From the Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
Received for publication, September 12, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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Covalent lipid modifications mediate
protein-membrane and protein-protein interactions and are often
essential for function. The purposes of this study were to examine the
Cys residues of the transmembrane domain of metallocarboxypeptidase D
(CPD) that could be a target for palmitoylation and to clarify the
function of this modification. CPD is an integral membrane protein that cycles between the trans Golgi network and the
plasma membrane. We constructed AtT-20 cells stably expressing various
constructs carrying a reporter protein (albumin) fused to a
transmembrane domain and the CPD cytoplasmic tail. Some of the
constructs contained the three Cys residues present in the CPD
transmembrane region, while other constructs contained Ala in place of
the Cys. Constructs carrying Cys residues were palmitoylated, while
those constructs lacking the Cys residues were not. Because
palmitoylation of several proteins affects their association with
cholesterol and sphingolipid-rich membrane domains or caveolae, we
tested endogenous CPD and several of the reporter constructs for
resistance to extraction with Triton X-100. A construct containing the
Cys residues of the CPD transmembrane domain was soluble in Triton
X-100 as was endogenous palmitoylated CPD, indicating that
palmitoylation does not target CPD to detergent-resistant membrane
rafts. Interestingly, constructs of CPD that lack palmitoylation sites
have an increased half-life, a slightly more diffuse steady-state localization, and a slower rate of exit from the Golgi as compared with
constructs containing palmitoylation sites. Thus, the covalent attachment of palmitic acid to the Cys residues of CPD has a functional significance in the trafficking of the protein.
Carboxypeptidases perform a wide variety of roles, ranging from
digestion of food to the selective biosynthesis of hormones and
neuropeptides (1, 2). Carboxypeptidase D
(CPD)1 is a member of the N/E
subfamily of metallocarboxypeptidases (named for the first two members
of this family that were identified, carboxypeptidase N and
carboxypeptidase E (CPE)). Among all members of this N/E
subfamily, CPD has a number of unique properties. Unlike the other
metallocarboxypeptidases, CPD is broadly distributed among mammalian
tissues where it is enriched in the trans Golgi network
(TGN) (3-5). CPD is also the only metallocarboxypeptidase that is a
transmembrane protein (3, 4, 6-8). In addition, CPD consists of
multiple carboxypeptidase domains that are present in the lumen of the
TGN; this multiple catalytic domain structure is found in CPD homologs
in Drosophila, Aplysia, duck, and all mammalian
species investigated (3, 4, 6, 9, 10). As a result of its broad
distribution, subcellular localization, and specificity for C-terminal
Lys and Arg residues, CPD is thought to function following the action
of furin, proprotein convertase 7 (PC7, also known as lymphoma
proprotein convertase), and related endopeptidases in the processing of
proteins that transit the secretory pathway (11, 12). Likely substrates
include precursors for neuroendocrine peptides, growth factors, and
some growth factor receptors (13).
As with many proteins that are localized to the TGN, the cytosolic tail
region of CPD has been found to contain domains that affect the
intracellular trafficking of this protein. These domains include two
acidic clusters, a casein kinase-2 consensus sequence, a di-Leu motif,
and a Phe-Xaa-Xaa-Leu sequence that may function as a
Tyr-Xaa-Xaa-Leu-like element (14, 15). None of the previous studies on
CPD has specifically examined sequences within the transmembrane
domain, which is highly conserved among species. The 26 hydrophobic
residues in this domain are identical in human, rat, and mouse CPD
(Fig. 1). Duck CPD has only 4 amino acid
differences in this 26-residue region, and all of these differences are
conservative substitutions (such as Ser versus Thr or Ala,
and Ile versus Val). The Drosophila CPD homolog
also contains a hydrophobic segment of the same overall length.
Interestingly, CPD from mammals, duck, and Drosophila
contains 3 Cys residues within the hydrophobic region, and except for 1 Cys residue in Drosophila CPD, all of these Cys residues are
located within the cytosolic side of the transmembrane segment. This
strong conservation of the Cys residues implies an important
function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Top, comparison of the transmembrane
domains of CPD from human (4), rat (3), mouse (9), duck (6), and
Drosophila (32). Bottom, schematic diagram of CPD
indicating the three carboxypeptidase-like (CP) domains, the
transmembrane region, and the cytosolic tail. The signal peptide and
the transmembrane domains are shaded black.
Cys residues located near transmembrane regions are often sites for the
reversible attachment of the fatty acid palmitate (16-18). In many
cases palmitoylation has been found to affect the intracellular
localization and/or the rate of trafficking between subcellular
compartments (16-18). For example, palmitoylation of the endopeptidase
PC7 prolongs the half-life of the protein but not the localization to
the TGN (19). In the present study, CPD was found to be palmitoylated
in a variety of cell lines, and this palmitoylation required the
presence of the 3 Cys residues within the hydrophobic region near the
cytosolic domain. Potential functions of this modification were
investigated by comparing reporter constructs that differed only in the
presence of the 3 Cys residues. The finding that the absence of Cys
residues prolongs the half-life of the protein, presumably by
decreasing the rate at which the newly synthesized protein exits the
Golgi, suggests a role for palmitoylation in the intracellular
trafficking of CPD.
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MATERIALS AND METHODS |
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Constructs--
Human albumin C-terminally fused with the
transmembrane and cytosolic domains of duck CPD (construct 1, Fig.
2) was cloned as described in Ref. 20.
The generation of this and the subsequent constructs required the
introduction of an AflII site between the transmembrane
domain and the cytosolic tail; this point mutation converts the Ile at
the junction of these two domains into a Leu. Human albumin
C-terminally fused with the vesicular stomatitis virus G protein (VSVG)
transmembrane and the cytosolic domain of duck CPD (construct 2, Fig.
2) was cloned using the pcDNA3 plasmid with the coding region of
human albumin (Alb/pcDNA3). This plasmid contains a
Bsu36I site near the C terminus of the coding region of
albumin and an ApaI site in the 3' non-coding region (21).
Two synthetic oligonucleotides
(5'-TTAGGCCTGGTTCCTCGAGCGCTTAAGGAGAGGTAGG GCC and
5'-CTACCTCTCCTTAAGCGCTCGAGGAACCAGGCC) containing XhoI, AflII, and ApaI sites and encoding a cleavage
site for thrombin were annealed and subcloned into the
Bsu36I/ApaI sites of the Alb/pcDNA3
expression vector. A PCR fragment encoding the VSVG transmembrane
domain was digested with XhoI/AflII and subcloned into the XhoI/AflII sites of the Alb/pcDNA3
plasmid containing the linker described above. The PCR product encoding
the cytosolic domain of duck CPD was subcloned into the
AflII/ApaI sites of the Alb/pcDNA3 plasmid.
To generate constructs 3 and 4 (Fig. 2), an 8-amino acid stretch of
duck CPD (CIIWCVCS) was inserted into the AflII site of
Alb/pcDNA3 expression vector containing the VSVG transmembrane and
CPD cytosolic domains. For construct 3, the two oligonucleotides were
based on the wild-type CPD sequence (5'-TTAACTGTATCATCTGGTGTGTCTGCTCAC
and 5'-TTAAGTGAGCAGACACACCAGATGATACAG). For construct 4, the two
oligonucleotides had changes (underlined) to create Cys to Ala
substitutions
(5'-TTAACGCTATCATCTGGGCTGTCGCTTCAC and
5'-TTAAGTGAAGCGACAGCCCAGATGATAGCG.
The complementary oligonucleotides were annealed and subcloned into the
AflII site of construct 2. All constructs were verified by
sequencing.
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Cell Lines and Transfection-- The BRL-3A cell line (CRL-1442) was maintained in F12 medium containing 5% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. The NIH3T3 (HB-11602), NIT3, C57 (TIB 157), and AtT-20 cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 5% humidified CO2 atmosphere. AtT-20 cells were transfected with 5 µg of DNA and 30 µl of LipofectAMINE PlusTM reagent (Invitrogen) according to the manufacturer's procedure. Stable cell lines expressing the various constructs were selected using 1 mg/ml Geneticin (G418) and then identified by Western blot using an antiserum to human serum albumin (Calbiochem). Five positive clonal cell lines for each construct were additionally screened for expression levels by immunoblotting. Three lines of each construct that expressed Alb protein levels comparable to the other constructs were selected for further study.
Immunofluorescence-- AtT-20 cells expressing albumin-fused proteins were grown on 18-mm poly-L-lysine-coated glass coverslips overnight and processed for immunofluorescent analysis as described previously (15). Briefly, cells were incubated with primary antibody to human albumin (dilution 1:1,000, Calbiochem) for 1 h at room temperature followed by fixation and then stained with the fluorescein isothiocyanate-labeled secondary antibody to rabbit IgG.
Metabolic Labeling with [35S]Met/Cys and [3H]Palmitate-- For labeling with [3H]palmitate, cells grown in 100-mm dishes were washed twice with DMEM, starved for 30 min in DMEM containing 20 mM Hepes (pH 7.4), and then labeled in DMEM containing 10% fetal bovine serum, 20 mM Hepes (pH 7.4), and 0.1 mCi/ml [9,10-3H]palmitic acid (specific activity 30-60 Ci/mmol, PerkinElmer Life Sciences) for 5 h at 37 °C. For labeling with [35S]Met/Cys, the cells were rinsed twice and preincubated in 5 ml of Met- and Cys-free DMEM containing 20 mM Hepes (pH 7.4) for 30 min and then pulsed for 3 h with 0.35 mCi/ml [35S]Met/Cys in DMEM containing 20 mM Hepes (pH 7.4). After four washes with ice-cold DMEM and one wash with phosphate-buffered saline, cells were scraped in 1 ml of lysis buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 2 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml leupeptin, and 1 µg/ml pepstatin A), sonicated for 10 s, and then precleared by centrifugation at 4 °C for 15 min at 16,000 × g. Proteins were immunoprecipitated from supernatants with 5 µl of a rabbit polyclonal antiserum directed against human albumin (Calbiochem) or 20 µl of rabbit polyclonal antiserum raised against rat CPD (AE142) (8) and protein A-Sepharose (70 µl of slurry protein, Sigma) by overnight incubation at 4 °C followed by centrifugation at 8,000 × g for 1 min. The beads were washed five times with lysis buffer and boiled in SDS gel loading buffer. Immunoprecipitated proteins were separated on denaturing 10% polyacrylamide gels. The gels were fixed with 40% methanol and 10% acetic acid for 90 min and rinsed in water three times for 10 min. One gel was treated overnight with 1 M Tris (pH 7.4) as a control, while a duplicate gel was soaked in 1 M hydroxylamine (pH 7.4). Both gels were then treated with Fluoro-Hance (Research Products International) and exposed to x-ray film (Kodak).
Determination of Triton X-100 Solubility-- Clonal AtT-20 cell lines expressing construct 1 or construct 2 were grown in 100-mm dishes. After three washes with ice-cold phosphate-buffered saline, the cells were scraped in buffer A (50 mM Tris/HCl, pH 7.4, 2 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml leupeptin, and 1 µg/ml pepstatin A), homogenized using a Brinkman Polytron, and then centrifuged at 4 °C for 2 h at 50,000 × g in a Ti 60 rotor (Beckman L8-70 centrifuge). Following centrifugation, the pellets were homogenized again in buffer A containing 1% Triton X-100, incubated on ice for 30 min, and then centrifuged at 4 °C for 3 h at 50,000 × g. The supernatant from this centrifugation was termed the Triton X-100-soluble fraction. The pellet, designated the Triton X-100-insoluble fraction, was resuspended in SDS-containing gel loading buffer. Equal volumes of both fractions were separated on a denaturing 10% polyacrylamide gel and analyzed by Western blot using a 1:1,000 dilution of polyclonal anti-albumin serum (Calbiochem).
To examine endogenous CPD, detergent-resistant membranes were prepared from NIT3 cells as described in Ref. 19. Briefly, the cells were labeled with [35S]Met/Cys or [3H]palmitic acid for 5 h and lysed on ice in 1 ml of extraction buffer containing 25 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X-100, and a protease inhibitor mixture (Sigma). After centrifugation at 4 °C for 10 min at 16,600 × g, the supernatant (900 µl) was combined with 100 µl of lysis buffer (50 mM Tris/HCl, pH 8.8, 5 mM EDTA, 1% SDS). The pellet was resuspended in 100 µl of lysis buffer, and 900 µl of extraction buffer was added. Both supernatant and pellet fractions were incubated overnight at 4 °C with 20 µl of a rabbit polyclonal antiserum raised to CPD and protein A-Sepharose as described above. Following centrifugation at 8,000 × g for 1 min, the supernatant was collected for reimmunoprecipitation with 10 µl of a rabbit polyclonal antiserum to caveolin-1 (N20, Santa Cruz Biotechnology) for 3 h at room temperature. Immunoprecipitated proteins were separated on denaturing 8 or 15% polyacrylamide gels.
Pulse-Chase Analysis and Budding from TGN-- Pulse-chase analysis was performed as described previously (15). In brief, AtT-20 cells expressing the Alb-CPD tail fusion protein were metabolically labeled for 20 min with [35S]Met/Cys, washed with DMEM, and incubated for different time points at 37 °C. The cells were lysed and subjected to immunoprecipitation using an antiserum to human albumin (Calbiochem).
The in vitro vesicle budding assay was performed as
described previously (22). The resulting fractions were subjected to immunoprecipitation using an antiserum to human albumin
(Calbiochem) or CPE C-terminally directed antiserum AE139 (23).
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RESULTS |
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AtT-20 cells expressing construct 1 (the transmembrane domain and
cytosolic tail of duck CPD attached to the C terminus of human albumin)
were labeled with [3H]palmitic acid and then extracted
and immunoprecipitated with an antiserum to human albumin. Gel
electrophoresis followed by fluorography revealed a major band of ~78
kDa, corresponding to the expected molecular mass of the construct
(Fig. 3, left lane). No signal
was detected when AtT-20 cells expressing construct 2 were similarly
analyzed with an antiserum to albumin (not shown), suggesting that the
presence of the CPD transmembrane region is required for this labeling.
When wild-type AtT-20 cells were labeled with
[3H]palmitic acid and immunoprecipitated with an
antiserum that recognizes mammalian CPD, a faint band was detected
around 180 kDa, corresponding to the molecular mass of CPD (Fig. 3,
lane 2). Similar analysis of BRL3A (a rat liver cell line),
NIH3T3 (a mouse fibroblast cell line), C57 (a mouse lymphoblast cell line), and NIT3 (a mouse pancreatic beta cell line) showed detectable signals in the 180-kDa range. The AtT-20, BRL3A, NIH3T3, and NIT3 cell
lines are known to contain CPD although at different expression levels
(3, 24). When the various cell lines were labeled with
[35S]Met/Cys and subjected to immunoprecipitation with
the anti-CPD antiserum, the amount of CPD protein in each line
generally correlated with the signal detected for palmitoylated CPD
(data not shown), indicating that a comparable fraction of CPD is
palmitoylated in each of these cell lines.
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To explore whether the labeling of the albumin/CPD construct with
[3H]palmitic acid requires the 3 Cys residues in the CPD
region, two additional constructs were created in which 8 additional
residues were added to the VSVG transmembrane portion of construct 2. These new constructs either contained the Cys residues as in CPD
(construct 3) or had substitutions of Ala for these Cys residues
(construct 4). AtT-20 cells expressing these two constructs were
labeled with either [3H]palmitic acid or
[35S]Met/Cys and immunoprecipitated with an antiserum to
albumin. Both the cell line expressing construct 4 (A16) and the cell
line expressing construct 3 (C21) showed equal levels of expression of
the [35S]Met/Cys-labeled albumin/CPD construct (Fig.
4, top). In contrast, only the
cell line expressing construct 3 containing the Cys residues showed
labeling with [3H]palmitic acid (Fig. 4,
bottom left). Treatment of the gel with hydroxylamine
reduced the amount of [3H]palmitic acid in the
albumin/CPD band (Fig. 4, bottom right). The reduction in
the signal with hydroxylamine is consistent with the palmitoylation of
Cys residues and not Ser or Thr.
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The steady-state localization of the albumin/CPD constructs was
investigated in three different clonal lines expressing either construct 3 or 4. Although the overall localization of both constructs was generally similar in all six clonal lines, the Cys-containing construct 3 tended to show a more defined perinuclear localization, whereas the Ala-containing construct 4 showed a more diffuse
distribution (Fig. 5). Double labeling of
the cell lines with a monoclonal antibody to Syntaxin-6 showed that the
major perinuclear staining of both constructs largely overlapped with
this TGN marker (data not shown) as previously reported for endogenous
CPD and other constructs containing the CPD transmembrane domain and
cytosolic tail (14, 15, 20).
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The addition of palmitic acid to some proteins affects their
association with lipid rafts (16-18). To determine whether this was
the case for the albumin/CPD tail constructs, the ability of Triton
X-100 to solubilize either construct 1 or 2 was examined. Both
constructs were predominantly soluble in Triton X-100 (Fig. 6), indicating that the presence or
absence of the Cys-containing region had no impact on the lipid raft
association of the constructs. Because CPD has been found to undergo
multimerization (25), which is presumably due to the luminal portion of
CPD that is not present in the constructs used in the present study,
the targeting of endogenous CPD to detergent-resistant membranes was
investigated. NIT3 cells were chosen for this analysis based on the
higher levels of endogenous palmitoylated CPD in this line compared
with the AtT-20 cell line (Fig. 3). When labeled with either
[35S]Met/Cys or with [3H]palmitic acid, the
endogenous CPD was detected in the Triton X-100-extractable fraction
and not in the detergent-resistant fraction (Fig.
7, left panels).
Caveolin-1, which is present in detergent-resistant membranes (26), was
detected only in the Triton X-100-insoluble fraction (Fig. 7,
right panels). Thus, neither the albumin/CPD constructs nor
endogenous palmitoylated CPD are detectable in the detergent-resistant
fraction of the NIT3 cells.
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Palmitoylation is known to affect the half-life of proteins such as PC7
(19). The three clonal cell lines expressing construct 3 and the three
clonal cell lines expressing construct 4 were examined by pulse-chase
analysis with [35S]Met/Cys. The Cys-containing construct
3 had a half-life of ~5 h (Fig. 8),
consistent with previous studies investigating similar constructs
containing reporter proteins with the transmembrane domain and
cytosolic tail of CPD (15). In contrast, the Ala-containing construct 4 was substantially more stable at every time point investigated with a
half-life of ~10 h (Fig. 8). To verify this result, a larger number
of replicates was performed at the single time point of 4 h (Fig.
8, inset). The Ala-containing construct was significantly
more stable than the Cys-containing construct (p < 0.001 using Student's t test).
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To test whether the additional stability of the Ala-containing
construct could be due to a reduced rate of exit from the Golgi, we
used a "budding" assay in which newly synthesized proteins are
trapped in the TGN by a 20 °C block, and then cells are
permeabilized and warmed to 37 °C in the presence (or absence) of an
energy-generating system. After incubation at 37 °C and
centrifugation, the supernatant represents newly packaged material,
whereas the pellet represents the material remaining in the Golgi/TGN.
There is substantially less of the Ala-containing construct in newly
packaged vesicles as compared with the Cys-containing construct (Fig.
9). To control for general differences in
the packaging efficiency of the two cell lines, we also examined the
same samples for endogenous CPE. Although the A16 cell line packaged
somewhat less of the CPE than the C21 cell line, this difference was a
smaller percentage of the total amount packaged than the difference in
the Alb-containing construct (Fig. 9). Taken together, these results
indicate that the packaging efficiency of the Ala-containing construct
into newly synthesized vesicles is reduced compared with the
Cys-containing construct.
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DISCUSSION |
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A major finding of the present study is that CPD is palmitoylated
on the Cys residues within the extended transmembrane domain. The
length of this transmembrane region (26 residues) is longer than the
typical 18 residues in a membrane-spanning helix, and it is
possible that the CPD sequence spans the lipid bilayer and then the
extra residues fold back into the bilayer as suggested for other
proteins (16, 27). If so, then the Cys-rich region would be contained
within this folded-back region and not within the membrane-spanning
helical region. It is interesting that both the length of the
hydrophobic segment and the number of Cys residues in this region
(three) have been conserved from Drosophila to mammals.
Additionally, a Caenorhabditis elegans gene encodes a
protein with two carboxypeptidase-like domains followed by a transmembrane segment of 27 residues that contains 3 Cys residues. Although it has not yet been shown whether this C. elegans
protein is a CPD homolog, it is the only transmembrane-bound
metallocarboxypeptidase in the genome, and so it is likely to represent
the CPD homolog. Taken together, the high conservation of the Cys
residues in the transmembrane segment of CPD and the finding that these
Cys residues are palmitoylated implies an important function for this modification.
The intracellular localization of a reporter construct lacking the CPD palmitoylation site was generally similar to that of the construct with the three Cys residues, although there was a subtle difference; the Cys-containing construct showed a more pronounced perinuclear distribution than the more diffuse localization of the Ala-containing construct. More quantitative analyses, such as the half-life and the rate of exit from the Golgi into vesicles, showed large differences between the two constructs. The findings that the Ala-containing construct has a longer half-life and slower rate of exit from the Golgi compared with the Cys-containing construct are consistent with each other and with previous studies that correlated turnover with Golgi exit (14, 15). CPD cycles between the Golgi and the cell surface via the constitutive secretory pathway and various endosomal compartments (5, 14, 15, 28). During this trafficking, a portion of the CPD ends up in the lysosomes where it gets degraded. Deletions of the CPD cytosolic tail that increase the rate of exit from the Golgi and the subsequent amount of CPD in the secretory and endosomal pathways lead to increased degradation of the protein (14, 15). The current hypothesis is that the CPD tail has positive signals that are required for the efficient movement of CPD through this cycling pathway as well as retention signals that serve to anchor the protein within the TGN (15). The present finding suggests that palmitoylation is one of the positive signals that increase the efficiency of trafficking of CPD throughout the secretory pathway.
Because CPD and PC7 are in the same enzymatic pathway, it was anticipated that palmitoylation would affect both proteins in the same manner. Interestingly, the mutation of the palmitoylation site in PC7 reduces the stability of this protein (19), the opposite of the effect on CPD. Similarly, palmitoylation has been found to direct many but not all proteins into detergent-resistant membrane rafts (16-18, 29, 30) but does not appear to affect the Triton X-100 solubility of CPD constructs or of endogenous CPD at neutral pH. Previous studies have found that endogenous bovine pituitary CPD is largely resistant to solubilization with Triton X-100 at pH 5.5 (7) but not at neutral pH values.2 It is possible that multimerization of CPD, which has been proposed to occur (25), increases the net amount of palmitoyl groups per protein complex and enables the protein to enter detergent-resistant membrane rafts at low pH. Although pH dependence of the multimerization of CPD has not been tested, the related CPE is known to form aggregates at mildly acidic pH values and at millimolar Ca2+ levels (31). Thus, it is possible that palmitoylation of full-length CPD present in multimeric complexes affects the interaction of the protein with lipids under physiological conditions in the secretory pathway. Multimerization is found for other palmitoylated proteins such as influenza hemagglutinin. When each of the subunits has three palmitoyl groups, the trimeric complex has a total of nine palmitoyl groups, and ~30% enters detergent-resistant membrane rafts (29). However, when each subunit has only two palmitoyl groups, or six per complex, less than 3% of the complex is Triton X-100-insoluble (29). Thus, the total number of palmitoyl groups per protein complex is critical for driving the complex into detergent-resistant membrane rafts.
A general problem in interpreting studies involving mutations is that
the particular mutation may affect multiple processes. For example, the
replacement of Cys by Ala could alter the conformation of the protein
or disrupt a consensus site for binding to some factor other than
palmitate. In the CPD tail sequence, a protein phosphatase 2A binding
site maps to the region of the cytosolic tail that is adjacent to the
Cys-rich region in the proximal portion of the transmembrane domain. In
the present study, it is clear that Cys residues are required for
palmitoylation and are also required for efficient trafficking of CPD
out of the Golgi. While it is likely that palmitoylation of these Cys
residues is therefore important for trafficking, other possibilities
cannot be excluded.
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ACKNOWLEDGEMENT |
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Microscopy was performed in the laboratory of Dr. Jonathan Backer (Molecular Pharmacology, Albert Einstein College of Medicine).
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FOOTNOTES |
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* This work was supported primarily by National Institutes of Health Grant R01 DK55711 and also by Research Scientist Development Award K02 DA00194 (to L. D. F.). The DNA sequencing facility of the Albert Einstein College of Medicine was supported in part by Cancer Center Grant CA13330.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
Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park
Ave., Bronx, NY 10461. Tel.: 718-430-4225; Fax: 718-430-8954; E-mail:
fricker@aecom.yu.edu.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M209379200
2 E. V. Kalinina and L. D. Fricker, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: CPD, carboxypeptidase D; PC7, proprotein convertase 7; VSVG, vesicular stomatitis virus G protein; Alb, albumin; CPE, carboxypeptidase E; DMEM, Dulbecco's modified Eagle's medium; TGN, trans Golgi network.
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