Palmitoylation of Carboxypeptidase D

IMPLICATIONS FOR INTRACELLULAR TRAFFICKING*

Elena V. Kalinina and Lloyd D. FrickerDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2.   Schematic representation of the various CPD transmembrane domain (CPDTM) and VSVG transmembrane domain (VSVGTM) mutants attached to the C terminus of albumin (ALB). The Alb and CPD-tail regions are not drawn to scale. Bottom, the sequence of the rat CPD tail portion of the various constructs with the Ile at the junction of the transmembrane domain and the cytoplasmic domain changed to Leu to create an AflII site (used for constructing the plasmids). Domains previously described in the CPD tail (15) are indicated including the YXXL-like motif, two acidic clusters, a di-Leu sequence, and a casein kinase 2 (CK-2) consensus site.

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 3.   Palmitoylation of CPD in various wild-type cell lines and in AtT-20 cells expressing Alb-CPD tail (construct 1). Cells were incubated with [3H]palmitic acid for 5 h and solubilized, and Alb-tail or CPD proteins were isolated by immunoprecipitation using an antiserum to human albumin (lane 1) or to CPD (lanes 2-6) and subjected to denaturing gel electrophoresis and fluorography as described under "Materials and Methods." The position and molecular masses of prestained markers (Invitrogen) are indicated.

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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Fig. 4.   CPD is palmitoylated on Cys residues. AtT-20 cells stably expressing construct 3 (clone C21) or construct 4 (clone A16) were labeled with [35S]Met/Cys (A) or [3H]palmitic acid (B) and immunoprecipitated with an anti-albumin antiserum as described under "Materials and Methods." Duplicate samples of the immunoprecipitates were separated on denaturing polyacrylamide gels. One gel was subsequently soaked overnight in 1 M Tris (pH 7.4) as a control, while the other gel was treated overnight with 1 M hydroxylamine (NH2OH, pH 7.4) followed by fluorography. The positions and molecular masses of prestained protein standards (Invitrogen) are indicated.

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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Fig. 5.   Steady-state localization of CPD constructs by immunofluorescence. AtT-20 cells stably expressing construct 3 carrying the C/C/C insert (clones C21, C24, and C36; right panel) or construct 4 carrying A/A/A insert (clones A16, A17, and A21; left panel) were stained with an antiserum to human albumin and visualized using a fluorescein isothiocyanate-labeled goat anti-rabbit IgG. Scale bar, 10 µm.

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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Fig. 6.   Constructs 1 and 2 are not resistant to extraction with Triton X-100. AtT-20 cells stably expressing the palmitoylation-positive CPD transmembrane domain (CPD TM) (construct 1, left panel) or palmitoylation-negative VSVG transmembrane domain (VSVG TM) (construct 2, right panel) were solubilized with Triton X-100 at 4 °C and then separated by centrifugation into Triton X-100-soluble (S) and -insoluble (I) fractions. Proteins of the two fractions were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with an anti-albumin antibody. The positions and molecular masses of prestained protein standards are indicated.


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Fig. 7.   Endogenous CPD is not resistant to extraction with Triton X-100. NIT3 cells were labeled with [35S]Met/Cys or [3H]palmitic acid and extracted with Triton X-100 as described under "Materials and Methods." CPD (left panels) and caveolin-1 (right panel) were immunoprecipitated with the appropriate antisera. S, supernatant containing Triton X-100-soluble material; P, pellet containing detergent-resistant membranes. The positions and molecular masses of prestained protein standards are indicated. Arrows show the expected position of CPD and caveolin-1 (Cav-1).

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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Fig. 8.   Pulse-chase analysis of cell lines expressing constructs 3 and 4. Three independent clones of AtT-20 cells stably expressing construct 3 and three clones expressing construct 4 were labeled for 15 min with [35S]Met/Cys and then chased for the indicated periods of time. Proteins were isolated by immunoprecipitation with an antiserum to human albumin (Calbiochem) and then separated on a denaturing polyacrylamide gel. Error bars represent S.E., n = 3. Inset, a second experiment was performed for a single time point of 4 h. *, p < 0.001 (Student's t test).

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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Fig. 9.   Packaging of constructs 3 and 4 into post-TGN secretory vesicles. The in vitro vesicle budding assay was performed using AtT-20 cells expressing construct 3 (clone C21) or construct 4 (clone A16). Permeabilized cells were incubated in the absence (-) or presence (+) of an energy-generating system. Following incubation, the samples were centrifuged to generate supernatants (containing nascent vesicles) and pellets (containing the TGN) and then subjected to immunoprecipitation using an antibody to human albumin or CPE (as control). Upper panel, representative autoradiograms of the immunoprecipitate with antisera to Alb (left) or CPE (right). The positions and molecular masses of prestained protein standards are indicated. Lower panel, quantitation of the TGN budding assay. The efficiency of budding was calculated as the ratio of radiolabeled albumin immunoreactivity in the vesicle fraction (after subtraction of the background measured in the absence of energy) relative to the total amount of radiolabeled albumin immunoreactivity. Error bars indicate the range of duplicate determinations. sup, supernatant.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha  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 alpha  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.

    ACKNOWLEDGEMENT

Microscopy was performed in the laboratory of Dr. Jonathan Backer (Molecular Pharmacology, Albert Einstein College of Medicine).

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Barrett, A. J., Rawlings, N. D., and Woessner, J. F. (1998) in Handbook of Proteolytic Enzymes (Barrett, A. J. , Rawlings, N. D. , and Woessner, J. F., eds) , pp. 1318-1320, Academic Press, San Diego
2. Reznik, S. E., and Fricker, L. D. (2001) Cell. Mol. Life Sci. 58, 1790-1804[Medline] [Order article via Infotrieve]
3. Xin, X., Varlamov, O., Day, R., Dong, W., Bridgett, M. M., Leiter, E. H., and Fricker, L. D. (1997) DNA Cell Biol. 16, 897-909[Medline] [Order article via Infotrieve]
4. Tan, F., Rehli, M., Krause, S. W., and Skidgel, R. A. (1997) Biochem. J. 327, 81-87[Medline] [Order article via Infotrieve]
5. Varlamov, O., and Fricker, L. D. (1998) J. Cell Sci. 111, 877-885[Abstract/Free Full Text]
6. Kuroki, K., Eng, F., Ishikawa, T., Turck, C., Harada, F., and Ganem, D. (1995) J. Biol. Chem. 270, 15022-15028[Abstract/Free Full Text]
7. Song, L., and Fricker, L. D. (1995) J. Biol. Chem. 270, 25007-25013[Abstract/Free Full Text]
8. Song, L., and Fricker, L. D. (1996) J. Biol. Chem. 271, 28884-28889[Abstract/Free Full Text]
9. Ishikawa, T., Murakami, K., Kido, Y., Ohnishi, S., Yazaki, Y., Harada, F., and Kuroki, K. (1998) Gene (Amst.) 215, 361-370[CrossRef][Medline] [Order article via Infotrieve]
10. Fan, X., Qian, Y., Fricker, L. D., Akalal, D. B., and Nagle, G. T. (1999) DNA Cell Biol. 18, 121-132[CrossRef][Medline] [Order article via Infotrieve]
11. Fricker, L. D. (1998) in Handbook of Proteolytic Enzymes (Barrett, A. J. , Rawlings, N. D. , and Woessner, J. F., eds) , pp. 1349-1351, Academic Press, San Diego
12. Fricker, L. D. (2002) in The Enzymes: Co- and Posttranslational Proteolysis of Proteins (Dalbey, R. E. , and Sigman, D. S., eds), Vol. 23 , pp. 421-452, Academic Press, San Diego
13. Nakayama, K. (1997) Biochem. J. 327, 625-635[Medline] [Order article via Infotrieve]
14. Eng, F. J., Varlamov, O., and Fricker, L. D. (1999) Mol. Biol. Cell 10, 35-46[Abstract/Free Full Text]
15. Kalinina, E., Varlamov, O., and Fricker, L. D. (2002) J. Cell. Biochem. 85, 101-111[CrossRef][Medline] [Order article via Infotrieve]
16. Milligan, G., Parenti, M., and Magee, A. I. (1995) Trends Biochem. Sci. 20, 181-186[CrossRef][Medline] [Order article via Infotrieve]
17. Mumby, S. M. (1997) Curr. Opin. Cell Biol. 9, 148-154[CrossRef][Medline] [Order article via Infotrieve]
18. Resh, M. D. (1999) Biochim. Biophys. Acta 1451, 1-16[Medline] [Order article via Infotrieve]
19. Van de Loo, J. H. P., Teuchert, M., Pauli, I., Plets, E., van de Ven, W. J. M., and Creemers, J. W. M. (2000) Biochem. J. 352, 827-833[CrossRef][Medline] [Order article via Infotrieve]
20. Varlamov, O., Kalinina, E., Che, F., and Fricker, L. D. (2001) J. Cell Sci. 114, 311-322[Abstract/Free Full Text]
21. Mitra, A., Song, L., and Fricker, L. D. (1994) J. Biol. Chem. 269, 19876-19881[Abstract/Free Full Text]
22. Varlamov, O., Wu, F., Shields, D., and Fricker, L. D. (1999) J. Biol. Chem. 274, 14040-14045[Abstract/Free Full Text]
23. Fricker, L. D., Berman, Y. L., Leiter, E. H., and Devi, L. A. (1996) J. Biol. Chem. 271, 30619-30624[Abstract/Free Full Text]
24. Varlamov, O., Fricker, L. D., Furukawa, H., Steiner, D. F., Langley, S. H., and Leiter, E. H. (1997) Endocrinology 138, 4883-4892[Abstract/Free Full Text]
25. Urban, S., Kruse, C., and Multhaup, G. (1999) J. Biol. Chem. 274, 5707-5715[Abstract/Free Full Text]
26. Kurzchalia, T. V., Dupree, P., Parton, R. G., Kellner, R., Virta, H., Lehnert, M., and Simons, K. (1992) J. Cell Biol. 118, 1003-1014[Abstract]
27. Hawtin, S. R., Tobin, A. B., Patel, S., and Wheatley, M. (2001) J. Biol. Chem. 276, 38139-38146[Abstract/Free Full Text]
28. Varlamov, O., Eng, F. J., Novikova, E. G., and Fricker, L. D. (1999) J. Biol. Chem. 274, 14759-14767[Abstract/Free Full Text]
29. Melkonian, K. A., Ostermeyer, A. G., Chen, J. Z., Roth, M. G., and Brown, D. A. (1999) J. Biol. Chem. 274, 3910-3917[Abstract/Free Full Text]
30. Schweizer, A., Loffler, B., and Rohrer, J. (1999) Biochem. J. 340, 649-656[CrossRef][Medline] [Order article via Infotrieve]
31. Song, L., and Fricker, L. D. (1995) J. Biol. Chem. 270, 7963-7967[Abstract/Free Full Text]
32. Sidyelyeva, G., and Fricker, L. D. (2002) J. Biol. Chem. 277, 49613-49620[Abstract/Free Full Text]


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