From the
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 [
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
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).
Oligonucleotides were synthesized with a solid phase
synthesizer (380A, Applied Biosystems) by the Protein Chemistry
Facility of Washington University.
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
SK
The construction of the
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 KS
To create the
For the construction of mutants
P1-P12 (see Figs. 2, 4, 6, and 8), plasmid pBKS-
The p63-DPPIV chimera
All mutants were verified by
sequencing at the level of the final plasmid.
Quantitation of fluorograms was carried out by means of a Molecular
Dynamics Personal Densitometer. The amount of
[
The correct subcellular localization of P-A100 and
Taken together, these data demonstrated that p63 is
palmitoylated at Cys
As expected for a thioester linkage
(Schlesinger et al., 1980; Kaufman et al., 1984), the
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 Cys
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
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
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.
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
We thank Dr. M. Linder and Dr. M. Schlesinger for
helpful discussion and critical reading of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H]palmitate, immunoprecipitation,
and SDS-polyacrylamide gel electrophoresis. By investigating deletion
and point mutations, Cys
in 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 Cys
are 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.
(Magee et al., 1987), a neuronal growth cone protein
(GAP-43) (Skene and Virag, 1989), erythrocyte ankyrin (Staufenbiel,
1987), as well as the BC
H1 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.
(
)(
)
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
Cys
as the site of palmitoylation in the p63 protein. The
acylation of Cys
appeared 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.
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.; EXPRE
S
S protein labeling
mixture from DuPont NEN; protein A-Sepharose beads from Repligen Corp.
(Cambridge, MA); and cell culture dishes from Falcon (Becton Dickinson
Co.).
Recombinant DNA Procedures
All basic DNA
procedures were as described (Sambrook et al., 1989).
or KS
vector (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.
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.
vector 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.
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
KS
vector 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.
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.
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.
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 EXPRE
S
S 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.
H]palmitate incorporated was calculated relative
to the expression found with [
S]methionine.
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.
Cys
The entire p63 sequence (Schweizer et al.,
1993b) contains only two cysteine residues. CysIs the Site of Palmitoylation in
p63
is
located in the 106-amino acid cytoplasmic tail close to the membrane
spanning segment (Fig. 2), while Cys
is 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
Cys
with 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
Cys
by alanine completely abolished palmitoylation.
24-98 was
confirmed by immunofluorescence analysis of transfected cells (data not
shown).
while Cys
is not
modified by this fatty acid.
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 Cys
We
next tested whether the amino acids near the palmitoylation site form a
signal needed for efficient palmitoylation. To this end, the region
surrounding CysDo Not
Contain a Specific Signal for the Palmitoylation of p63
was 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 Cys
were 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 Cys
We next analyzed the importance of the spacing between
Cysand the
Transmembrane Domain Is Critical for the Palmitoylation of
p63
and 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 Cys
and 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, Asn
was replaced by a
cysteine while Cys
was 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 Cys
and 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.
and 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 Cys
and the predicted transmembrane
segment present in p63wt allowed efficient palmitoylation, while both
increasing and decreasing this distance strongly impaired acylation.
-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.
-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)).
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.
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.