to boiling under nonreducing conditions (not shown). Since immunoprecipitated p63 gives a complex pattern of monomeric, dimeric, and multimeric forms when subjected to SDS-PAGE under nonreducing conditions (Schweizer et al., 1993a), all of the gels shown were run under reducing conditions. However, the relative amounts of [H]palmitate labeling of p63wt and mutant proteins were found to be identical under reducing and nonreducing conditions.

The Amino Acids Surrounding CysDo Not Contain a Specific Signal for the Palmitoylation of p63

We next tested whether the amino acids near the palmitoylation site form a signal needed for efficient palmitoylation. To this end, the region surrounding Cyswas subjected to extensive site-directed mutagenesis. Since deletions upstream of amino acid 99 did not prevent palmitoylation of p63 (see Fig. 3), we concentrated on amino acids downstream of this position. First, the serines on both sides of the Cyswere changed to alanines ( construct P1, Fig. 4 ). In addition, amino acids 99-103 ( construct P2, Fig. 4) and amino acids 99-105 ( construct P3, Fig. 4) were substituted by alanines except for the cysteine at position 100. Surprisingly, these substitutions had no detectable effect on the palmitoylation of p63 when compared to p63wt (Fig. 5).


Figure 4: Point mutations within the cytoplasmic tail of 24-98. Selected amino acids of the cytoplasmic tail of p63wt are given in single-letter code. Boxes represent the single p63 transmembrane domain. Amino acids in the cytoplasmic tail of 24-98 replaced by alanines are shown.




Figure 5: Amino acids surrounding Cys are not critical for the palmitoylation of the p63 protein. COS cells transfected with wt or mutant p63 were labeled with [S]methionine and [H]palmitate in the presence of 10 µg/ml brefeldin A and further analyzed as described in Fig. 2.



These data demonstrate that Cysis efficiently palmitoylated independent of its surrounding amino acids.

The Distance between Cysand the Transmembrane Domain Is Critical for the Palmitoylation of p63

We next analyzed the importance of the spacing between Cysand the transmembrane domain for palmitoylation. To address this point, we constructed a series of mutants that had alanine spacers of various lengths introduced between amino acids 101 and 102. As indicated in Fig. 6, the distance between Cysand the first amino acid of the predicted transmembrane domain, which is six amino acids in p63wt, was systematically increased by one (P8), two (P7), three (P6), four (P5), and five (P4) alanines, respectively. When cells expressing these constructs were analyzed by [H]palmitate labeling, all of the mutants showed dramatic decreases in the levels of palmitoylation (Fig. 7). Compared to p63wt, incorporation of [H]palmitate into P5, P6, and P8 was reduced to 2%, 8%, and 5%, respectively ( n = 2), while P4 and P7 were not labeled at all. As shown in Fig. 7 , lanes 7-12, the mutant proteins were expressed to the same extent as p63wt as judged by [S]methionine incorporation.


Figure 6: Schematic diagram of p63wt and 24-98 cytoplasmic tail mutants that have alanine spacers of various lengths (1 to 5) introduced between amino acids 101 and 102. Selected amino acids of the cytoplasmic tails are shown ( single-letter code). Boxes represent the single transmembrane domain of p63.




Figure 7: Increasing the distance between Cys and the transmembrane domain drastically affects palmitoylation of p63. COS cells transfected with wt or mutant p63 were labeled with [S]methionine and [H]palmitate in the presence of 10 µg/ml brefeldin A and further analyzed as described in Fig. 2.



Another set of mutants was created in which the spacing of Cysfrom the transmembrane segment was decreased from the original six amino acids to five (P9), three (P10), or one (P11) residue (Fig. 8). In each of these constructs, amino acids 99-105 were replaced by alanines except for the cysteine in the appropriate position. These alanine substitutions are comparable to those in the P3 mutant which shows normal palmitoylation. In an additional construct (P12), the cysteine was moved to the third position of the transmembrane domain (Fig. 8). For this purpose, Asnwas replaced by a cysteine while Cyswas mutated to alanine. When cells transfected with the various constructs were tested for [H]palmitate incorporation, palmitoylation of all of these mutants was strongly impaired (Fig. 9). Compared to P3 ( lane 1), labeling of P9 ( lane 2), P10 ( lane 3), and P11 ( lane 4) was reduced to 7%, 13%, and 6%, respectively ( n = 2), while P12 ( lane 5) showed no incorporation at all. Lanes 6-10 of Fig. 9 show that the cells expressing the different mutants efficiently incorporated [S]methionine into the p63 protein. The upper band in lane 10 most likely represents dimers of P12 which could be diminished by the combined presence of dithiothreitol and -mercaptoethanol (not shown).


Figure 8: Schematic illustration of 24-98 cytoplasmic tail and transmembrane mutants that have a cysteine at decreasing distance from the transmembrane domain or in the transmembrane domain itself. Selected amino acids of the cytoplasmic tail and the first four amino acids of the transmembrane domain are shown ( single-letter code). Boxes represent the single transmembrane domain of p63.




Figure 9: Decreasing the distance between Cys and the transmembrane domain drastically affects palmitoylation of p63. COS cells transfected with the p63 mutants P3, P9, P10, P11, and P12 were labeled with [S]methionine and [H]palmitate in the presence of 10 µg/ml brefeldin A and further analyzed as described in Fig. 2. Note that P12 which has the cysteine in the third position of the transmembrane domain shows no palmitoylation.



Taken together, these results show that the spacing of Cysrelative to the transmembrane domain is critical for efficient palmitoylation of p63. Only the original distance of six amino acids between Cysand the first amino acid of the predicted transmembrane segment as occurs in p63wt allows normal palmitoylation.

The Transmembrane Domain of p63 Is Not Essential for Palmitoylation

Given the importance of the distance of the palmitoylation site to the transmembrane domain, we next tested whether this domain is a necessary component for p63 palmitoylation. To this end, a chimeric construct in which the transmembrane domain of 24-98 was replaced by that of DPPIV (24-98PDP; Fig. 10) was created. The serine protease DPPIV is a type II integral membrane protein localized at the plasma membrane and is found on a variety of epithelial, endothelial, and lymphocytic cell types (Hong and Doyle, 1987; 1990; Ogata et al., 1989). Fig. 11shows that 24-98PDP expressed in COS cells was strongly labeled with both [S]methionine ( lane 2) and [H]palmitate ( lane 1). This result indicates that the p63 transmembrane domain is not necessary for proper palmitoylation of the protein.


Figure 10: Schematic diagram of p63wt, DPPIVwt, and the 24-98PDP chimera. p63 sequence is indicated as striated bars, while sequence derived from DPPIV is shown as dotted bars. Numbers indicate amino acid positions at the beginning or end of topological domains. The chimera is basically named by a three-letter code indicating the origin of its cytoplasmic, transmembrane, and lumenal domain ( left margin of figure).




Figure 11: The transmembrane domain of p63 is not essential for its palmitoylation. COS cells transfected with 24-98PDP were labeled with [S]methionine and [H]palmitate in the presence of 10 µg/ml brefeldin A and further analyzed as described in Fig. 2.




DISCUSSION

Previous work has established that palmitoylation of p63 is greatly enhanced by the drug brefeldin A (Schweizer et al., 1993b). In the present study, we have identified the palmitoylation site in p63 as Cysand have analyzed the structural requirements for p63 palmitoylation using site-directed mutagenesis. p63 was found to be efficiently palmitoylated independent of the amino acids that surround Cys, suggesting that the acylation occurs without a primary sequence motif. This notion was supported by the finding that amino acids of the transmembrane domain also did not contribute to the specificity of palmitoylation. The most striking characteristic of the p63 palmitoylation is that only the six-amino acid spacing between Cysand the predicted transmembrane segment present in p63wt allowed efficient palmitoylation, while both increasing and decreasing this distance strongly impaired acylation.

Although similar studies on the structural requirements for the palmitoylation of a particular cysteine residue have not been reported for other proteins, some general conclusions can be drawn from the sequence data available. When the localization of the palmitoylation site in p63 is compared to that in other acylated integral membrane proteins, it becomes obvious that the six-amino acid distance of the p63 acylation site from its transmembrane domain is not universal. In a number of instances, including the transferrin receptor (Jing andTrowbridge, 1987, 1990) and the cell surface glycoprotein CD4 (Crise and Rose, 1992), the palmitoylation sites are localized within the transmembrane domains of the polypeptides. Among the proteins containing palmitoylated cysteines located in their cytosolic tails, there is a variable distance from the transmembrane junctions. For example, the second palmitoylation site of CD4 is located one amino acid from the transmembrane domain (Crise and Rose, 1992), whereas this distance is two amino acids in the HLA-D-associated invariant chain (Koch and Hämmerling, 1986). The two fatty acylated cysteine residues of bovine opsin and bovine rhodopsin, on the other hand, are located 11 and 12 amino acids (Karnik et al., 1993) and 12 and 13 amino acids (O'Brien et al., 1987; Ovchinnikov et al., 1988; Papac et al., 1992), respectively, into the cytoplasm. Similarly, a 12-amino acid distance between the acylation site and the transmembrane domain was found for the human -adrenergic receptor (O'Dowd et al., 1989). In fact, as far as we are aware, only in the cases of the vesicular stomatitis virus G protein (Rose et al., 1984) and some subtypes of influenza virus hemagglutinin (Veit et al., 1991) has the spacing between the palmitoylated cysteine and the first amino acid of the transmembrane domain been shown to be six amino acids.

How can proteins with palmitoylation sites at many different distances from the transmembrane domain or even within the transmembrane segment be palmitoylated when acylation of p63 is critically dependent on its six-amino acid spacing between the acylated cysteine and the transmembrane domain? One possible explanation is that the mere number of amino acids between the palmitoylation site and the transmembrane domain is not an appropriate measure for the actual distance present in the mature protein. Due to different structural conformations of the individual primary sequences, similar distances might be achieved with a variable number of residues as spacer. It is, however, difficult to imagine how this mechanism could apply to cysteine residues that are part of transmembrane domains. Alternatively, but not necessarily to the exclusion of the first possibility, the acylation of different subsets of proteins could be mediated by multiple palmitoyltransferases that have specific but diverse structural requirements. The specificity could be determined by differences in the required spacing of cysteine residues relative to the transmembrane bilayers, as suggested by the p63 analysis, or may also involve primary sequence motifs for some of the acyltransferases. The few examples of protein motifs that have been proposed as consensus sequences for palmitoylation include the sequence hydrophobic-Leu-Cys-Cys- X-basic-basic present in GAP-43, the human -adrenergic receptor, and in modified forms in other G protein-coupled receptors (Strittmatter et al., 1990) as well as the NH-terminal motif Met-Gly-Cys in several -subunits of trimeric G proteins and in members of the Src family of protein tyrosine kinases (Parenti et al., 1993; Resh, 1994; Koegl et al., 1994). Since the Met-Gly-Cys motif is part of a consensus sequence for the attachment of the 14-carbon saturated fatty acid myristate (Towler et al., 1988), its importance for palmitoylation could be indirect by allowing myristoylation which leads to membrane association (Koegl et al., 1994; Galbiati et al., 1994). In fact, a nonmyristoylated mutant of a G protein -subunit (glycine to alanine) was palmitoylated when membrane attachment was achieved by an alternative mechanism (Degtyarev et al., 1994). The proposed existence of multiple palmitoyltransferases is also suggested by the finding that protein palmitoylation can occur at different subcellular locations including organelles of the early secretory pathway and the plasma membrane (for a review, see Schlesinger et al. (1994)).

An interesting property of the p63 palmitoylation that was observed both in Vero (Schweizer et al., 1993b) and COS cells is its strong induction in the presence of the fungal metabolite BFA. In many different cell types, BFA causes disassembly of the Golgi apparatus and redistribution of Golgi resident proteins into the ER (reviewed in Klausner et al. (1992)). The p63 protein has recently been localized to the rough ER by subcellular fractionation and immunoelectron microscopy.In the presence of BFA, p63 could therefore be palmitoylated by a palmitoyltransferase that is normally localized in a post-ER compartment but is redistributed into the ER upon addition of BFA. Consistent with this hypothesis, the different intracellular locations where palmitoylation has been found to occur include the Golgi apparatus (Gutierrez and Magee, 1991) and a pre-/early Golgi compartment (Bonatti et al., 1989; Veit and Schmidt, 1993). Of particular interest, the two proteins that have palmitoylated cysteine residues located at the same distance from the transmembrane domain as the palmitoylated cysteine of p63, e.g. the vesicular stomatitis virus G protein and the influenza virus hemagglutinin (H7 subtype), have been proposed to be palmitoylated in a pre-/early Golgi compartment (Bonatti et al., 1989; Veit and Schmidt, 1993). Alternatively, the induction of p63 palmitoylation upon BFA treatment could result from an accumulation of a palmitoyltransferase in the rough ER as a consequence of the block in anterograde intracellular protein transport that is caused by BFA (reviewed in Klausner et al. (1992)). Another possibility raised by Mundy et al. (1992) is that the inhibition of protein transport induced by BFA is itself a stimulus for palmitoylation. Finally, a less likely explanation for the effect of BFA on the acylation of p63 is an inactivation of the protein thioesterase that is responsible for the removal of palmitate from p63.

The functional significance of the covalent attachment of fatty acid to p63 remains unknown. For some of the palmitoylated cellular proteins, mutations that altered the acylation sites were found to have significant effects (for a review, see Schlesinger et al. (1994)). A nonpalmitoylated form of the human -adrenergic receptor, for example, was markedly impaired in its ability to mediate agonist stimulation of adenylylcyclase (O'Dowd et al., 1989). For some forms of p21(Hancock et al., 1989) and for the neuronal growth cone protein GAP (Skene and Virag, 1989; Zuber et al., 1989; Liu et al., 1993), palmitoylation has been shown to enhance membrane binding while the attachment of palmitate to G influenced both its membrane association and its ability to mediate hormonal stimulation of adenylylcyclase (Wedegaertner et al., 1993). In addition, fatty acylation has been suggested to play a role in vesicle-mediated transport (Glick and Rothman, 1987; Pfanner et al., 1989, 1990). Appropriate model systems and the mutants generated for this study should now help to unravel putative functions of the dynamic acylation of the p63 protein.


FOOTNOTES

*
This work was supported in part by United States Public Service Grant CA08759. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a W. M. Keck fellowship.

Recipient of a Damon Runyon-Walter Winchell cancer postdoctoral fellowship.

The abbreviations used are: ER, endoplasmic reticulum; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; BFA, brefeldin A; DPPIV, dipeptidylpeptidase IV; wt, wild-type.

Schweizer, A., Rohrer, J., Geuze, H. J., Slot, J. W., and Kornfeld, S., J. Cell Sci., in press.


ACKNOWLEDGEMENTS

We thank Dr. M. Linder and Dr. M. Schlesinger for helpful discussion and critical reading of the manuscript.


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