©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Biochemical Heterogeneity and Phosphorylation of Coatomer Subunits (*)

(Received for publication, August 4, 1995; and in revised form, January 9, 1996)

David Sheff (§) Martin Lowe (1)(¶) Thomas E. Kreis (1) Ira Mellman (**)

From the Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002 and the Department of Cell Biology, University of Geneva, Sciences III, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The coat protomer complex I (COPI) family of coat proteins are involved in the assembly of membrane-associated coats thought to mediate vesicular transport between the endoplasmic reticulum and the Golgi complex, between adjacent Golgi cisternae, and possibly in the endocytic pathway. We investigated whether this heterogeneity in the sites of COPI action might be reflected in biochemical heterogeneity of one or more COPI subunits. A simplified method was devised to purify the cytosolic COPI precursor complex, coatomer, from rat liver cytosol. The individual subunits were analyzed by high resolution two dimensional gel electrophoresis and mass spectroscopic analysis of tryptic peptides. Considerable charge heterogeneity was observed, particularly for the beta-COP and -COP subunits. The multiple species detected, however, did not appear to reflect the presence of distinct translation products but rather a significant degree of protein phosphorylation. The observed pI of beta-COP was sensitive to alkaline phosphatase digestion. Moreover, isolation of coatomer from metabolically labeled tissue culture cells demonstrated directly that both beta-COP and -COP, but no other coatomer subunits, were serine-phosphorylated. COPI phosphorylation may regulate coatomer assembly, membrane recruitment, or the specificity of coatomer-organelle interaction.


INTRODUCTION

Transport of proteins between organelles is dependent on the formation of vesicular carriers. Often, these transport vesicles are formed in conjunction with the assembly of specific membrane-associated coat proteins derived from cytosolic precursors. At least three types of coat proteins are known, each associated with one or more transport steps. Clathrin coats act to selectively accumulate plasma membrane receptors at clathrin-coated pits, resulting in receptor endocytosis (1) and the transport of proteins from the trans-Golgi network (TGN) (^1)to endosomes and perhaps other destinations(2) . In addition, two distinct coat complexes, designated COPI and COPII, play important roles early in the secretory pathway (3, 4, 5, 6) . Both COP complexes are well known to mediate transport among the ER, intermediate compartment, and/or the Golgi complex by facilitating the formation of COP-coated vesicles at one or more of these three sites(6, 7, 8, 9) . Unlike clathrin-coated vesicles, it is less clear whether COPI and/or COPII vesicles selectively accumulate their intended cargo at the time of vesicle budding. However, like clathrin-coated vesicles, COPI vesicles do mediate multiple transport steps, being active not only in transport among ER, intermediate compartment, and the Golgi complex, but also between Golgi cisternae (2, 3, 4) . The role of COPI in transport between the ER and the Golgi complex, as well as in endosome function, has been established by inhibition of transport by microinjected anti-beta-COP antibody (10) as well as by the isolation of COPI mutants in yeast and Chinese hamster ovary cells(8, 11) . There is also considerable evidence from in vitro reconstitution experiments that at least COPI coats are involved in transport through the Golgi stack(12) .

COPI is recruited to membranes as a complex derived from a cytosolic precursor called coatomer(13) . The coatomer complex is a high molecular weight heterooligomer composed of seven proteins, alpha-COP (160 kDa), beta-COP (110 kDa), beta`-COP (102 kDa), -COP (98 kDa), -COP (61 kDa), -COP (35 kDa), and -COP (20 kDa), each present in equimolar amounts(13, 14) . Binding of coatomer to membranes is dependent on adp-RIBOSYLATION FACTOR, a small myristoylated GTPase that is thought to bind GTP upon interaction with a membrane-associated guanine nucleotide exchange factor(15, 16, 17) . Exchange of GDP for GTP facilitates coatomer binding by an as yet unknown mechanism that is sensitive to the drug brefeldin A(18) . Assembly of the COPI complex is thought to drive vesicle budding(19) . COPI assembly in the ER or Golgi may also provide for the selective inclusion in Golgi-derived recycling vesicles of resident ER proteins bearing the KKXX retrieval motif(20) .

Although COPI in vivo is concentrated in the intermediate compartment and cis-Golgi(21) , immunolocalization experiments also suggest that at least some COPI is also found elsewhere in the Golgi stack including the TGN(22, 23) . Thus, COPI may play a functional role at sites other than those between the ER and cis-Golgi. Evidence is accumulating that COPI components are involved in the endocytic pathway, apparently at the level of endosomes. A CHO cell line bearing a temperature-sensitive mutation in -COP exhibits a defect in the recycling of low density lipoprotein receptors internalized from the plasma membrane(11) . Recent evidence also demonstrates that COPI components bind specifically to endosomes in vitro and that antibodies directed against beta-COP inhibit endosome function(24) .

How might a single protein complex act at multiple intracellular sites? One possibility is that the COPI proteins are structurally heterogeneous, with multiple isoforms of coatomer proteins combining to produce functionally distinct COPI complexes. Such heterogeneity might also be achieved by differential post-translational modification of one or more COPI subunits, with isoforms of COPI possibly associating with different organelles. Since little was known about the structure and composition of coatomer complex(es), we decided to determine whether individual members of the coatomer complex exhibited any biochemical heterogeneity. Indeed, a surprising degree of heterogeneity was observed, particularly in the beta and subunits. We also found that both subunits are phosphorylated, suggesting that phosphorylation may be responsible for at least some of these differences.


MATERIALS AND METHODS

Antibodies

Polyclonal antibodies against beta-COP peptides (EAGE, KLVE, and E-1) were developed in rabbits(10) . An antibody against beta`-COP was produced in rabbits(25) . Polyclonal antibodies against alpha-, -, -, and -COP were generous gifts from J. Rothman and C. Harter. Goat anti-rabbit-IgG and goat anti-mouse IgG horseradish peroxidase conjugates were purchased from Pierce.

Other Reagents

Chemiluminescent detection system and [P] orthophosphate (1000 Ci/mM) were purchased from Amersham Corp. All protease inhibitors, phosphatase inhibitors, ammonium sulfate, polyethylene glycol 3350, and L-1-tosylamido-2-phenylethyl chloromethyl ketone/trypsin were purchased from Sigma. Protein A-Sepharose CL-4B was purchased from Pharmacia Biotech Inc. All other reagents were reagent grade or cell culture grade as required.

Purification of Rat Liver Coatomer (COPI)

Purification of rat liver coatomer was accomplished through a substantial modification of the method of Waters and Rothman(13) . Unless otherwise noted, all operations were performed at 4 °C. Approximately 250 g of fresh liver from 10-15 adult Sprague-Dawley rats (Harlan Sprague-Dawley) were homogenized in 2 volumes of buffer (25 mM Tris, pH 7.5, 320 mM sucrose, 500 mM KCl, 2 mM EDTA, 1 mM dithiothreitol) containing protease inhibitors (2 µg/ml pepstatin A, antipain, and leupeptin; 1 mM phenylmethylsulfonyl fluoride) using a polytron homogenizer with 1.5-cm cutter assembly at maximum speed for three 1-min bursts on ice with 1-min rests. The lysate was cleared by sequential centrifugation at 9000 times g for 15 min followed by centrifugation of the supernatant at 100,000 times g for 1 h. This material (S100) was stored at -70 °C for up to 4 months. For a typical purification, 150 ml of S100 was diluted 6-fold with cytosol buffer (25 mM Tris, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA plus protease inhibitors as above). Protein concentration was 5 mg/ml. Ammonium sulfate was added to 25% of saturation and stirred for 15 min on ice, and then precipitate was removed by centrifugation, and the supernatant was brought to ammonium sulfate at 45% of saturation with stirring on ice. The precipitate was collected by centrifugation and redissolved in 150 ml of cytosol buffer. An additional 120 ml of cytosol buffer was added and then 30 ml of 60% (w/v) polyethylene glycol 3350 in distilled H(2)O with gentle stirring. The mixture was incubated at 4 °C for 30 min, and the precipitate was collected by centrifugation at 10,000 times g for 15 min. The precipitate was resuspended in 20 ml of G buffer (10 mM Tris, pH 7.5, 0.2 mM ATP, 0.2 mM CaCl(2)), the insoluble material was removed by centrifugation, and the supernatant was passed over a 20-ml column containing 250 mg of DNase-I (Sigma) coupled to agarose (Affi-Gel-10, Bio-Rad, prepared according to the manufacturer's directions) to remove contaminating actin and actin binding proteins. Eluent was desalted into cytosol buffer using 10DG desalting columns (Bio-Rad) and applied to a 50-ml DEAE cellulose column (DE52, Whatman) equilibrated in cytosol buffer. COPI was eluted with a 100-400 mM KCl gradient over 200 ml, with the elution of COPI followed by spot blot on nitrocellulose using EAGE antibody. In a final step, peak COPI fractions were pooled, diluted 1:1 with cytosol buffer, and applied to a 1-ml Mono-Q column (Pharmacia) equilibrated in cytosol buffer and mounted on a fast protein liquid chromatography apparatus (Pharmacia). The column was washed with 300 mM NaCl and then eluted with a 350-400 mM NaCl gradient over 20 ml. COPI, as assayed by the presence of beta-COP on a spot blot using EAGE antibody, eluted as a single peak. The presence and purity of COPI was confirmed by SDS-PAGE. An alternative final step was employed in preparing samples for two-dimensional gels. Here, DEAE eluent was concentrated in a Centricon-30 microconcentration (Amicon) to 400 µl and applied to a 24-ml Superose-6 (Pharmacia) column equilibrated in cytosol buffer with 50 mM KCl. As with Mono-Q, COPI eluted in a single peak. This final step produces a somewhat lower yield and contains some contaminants between 30 and 100 K(D) by SDS-PAGE. For copurification of labeled CHO cytosol and rat liver COPI, all quantities were divided by 3, 1 ml of labeled cytosol was added to 50 ml of rat liver S100, and the Mono-Q column was used as the final step.

Two-dimensional Gel Electrophoresis

Two-dimensional gels were run by a modification of the method of Laemmli(26) . Samples were prepared by first precipitating with 10% trichloroacetic acid. Pellets sufficient to run five minigels or two large gels were dissolved in 20 µl of 2% SDS, 4 µl of 1 M dithiothreitol and solubilized for 30 min at room temperature (some samples were boiled). Then 60 mg of urea, 10 µl of 80% CHAPS, and 5 µl of ampholines 7-9 (Bio-Rad) were added. The final volume was adjusted to 100 µl with distilled H(2)O. Approximately 20 µl of this solution was used for each 5-cm isoelectric focusing. First dimension IEF gels were run in 50 times 1-mm gels or in 120 times 1.5-mm gels. IEFs gels contained 6 M urea and 2% (w/v) CHAPS (Sigma) instead of Nonidet P-40 and were typically pH 5-7 or pH 5-9 using 2% ampholines with a ratio of 4:1 narrow range ampholines to pH 3-10 broad range ampholines. Overlay buffer was omitted from the two-dimensional gel procedure. Second dimension SDS-PAGE was 8% polyacrylamide using described methods. SDS gels were stained with silver stain or transferred to Immobilon-P for Western blot or analysis by peptide mass spectroscopy. For Western blot analysis, second dimension SDS-PAGE gels were placed directly onto either nitrocellulose (0.2 µM pore) or Immobilon-P membranes in a semidry blotter (Millipore). Transfer was accomplished at room temperature using 250-mA run for 20 min/minigel.

Alkaline Phosphatase Treatment

100 µg of coatomer in 100 µl of 25 mM Tris, pH 7.5, 1 M NaCl, 1 mM EDTA was diluted to 1 ml with 50 mM NaHCO(3) pH 9.8, 1 mM MgCl(2) (for negative controls, 1 mM EDTA was substituted for 1 mM MgCl(2)). 33.6 or 3.36 µg of calf intestine alkaline phosphatase (3000 units/mg Sigma) was added, and the reactions were incubated at 37 °C for 1 h and then precipitated with trichloroacetic acid and applied to two-dimensional gels.

Mass Spectroscopy

Purified rat liver coatomer was applied to 150 times 150 times 1.5-mm two-dimensional gels (first dimension pH 5-9 gradient). The second dimension gel was blotted onto Immobilon-P membranes and used for tryptic peptide digestion and mass spectroscopy analysis by D.J.C. Pappin (Imperial Cancer Research Fund, London), as described(27) .

P and S Labeling

CHO cells were maintained in spinner flasks using alpha-MEM (Sigma) with 10% heat inactivated fetal calf serum (Intergen), 5% CO(2). For labeling, a modified phosphate-free alpha-MEM medium was made from phosphate-free MEM by adding (in g/liter) 0.025 alanine, 0.05 asparagine, 0.03 aspartate, 0.1 cysteine, 0.75 glutamate, 0.05 glycine, 0.04 proline, 0.025 serine, and 0.11 pyruvate. Sulfate-free alpha-MEM was purchased from Sigma. Heat inactivated fetal calf serum was dialyzed extensively against 25 mM Tris, pH 7.4 (1000 M(r) cut-off). Cells were seeded at 1 times 10^5 cells/ml in 100 ml of labeling medium with 5 ml of unmodified alpha-MEM and 10 mCi of P orthophosphate and allowed to grow under standard conditions in a spinner flask for 14 h. CHO cells were also labeled under these conditions with 3 mCi of S translabel (Amersham) in sulfate-free media. Cells were collected by centrifugation and washed in Tris-buffered saline prior to lysis. Alternatively, CHO cells were grown to 80% confluence in a 10-cm culture dish, washed with labeling media, and labeled for 4 h with 1 mCi of P orthophosphate in 4 ml of labeling medium. HeLa cells were maintained in 10-cm culture dishes using Dulbecco's modified Eagle's medium, 5% heat-inactivated fetal calf serum, 5% CO(2). Labeling of HeLa cells was as for short term CHO cell labeling.

To prepare cell lysates, spinner culture cells were lysed in 4 volumes of wash buffer (50 mM Tris, pH 7.4, 300 mM NaCl, 0.5% Triton X-100) brought to 1% Triton X-100 with protease inhibitors (2 µg/ml pepstatin A, antipain, aprotinin, and leupeptin, 0.5 mM 1, 10 phenanthroline, 1 µM chymostatin, 1 mM phenylmethylsulfonyl fluoride) and phosphatase inhibitors (100 µM NaVO(4), 20 mM sodium glycerophosphate, 10 mM NaF). The cell pellet was agitated on ice for 15 min and then centrifuged at 10,000 times g for 15 min. The supernatant was cleared by centrifugation at 100,000 times g for 20 min. Supernatants were kept at -70 °C for up to 1 week.

Cytosols were prepared from labeled cell pellets without detergent by suspension of the pellet in 2 volumes of TEAS (10 mM trienthanolamine/acetic acid, pH 7.3, 1 mM EDTA, 250 mM sucrose) followed by disruption using a ball bearing cell homogenizer (0.2496-inch ball) until >80% cell lysis was achieved (assayed by trypan blue exclusion).

Immunoprecipitations and Autoradiography

Immunoprecipitations were used to recover intact coatomer from cell lysates. Affinity-purified rabbit polyclonal antibodies (0.4-1 µg of EAGE, KLVE, E-1, or anti beta`) were incubated overnight at 4 °C with 10 µl of protein A-Sepharose CL-4B beads. Lysate was prepared by preclearing with 250 µl of labeled lysate with 10 µl of uncoupled beads and incubating overnight at 4 °C. The uncoupled beads were removed and washed, and then the immunoprecipitation was started with the addition of antibody-coupled beads for 4-6 h at 4 °C. Beads were washed in the presence of Triton X-100 unless otherwise noted. Beads were eluted by boiling in SDS-PAGE sample buffer. Immunoprecipitated material was applied to 20-cm SDS-PAGE gels (6.5, 8, or 10% to resolve beta,beta`,; ,; or COPs, respectively), and blotted onto nitrocellulose or Immobilon-P as was done for two-dimensional gel blots. Blots were used for both autoradiography and Western blot detection of individual coatomer subunits.

Phosphoamino Acid Analysis

P-Labeled coatomer was immunoprecipitated from HeLa cells with EAGE antibodies and subjected to SDS-PAGE on a 10% gel. The gel was agitated in five successive changes of distilled water and dried under vacuum without protein fixation, and labeled COPs were detected by autoradiography. The 100-kDa COPs were excised from the gel, and the gel slice was washed once with 10% acetic acid, 30% methanol and then 3 times 1 h with 50% methanol. The gel slice was then dried under vacuum and swollen in 0.5-ml ammonium acetate for trypsin digestion. 25 µl of 1 mg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone/trypsin in the same buffer was added, and the COPs were digested for 6 h at 37 °C on a shaker. A further 10 µl of the trypsin solution was added, and the digestion continued for another 12 h. Released peptides were removed after centrifugation, and the gel slices were washed by two additions of 0.4 ml H(2)O and shaking to 25 min followed by centrifugation. Pooled supernatants were centrifuged at 150,000 times g for 1 h to clear. The resulting supernatant was lyophilized. The peptides were resuspended in 1 ml of 6 M HCl and hydrolyzed for 90 min at 110 °C. After drying, the samples were solubilized in 10 µl of the first dimension electrophoresis buffer, mixed with 5 µg standard phosphoamino acid mix, and analyzed by two dimensional thin layer electrophoresis on 100-µm-thick cellulose acetate plates (EM Science). The first dimension was run at pH 1.9 in 7.8% acetic acid, 2.5% formic acid, and 89.7% H(2)O at 750 V for 3.25 h at 4 °C. The second dimension was run at pH 3.5 in 5% acetic acid, 0.5% pyridine, and 94.5% H(2)O at 500 V for 2.5 h at 4 °C. P-labeled amino acids were detected by autoradiography, and standard phosphoamino acids were then detected by ninhydrin staining.


RESULTS

Heterogeneity of Coatomer Components

To determine whether heterogeneity existed among cytosolic COPI components, coatomer was isolated from rat liver using an optimized purification protocol. The method differed from that developed by Waters and Rothman (28) in that it avoids isoelectric precipitation as well as extensive dialysis. Purified coatomer ran as a single peak of 500 kDa on a Superose-6 column and as a single peak on a 1-ml Mono-Q anion exchange column (not shown). This procedure produced coatomer at a yield and purity comparable with previously published results, but the procedure was easier and generated fewer contaminants between 43 and 55 kDa. Purified material is shown in Fig. 1; individual COPI subunits were identified by Western blot with subunit-specific antibodies (kindly provided by J. Rothman and C. Harter). On our gel system, beta-COP was difficult to differentiate from -COP (except by immunologic means), while beta`-COP was found to migrate faster than the beta/-COP band. -COP typically migrated as a 61-kDa doublet, both components of which were immunoreactive. The lower band of the doublet increased with extended storage, suggesting it arose as a degradation product. alpha-, -, and -COPs were present as described previously(28) .


Figure 1: Purification of coatomer from rat liver. Purification of COP I was as described under ``Materials and Methods'' with Mono-Q column as the final step. An SDS-PAGE separation of the complex was performed and visualized with Coomassie Blue. The large gel is 6.5% polyacrylamide. Inset is 12% polyacrylamide. Positions of individual COPs are indicated at the right based upon Western blot identification with COP-specific antibodies. Hash marks indicate positions of molecular mass standards (205, 116.5, 80, and 49.5 kDa from top to bottom). beta` can be seen as a distinct band below the beta/ band. Doublet of delta COP is a degradation artifact in this particular sample, while the doublet of epsilon COP was seen in all samples.



We next analyzed the purified coatomer by high resolution two-dimensional IEF/SDS-PAGE. The first dimension was a denaturing pH 5-9 IEF; the second dimension was an 8% SDS gel. Proteins were visualized by silver stain (Fig. 2, right panel). To identify which spots corresponded to which coatomer subunit, a series of two-dimensional gels were run in pairs. One of each pair was transferred onto an Immobilon-P membrane. These membranes were stained with Coomassie Blue for accurate alignment and probed with subunit-specific antibodies. A composite of Western blots is shown in the left panel of Fig. 2. The gels used in this composite were selected for clarity and thus differ slightly in the amount of protein loaded from the silver-stained gel. There is also some intergel variability. To definitively identify a silver-stained spot, multiple pairs of silver-stained and Western blotted membranes were compared. It was thus possible to identify spots corresponding to almost all coatomer subunits with the exception of alpha-COP, which apparently entered the IEF gel poorly, if at all. -COP was not retained by the 6.5% gel used for SDS-PAGE. For some COPI species (e.g. -COP), the Western blotted pattern was often broader than that observed by silver stain; in this case -COP was identified by the M(r) and pI of the visualized spot and the absence of any neighboring spots even when the silver stain was overdeveloped. Several silver-stained spots were not reactive with any COPI antibody and presumably represented contaminants in the preparation.


Figure 2: Two dimensional IEF/SDS-PAGE reveals heterogeneity of individual coatomer subunits. Rat liver COP I purified as under ``Materials and Methods'' with Superose-6 column as a final step separated in a two-dimensional (IEF/SDS-PAGE) system, pH 5-7 in the first dimension and 8% SDS-PAGE in the second dimension. The left panel shows Western blots with COP-specific antibodies from a series of two-dimensional gels. Each was compared with a parallel silver-stained gel to identify the corresponding protein spot. A single silver-stained two-dimensional gel is shown in the right panel for comparison.



The two-dimensional gel analysis revealed considerable heterogeneity of the beta- and -COP spots. beta-COP existed as a series of spots at 110 kDa. Four to five separate species (pI 5.5-6.0) were consistently observed in multiple preparations of COPI analyzed by multiple two-dimensional gels, suggesting they reflected multiple beta-COP isoforms rather than deamidation or carbamylation in the denaturing IEF first dimension gel. Because COPI was purified to near homogeneity before two-dimensional gel analysis, it is unlikely that this is the result of a fortuitous cross-reactivity with a similarly sized group of unrelated proteins. This result is further confirmed by the finding of 3-4 beta-COP immunoreactive species in two-dimensional gels of total human keratinocyte extracts(29) .

-COP was consistently detected as three distinct 61-kDa species (pI 6.5-7.0) by both Western blot and silver stain (Fig. 2). As for beta-COP, multiple -COP species were observed upon two-dimensional gel electrophoresis. They were also widely spaced, suggesting the spots represented isoforms rather than IEF artifacts.

-COP was poorly soluble in the first dimension, resulting in a diminished spot in the silver-stained gel (and on Coomassie-stained filters); material precipitated at the basic end of the IEF gel was detected in the Western blot (Fig. 2, left edge of left panel). Nevertheless, sufficient -COP did enter the gel to be identified as pI 5.9 slightly smaller than beta-COP by both Western blot and silver stain. Occasionally, immunoreactive -COP appeared as a streak or two partially resolved spots (Fig. 2), but this result was inconsistent relative to the behavior of beta- and -COP.

-COP corresponded to a 36-kDa doublet or triplet (pI 4.6) by silver stain. This was consistent with a recent report of partial truncation of the -COP amino terminus(30) . Identification of -COP was made primarily on the basis of M(r) and calculated pI. Western blots for -COP typically revealed a much broader streak. The reason for this disparity is unclear.

An unknown 90-kDa protein (pI 5.2) was consistently found associated with even the most homogenous preparations of coatomer (Fig. 2, right panel, arrow). It was associated with a smaller spot of the same M(r) but a pI of 5.1. The identity of these spots may be of interest due to their clear propensity to be enriched through all stages of the coatomer purification. Other contaminants observed between 40 and 55 kDa varied between coatomer preparations.

Although the charge heterogeneity of immunoreactive beta- and -COP in highly purified coatomer preparations might have reflected multiple beta- and -COP gene products, further analysis strongly suggested this was not the case. Purified coatomer was separated on preparative two-dimensional gels, transferred to Immobilon-P membranes, and visualized with Coomassie Blue. Each of the multiple beta and spots were separately excised and subjected to tryptic digestion. Digested peptides were analyzed by mass spectroscopy to generate a tryptic peptide map. All large peptides obtained from each of the middle three (of five) beta spots had identical sizes, strongly suggesting that they did not arise from different gene products (not shown). The two most acidic -COP spots were also subjected to mass spectroscopy peptide analysis. As with beta-COP, a comparison of the 20 largest tryptic fragments from each spot was identical, suggesting that each set of peptides arose from the same protein and that modifications of the protein could not be detected among the larger peptide fragments.

We also attempted to identify closely related beta-COP cDNAs in a rat liver cDNA library by probing with authentic beta-COP cDNA probes at low stringency. Polymerase chain reaction was also performed using a variety of oligonucleotide primers corresponding to regions of high conservation between rat and yeast beta-COP sequences(23, 31) . Both approaches yielded only multiple cDNA clones identical to beta-COP. (^2)Taken together, these negative results suggested that the observed heterogeneity of beta-COP, and possibly of -COP, represented post-translational modification of both proteins.

Phosphorylation of beta-COP-We first determined whether beta-COP in purified coatomer was phosphorylated. COPI partially purified from rat liver was treated with alkaline phosphatase at various concentrations and times and analyzed by two-dimensional IEF/SDS-PAGE. Because the alkaline phosphatase preparations contained contaminants of similar molecular weight, beta-COP was identified by Western blot. As shown in Fig. 3, alkaline phosphatase treatment shifted the beta-COP spots from their characteristic positions (between pH 5.5 and 6.0) to the basic end of the gel (pH 9), suggesting that dephosphorylation had occurred. A similar shift was also observed for the corresponding silver-stained spots, although not for spots corresponding to -COP or -COP; the -COP spot was obscured by the alkaline phosphatase itself (not shown). The shift of immunoreactive beta-COP was also dependent on Mg and was inhibited by EDTA, indicating that the shift was the result of alkaline phosphatase activity and not proteolysis.


Figure 3: beta-COP pI is shifted by alkaline phosphatase treatment. Rat liver COPI (100 µg) was treated with various concentrations of alkaline phosphatase for 1 h and separated on two-dimensional gel as in Fig. 2, except that the first dimension was pH 5-9. The second dimension was blotted onto nitrocellulose and probed with EAGE for beta-COP. Dephosphorylated beta-COP appears at the base end of the gel. Lane 1, no added alkaline phosphatase; lane 2, 3.36 µg/ml alkaline phosphatase added; lane 3, 33.6 µg/ml alkaline phosphatase added.



The magnitude of the pI shift was greater than that expected for removal of a single phosphate group. In addition, the total amount of immunoreactive (and silver-stained) beta-COP also appeared to decrease, suggesting that the dephosphorylation reduced the ability of the protein to enter the IEF gel, perhaps by altering its solubility characteristics. For these reasons, we sought to demonstrate directly that beta-COP was phosphorylated.

CHO cells were labeled for 12 h with [P]orthophosphate and lysed in 1% Triton X-100 or homogenized in TEAS. Labeled lysates were immunoprecipitated with the anti-beta-COP peptide antibody E-1(23) , separated by 6.5 and 10% SDS-PAGE, and blotted onto polyvinylidene difluoride filters. The filters were first autoradiographed (Fig. 4, left two lanes of each panel). A single labeled band of 110 kDa was observed in the immunoprecipitate but not in a control precipitation using only protein A-Sepharose beads. To confirm that the labeled protein was indeed beta-COP, the filter was probed with a second mouse monoclonal antibody against beta-COP (M3A5)(32) . The same filter was stripped and reprobed with a rabbit antibody directed against beta`-COP. The P-labeled band comigrated with beta-COP but not with beta`-COP. -COP was also detected in the immunoprecipitate (Fig. 4, right panel, right lane), but no corresponding P-labeled band was detected in this position. Thus, neither the co-precipitated beta` nor -COP were phosphorylated in CHO cells. Western blots using antibodies directed against alpha-, -, -, and -COPs were negative (not shown), suggesting that the conditions used for precipitation led to the partial dissociation of the coatomer into smaller complexes, one of which contained beta-, beta`-, and -COP. Elimination of detergent from the immunoprecipitation did not enhance recovery of -COP, suggesting that dissociation of the COP complex may have been due to salt or freeze/thawing of COP samples prior to immunoprecipitation. Interestingly, partial coatomer complexes are also recovered under high salt conditions, although in this case -COP associated with alpha and beta` but not beta-COP(25) .


Figure 4: Immunoprecipitation of phosphorylated beta-COP from CHO cell lysates. Lysates from CHO cells, metabolically labeled with P, were immunoprecipitated with the anti-beta-COP antibody E1 (+) or with protein A-Sepharose alone(-). The precipitate was run on a 6.5% (left panel) or 10% (right panel) SDS-PAGE and analyzed by autoradiography (32P) and then by sequential Western blots (beta, beta`, and lanes). The Western blots were first visualized with the monoclonal antibody M3A5 directed against beta-COP. The same blots were stripped and reprobed with rabbit antibodies directed against beta` or -COPs as indicated in each lane. Positions of detected COPs are indicated. Positions of molecular mass standards are indicated.



Phosphorylation of Other COP Components

Because immunoprecipitations using antibodies to beta-COP were not effective in capturing all COPI components, we next purified coatomer from P-labeled CHO cells in order to determine if any other coatomer subunits were phosphorylated. The purification was performed in the presence of unlabeled rat liver cytosol as carrier, and the resulting coatomer preparation was analyzed by SDS-PAGE and transferred to Immobilon-P filters. As shown in Fig. 5A, two major P-labeled bands were detected (right lane). The first had an apparent molecular mass of 110 kDa and thus was likely to represent beta-COP; this was confirmed by Western blot (left lane). The second protein was 61 kDa and reacted with the anti--COP antibody. Quantification of PhosphorImager data revealed that the ratio of beta- to -COP associated P was 3-4:1. Western blots of the co-purified material confirmed that alpha-, beta-, beta`-, -, -, -, and -COPs were all present in this preparation, again suggesting that none of these proteins was phosphorylated.


Figure 5: Isolated coatomer contains phosphorylated beta-COP and -COP. A, COPI from CHO cells metabolically labeled with P was copurified with unlabeled rat liver COPI. The resulting purified COPI was separated by SDS-PAGE and autoradiographed (32P). A parallel lane containing whole CHO cytosol was analyzed by Western blot to locate beta-COP (upper left panel) and -COP (lower left panel). Positions of beta- and -COPs are indicated, respectively, by upper and lower arrows at right. Bands appearing in the lower left panel below 50 kDa were degradation products developed in whole cytosol only. Hash marks indicate positions of molecular mass standards (from top to bottom: 205, 116.5, 80, and 49.5 kDa). B, CHO cells labeled with [S]Met/Cys treated as above. Purified coatomer was separated on 8% (upper panels) and 12% (lower panels) SDS-PAGE, transferred to Immobilon-P, stained, and autoradiographed. All COPI subunits (labeled at right) were visible by both Coomassie Blue stain (left) and autoradiography (right).



The fact that coatomer complexes at least partly dissociated upon immunoprecipitation made it necessary to provide additional evidence that none of the subunits had been partially lost during purification: conditions similar to those used for immunoprecipitation were used to purify coatomer from P-labeled CHO cells. To confirm that each of the COPI subunits were recovered from CHO lysates, coatomer was purified from [S]methionine/cysteine-labeled cells in the presence of excess unlabeled purified rat liver coatomer. As shown in Fig. 5B, all of the subunits were detected as S-labeled bands comigrating with each of the seven unlabeled rat liver COP components. The presence of each COP component was also confirmed by Western blot analysis using subunit-specific antibodies (not shown). The copurified material thus contained all COP components. As phosphorylation was detected only on the beta- and -COPs, it is likely that only these components were phosphorylated, although beta-COP appeared to be phosphorylated more heavily.

Finally, to determine whether beta-COP phosphorylation occurred on serine, threonine, or tyrosine residues, beta-COP was immunoprecipitated from lysates of P-labeled HeLa cells and subjected to phosphoamino acid analysis. As shown in Fig. 6, only phosphoserine was detected, indicating that at least beta-COP was phosphorylated only on serines.


Figure 6: Phosphorylation of beta-COP is on serine. Partially purified COP I from HeLa cells metabolically labeled with P was separated by SDS-PAGE. The labeled band with an M(r) of 110,000 was eluted and subjected to mild acid hydrolysis. The products were separated by thin layer electrophoresis first at pH 1.9 and then in a second dimension at pH 3.5. Left panel, authentic unlabeled phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards were visualized with ninhydrin. Right panel, autoradiograph of labeled hydrolyzed sample.




DISCUSSION

We have found that several of the COPI subunits of the cytosolic coatomer complex exhibit significant structural heterogeneity. This heterogeneity is most obvious for beta-COP and -COP; beta-COP exists as three to five distinct species, while -COP exists as up to three species with differing pI but identical molecular weights. Because both beta and -COP are phosphorylated, the simplest conclusion would be that differential phosphorylation of beta- and -COP is responsible for at least part of the observed heterogeneity. Despite much effort, we have thus far been unable to detect novel cDNAs or transcripts encoding distinct proteins closely related to beta-COP. Using antibody screening of expression libraries, low stringency hybridization, and polymerase chain reaction, only ``authentic'' beta-COP cDNAs have been recovered. Similarly, closely related proteins have yet to emerge in the sequence data base. Thus it seems less likely that the existence of multiple beta-COP species reflects the existence of different gene products each sufficiently related so as to cross-react with a common set of anti-peptide antibodies.

Given the magnitude of the shift in pI upon dephosphorylation of beta-COP with alkaline phosphatase, it was impossible to determine the actual number of phosphates that exist per beta-COP or to establish that the phosphorylation is completely responsible for the heterogeneity observed after IEF/SDS-PAGE. Similar considerations apply to -COP. However, taking into account the presence of five beta-COP and three -COP spots and noting that beta- and -COP all exist in the coatomer in roughly equimolar amounts (by Coomassie Blue staining), the 3-4:1 ratio of beta-COP:-COP P-labeling suggests that there may be 1-5 phosphorylations/beta-COP molecule and 0-2 phosphorylations/-COP molecule. The different levels of phosphorylation are unlikely to be the result of differential turnover rates, as the ratio was preserved whether labeling was for 4 or 12 h.

What role then may phosphorylation of COPI components play? Phosphorylation could regulate the partitioning of COPI between cytosolic and membrane-bound states. This possibility has at least one precedent in the binding of the small GTPase Rab4 to endosomes. In this case, phosphorylation of rab4 by p34 kinase leads to its cytosolic accumulation in mitotic cells(33) .

Alternatively, COPI phosphorylation may control the specificity of organelle binding. It is becoming increasingly apparent that one or more COPI components bind to and/or function at multiple intracellular sites in both the secretory and endocytic pathways(4, 11, 12, 20) . Recruitment of COPI subunits to these various sites of action may, at least in part, be controlled by the phosphorylation state of beta-COP or -COP. Consistent with this view, beta-COP along with several other COPI subunits is recruited to endosomes but not - or -COP(24) . - and -COP may either be dissociated from a putative beta-, beta`-, -COP complex before or after binding; or they may be replaced in the holocomplex by novel non-cross-reactive isoforms. Conceivably, phosphorylation may, under certain conditions, help generate subcomplexes of coatomer. This possibility is consistent with our finding that antibodies to beta-COP co-precipitated with beta`- and -COP but not with alpha-, -, -, or -COP. In this case, antibody binding itself may cause a partial dissociation of the complex. Binding to the cognate COPI receptor on endosomes may have a similar effect.

Another intriguing possibility is suggested by a variety of observations indicating that the binding of coatomer, as well as membrane protein sorting and vesicle budding, are events that are regulated by molecules associated with signal transduction. Evidence is accumulating that G protein activators can enhance and/or alter specific steps in intracellular transport. Among these examples is the increase in ER to Golgi transport (34) and transport from the TGN or endosomes to the apical surface in polarized Madin-Darby canine kidney cells (35, 36) (^3)due to treatment with AlF(4), and cholera toxin. It is therefore interesting to note that AlF4 and mastoparan enhance COPI binding at least to ER/Golgi membranes, while conversely, the addition of G subunits inhibits binding(7) . G protein activators do not, however, enhance COPI binding to endosomes(24) . Nevertheless, COPI does interact with the inositol phosphates inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate(37) . Finally, binding of ADP-ribosylation factor to membranes activates phospholipase D activity, an event that is likely to directly or indirectly affect COPI recruitment(16) .

Phosphorylation of COPI subunits is a link between G protein enhancement of coatomer binding and transport. Conceivably, this link would be provided if COPI subunits were substrates for protein kinase C. Phorbol esters (direct activators of protein kinase C) activate transport between the ER and the Golgi (a COPI-mediated process)(34) , and binding of both ADP-ribosylation factor and beta-COP is regulated by protein kinase C activity in rat basophilic leukemia cells(38) . Although we have not identified the specific residue(s) phosphorylated in beta-COP, there are three consensus protein kinase C serine phosphorylation sites in the rat beta-COP sequence (KSVK, KKES and KKTS). Other sites for protein kinase C or other kinases whose activities are regulated by signal transduction events may also exist. Differential beta-COP phosphorylation in response to different signals may regulate the recruitment of COPI to specific intracellular sites in response to shifts in demand to vesicular traffic between compartments. This idea is also appealing in light of the behavior of protein kinase C itself in response to activation. Inactive protein kinase C is cytosolic, whereas activated protein kinase C becomes membrane-bound(39, 40) . Indeed, some activated isoforms of protein kinase C have been found to bind directly to Golgi membranes(41) . Conceivably, localization of activated protein kinase C may help define sites of vesicle budding by phosphorylating and locally recruiting coatomer complexes. These considerations each create a number of highly testable possibilities.


FOOTNOTES

*
This work was supported in part by research grants from the National Institutes of Health (to I. M.) (GM29765), from the Fonds National Suisse (to T. E. K.) (31-33546.92), and from the Human Frontier Science Foundation (to I. M. and T. E. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health National Research Service Award Fellowship F32GM16339.

Supported by a Travelling Fellowship from the Wellcome Trust.

**
To whom correspondence should be addressed: Dept. of Cell Biology, Yale University School of Medicine, 333 Cedar St., P.O. Box 208002, New Haven, CT 06520-8002. Tel.: 203-785-5058; Fax: 203-785-7226; Ira.Mellman{at}yale.edu.

(^1)
The abbreviations used are: TGN, trans-Golgi network; COP, coat protomer complex; COPI and COPII, coat protomer complexes I and II, respectively; ER, endoplasmic reticulum; S100, 100,000 times g supernatant; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing; CHO, Chinese hamster ovary; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MEM, minimal essential medium.

(^2)
M. Lu, M. Gomez, T. E. Kreis, and I. Mellman, unpublished data.

(^3)
M. Kim and I. Mellman, unpublished data.


REFERENCES

  1. Robinson, M. S. (1994) Curr. Opin. Cell Biol. 6, 538-544 [Medline] [Order article via Infotrieve]
  2. Matter, K., and Mellman, I. (1994) Curr. Opin. Cell Biol. 6, 545-554 [Medline] [Order article via Infotrieve]
  3. Rothman, J. E., and Orci, L. (1992) Nature 355, 409-413 [CrossRef][Medline] [Order article via Infotrieve]
  4. Peter, F., Plutner, H., Zhu, H., Kreis, T. E., and Balch, W. E. (1993) J. Cell Biol. 122, 1155-1167 [Abstract]
  5. Kreis, T. E., Lowe, M., and Pepperkok, R. (1996) Annu. Rev. Cell Dev. Biol ., in press
  6. Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M. F., Ravazzola, M., Amherdt, M., and Schekman, R. (1994) Cell 77, 895-907 [Medline] [Order article via Infotrieve]
  7. Robinson, M., and Kreis, T. E. (1992) Cell 69, 129-138 [Medline] [Order article via Infotrieve]
  8. Hosobuchi, M., Kreis, T., and Schekman, R. (1992) Nature 360, 603-605 [CrossRef][Medline] [Order article via Infotrieve]
  9. Aridor, M., Bannykh, S. I., Rowe, T., and Balch, W. E. (1995) J. Cell Biol. 131, 875-893 [Abstract]
  10. Pepperkok, R., Scheel, J., Horstmann, H. P., Hauri, H., Griffiths, G., and Kreis, T. E. (1993) Cell 74, 71-82 [Medline] [Order article via Infotrieve]
  11. Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213-1224 [Abstract]
  12. Orci, L., Palmer, D. J., Ravazzola, M., Perrelet, A., Amherdt, M., and Rothman, J. E. (1993) Nature 362, 648-652 [CrossRef][Medline] [Order article via Infotrieve]
  13. Waters, M. G., Serafini, T., and Rothman, J. E. (1991) Nature 349, 248-251 [CrossRef][Medline] [Order article via Infotrieve]
  14. Stenbeck, G., Harter, C., Brecht, A., Herrmann, D., Lottspeich, F., Orci, L., and Wieland, F. T. (1993) EMBO J. 12, 2841-2845 [Abstract]
  15. Serafini, T., Orci, L., Amherdt, M., Brunner, M., Kahn, R. A., and Rothman, J. E. (1991) Cell 67, 239-253 [Medline] [Order article via Infotrieve]
  16. Moss, J., and Vaughan, M. (1995) J. Biol. Chem. 270, 12327-12330 [Free Full Text]
  17. Donaldson, J. G., Cassel, D., Kahn, R. A., and Klausner, R. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6408-6412 [Abstract]
  18. Donaldson, J. G., Finazzi, D., and Klausner, R. D. (1992) Nature 360, 350-351 [CrossRef][Medline] [Order article via Infotrieve]
  19. Orci, L., Palmer, D. J., Amherdt, M., and Rothman, J. E. (1993) Nature 364, 732-734 [CrossRef][Medline] [Order article via Infotrieve]
  20. Cosson, P., and Letourner, F. (1994) Science 263, 1629-1631 [Medline] [Order article via Infotrieve]
  21. Griffiths, G., Pepperkok, R., Krijnse, L. J., and Kreis, T. E. (1996) J. Cell Sci. , in press
  22. Oprins, A., Duden, R., Kreis, T. E., Geuze, H. J., and Slot, J. W. (1993) J. Cell Biol. 121, 49-59 [Abstract]
  23. Duden, R., Griffiths, G., Frank, R., Argos, P., and Kreis, T. E. (1991) Cell 64, 649-665 [Medline] [Order article via Infotrieve]
  24. Whitney, J. A., Gomez, M., Sheff, D. R., Kreis, T. E., and Mellman, I. (1995) Cell 83, 703-713 [Medline] [Order article via Infotrieve]
  25. Lowe, M., and Kreis, T. E. (1995) J. Biol. Chem. 270, 31364-31371 [Abstract/Free Full Text]
  26. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  27. Pappin, D. J. C., Hojrup, P., and Bleasby, A. J. (1993) Curr. Biol. 3, 327-332
  28. Waters, M. G., Beckers, C. J., and Rothman, J. E. (1992) Methods Enzymol. 219, 331-337 [Medline] [Order article via Infotrieve]
  29. Celis, J. E., Rasmussen, H. H., Olsen, E., Leffers, H., Honore, B., Dejgaard, K., Gromov, P., Hoffmann, H. J., Nielsen, M., Vassilev, A., Vintermyr, O., Hao, J., Cells, A., Basse, B., Lauridsen, J. E., Ratz, G. P., Andersen, A. H., Walbum, E., Kjaergaard, I., Puype, M., Van Damme, J., and Vandekerckhove, J. S. (1993) Electrophoresis 14, 1091-1198 [Medline] [Order article via Infotrieve]
  30. Hara-Kuge, S., Kuge, O., Orci, L., Amherdt, M., Ravazzola, M., Wieland, F. T., and Rothman, J. E. (1994) J. Cell Biol. 124, 883-892 [Abstract]
  31. Duden, R., Hosobuchi, M., Hamamoto, S., Winey, M., Byers, B., and Schekman, R. (1994) J. Biol. Chem. 269, 24486-24495 [Abstract/Free Full Text]
  32. Allan, V. J., and Kreis, T. E. (1986) J. Cell Biol. 103, 2229-2239 [Abstract]
  33. Van der Sluijs, P., Hull, M., Male, P., Goud, B., and Mellman, I. (1992) EMBO J. 11, 4379-4389 [Abstract]
  34. Fabbri, M., Bannykh, S., and Balch, W. E. (1994) J. Biol. Chem. 269, 26848-26857 [Abstract/Free Full Text]
  35. Cardone, M. H., Smith, B. L., Song, W., and Mochly-Rosen, D. (1994) J. Cell Biol. 124, 717-727 [Abstract]
  36. Pimplikar, S. W., Ikonen, E., and Simons, K. (1994) J. Cell Biol. 125, 1025-1035 [Abstract]
  37. Fleischer, B., Xie, J., Mayrleitner, M., Shears, S. B., Palmer, D. J., and Fleischer, S. (1994) J. Biol. Chem. 269, 17826-17832 [Abstract/Free Full Text]
  38. De Matteis, M. A., Santini, G., Kahn, R. A., Di Tullio, G., and Luini, A. (1993) Nature 364, 818-821 [CrossRef][Medline] [Order article via Infotrieve]
  39. Mochly-Rosen, D., Khaner, H., and Lopez, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3997-4000 [Abstract]
  40. Mochly-Rosen, D., Khaner, H., Lopez, J., and Smith, B. L. (1991) J. Biol. Chem. 266, 14866-14868 [Abstract/Free Full Text]
  41. Lehel, C., Olah, Z., Jakab, G., and Anderson, W. B. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1406-1410 [Abstract]

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