Oligomeric State and Stoichiometry of p24 Proteins in the Early Secretory Pathway
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m206989200
ISSN1083-351X
AutoresNicole Jenne, Karolin Frey, Britta Brügger, Felix Wieland,
Tópico(s)Pancreatic function and diabetes
ResumoThe p24 proteins belong to a highly conserved family of membrane proteins that cycle in the early secretory pathway. They bind to the coat proteins of COPI and COPII vesicles, and are proposed to be involved in vesicle biogenesis, cargo uptake, and quality control, but their precise function is still under debate. Most p24 proteins form hetero-oligomers, essential for their correct localization and stability. Functional insights regarding the mechanisms of their steady state localization and the role of interaction with coat proteins has been hampered by a lack of data on their concentration and state of oligomerization within the endoplasmic reticulum, the intermediate compartment, and Golgi complex. We have determined for all mammalian p24 family members the size of the oligomers formed and their stoichiometric relation in each of these individual organelles. In contrast to earlier reports, we show that individual members exist as dimers and monomers and that the ratio between these two forms depends on both the organelle investigated and the p24 protein. We find unequal quantities, with p23 and p27 building up concentration gradients, ruling out a simple 1:1 stoichiometry. In addition, we show differential cycling of individual p24 members. These data point to a complex and dynamic system of altering dimerizations of the family members. The p24 proteins belong to a highly conserved family of membrane proteins that cycle in the early secretory pathway. They bind to the coat proteins of COPI and COPII vesicles, and are proposed to be involved in vesicle biogenesis, cargo uptake, and quality control, but their precise function is still under debate. Most p24 proteins form hetero-oligomers, essential for their correct localization and stability. Functional insights regarding the mechanisms of their steady state localization and the role of interaction with coat proteins has been hampered by a lack of data on their concentration and state of oligomerization within the endoplasmic reticulum, the intermediate compartment, and Golgi complex. We have determined for all mammalian p24 family members the size of the oligomers formed and their stoichiometric relation in each of these individual organelles. In contrast to earlier reports, we show that individual members exist as dimers and monomers and that the ratio between these two forms depends on both the organelle investigated and the p24 protein. We find unequal quantities, with p23 and p27 building up concentration gradients, ruling out a simple 1:1 stoichiometry. In addition, we show differential cycling of individual p24 members. These data point to a complex and dynamic system of altering dimerizations of the family members. The p24 family of type I transmembrane proteins consists of six members in mammalian cells which, referring to sequence homologies, can be divided in four subfamilies (1Dominguez M. Dejgaard K. Füllekrug J. Dahan S. Fazel A. Paccaud J.P. Thomas D.Y. Bergeron J.J. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (292) Google Scholar, 2Emery G. Grünberg J. Rojo M. Protoplasma. 1999; 207: 24-30Crossref Scopus (19) Google Scholar). Mammalian cells contain three members of the p26 subfamily, namely p26, p27, and tp24 and only one member each of the p23, p24, and p25 subfamilies. According to studies in yeast, p24 proteins may function as cargo receptors or adaptors since in knock-out mutants distinct cargo proteins show delayed transport kinetics (3Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 4Marzioch M. Henthorn D.C. Herrmann J.M. Wilson R. Thomas D.Y. Bergeron J.J. Solari R.C. Rowley A. Mol. Biol. Cell. 1999; 10: 1923-1938Crossref PubMed Scopus (154) Google Scholar, 5Schimmöller F. Singer-Krüger B. Schröder S. Krüger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (284) Google Scholar). Additional experiments in yeast andCaenorhabditis elegans implicate also an involvement of p24 proteins in ER 1The abbreviations used for: ER, endoplasmic reticulum; PDI, protein disulfide isomerase; POD, peroxidase; DSG, disuccinimidyl glutarate; IC, intermediate compartment; GST, glutathione S-transferase; POE, n-octyl-poly-oxyethylene; SNARE, soluble NSF attachment protein receptors. quality control (4Marzioch M. Henthorn D.C. Herrmann J.M. Wilson R. Thomas D.Y. Bergeron J.J. Solari R.C. Rowley A. Mol. Biol. Cell. 1999; 10: 1923-1938Crossref PubMed Scopus (154) Google Scholar, 6Elrod-Erickson M.J. Kaiser C.A. Mol. Biol. Cell. 1996; 7: 1043-1058Crossref PubMed Scopus (143) Google Scholar, 7Wen C. Greenwald I. J. Cell Biol. 1999; 145: 1165-1175Crossref PubMed Scopus (78) Google Scholar). For mammalian p23, it has been proposed that it serves as COPI receptor because it can bind to coat components (1Dominguez M. Dejgaard K. Füllekrug J. Dahan S. Fazel A. Paccaud J.P. Thomas D.Y. Bergeron J.J. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (292) Google Scholar, 8Sohn K. Orci L. Ravazzola M. Amherdt M. Bremser M. Lottspeich F. Fiedler K. Helms J.B. Wieland F.T. J. Cell Biol. 1996; 135: 1239-1248Crossref PubMed Scopus (181) Google Scholar) and belongs to the minimal machinery needed to bud COPI vesicles from liposomes (9Bremser M. Nickel W. Schweikert M. Ravazzola M. Amherdt M. Hughes C.A. Söllner T.H. Rothman J.E. Wieland F.T. Cell. 1999; 96: 495-506Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). In addition, a direct interaction of p24 family members has been shown with proteins involved in vesicle budding and cargo sorting, such as ARF-1 (ADP-ribosylation factor) (10Gommel D.U. Memon A.R. Heiss A. Reinhard C. Helms J.B. Nickel W. Wieland F.T. EMBO J. 2001; 20: 6751-6760Crossref PubMed Scopus (83) Google Scholar, 11Majoul I. Straub M. Hell S.W. Duden R. Söling H.-D. Dev. Cell. 2001; 1: 139-153Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), ARF-GAP (GTPase-activating protein) (10Gommel D.U. Memon A.R. Heiss A. Reinhard C. Helms J.B. Nickel W. Wieland F.T. EMBO J. 2001; 20: 6751-6760Crossref PubMed Scopus (83) Google Scholar) and Sar-1p (12Belden W.J. Barlowe C. J. Biol. Chem. 2001; 276: 43040-43048Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). p24 proteins are also effectively enriched in COPI (8Sohn K. Orci L. Ravazzola M. Amherdt M. Bremser M. Lottspeich F. Fiedler K. Helms J.B. Wieland F.T. J. Cell Biol. 1996; 135: 1239-1248Crossref PubMed Scopus (181) Google Scholar, 13Gommel D. Orci L. Emig E.M. Hannah M.J. Ravazzola M. Nickel W. Helms J.B. Wieland F.T. Sohn K. FEBS Lett. 1999; 447: 179-185Crossref PubMed Scopus (66) Google Scholar) and COPII vesicles (3Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 5Schimmöller F. Singer-Krüger B. Schröder S. Krüger U. Barlowe C. Riezman H. EMBO J. 1995; 14: 1329-1339Crossref PubMed Scopus (284) Google Scholar), and some of them bear signals in their cytoplasmic tails, which when fused to a cargo protein direct them either anterogradely or retrogradely (14Nickel W. Sohn K. Bünning C. Wieland F.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11393-11398Crossref PubMed Scopus (66) Google Scholar). Therefore, p24 proteins have been implicated to be involved in transport processes of the early secretory pathway, but their precise function is still unclear. All p24 proteins are found in membranes of the early secretory pathway (1Dominguez M. Dejgaard K. Füllekrug J. Dahan S. Fazel A. Paccaud J.P. Thomas D.Y. Bergeron J.J. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (292) Google Scholar, 8Sohn K. Orci L. Ravazzola M. Amherdt M. Bremser M. Lottspeich F. Fiedler K. Helms J.B. Wieland F.T. J. Cell Biol. 1996; 135: 1239-1248Crossref PubMed Scopus (181) Google Scholar, 13Gommel D. Orci L. Emig E.M. Hannah M.J. Ravazzola M. Nickel W. Helms J.B. Wieland F.T. Sohn K. FEBS Lett. 1999; 447: 179-185Crossref PubMed Scopus (66) Google Scholar, 15Emery G. Rojo M. Grünberg J. J. Cell Sci. 2000; 113: 2507-2516Crossref PubMed Google Scholar, 16Füllekrug J. Suganuma T. Tang B.L. Hong W. Storrie B. Nilsson T. Mol. Biol. Cell. 1999; 10: 1939-1955Crossref PubMed Scopus (117) Google Scholar), and there is evidence that they cycle constitutively between these membranes (13Gommel D. Orci L. Emig E.M. Hannah M.J. Ravazzola M. Nickel W. Helms J.B. Wieland F.T. Sohn K. FEBS Lett. 1999; 447: 179-185Crossref PubMed Scopus (66) Google Scholar, 14Nickel W. Sohn K. Bünning C. Wieland F.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11393-11398Crossref PubMed Scopus (66) Google Scholar, 16Füllekrug J. Suganuma T. Tang B.L. Hong W. Storrie B. Nilsson T. Mol. Biol. Cell. 1999; 10: 1939-1955Crossref PubMed Scopus (117) Google Scholar, 17Blum R. Pfeiffer F. Feick P. Nastainczyk W. Kohler B. Schäfer K.H. Schulz I. J. Cell Sci. 1999; 112: 537-548Crossref PubMed Google Scholar). Another outstanding property of p24 proteins is the formation of hetero-oligomers. As was shown in several immunoprecipitation studies, they interact with each other to a certain extent (3Belden W.J. Barlowe C. J. Biol. Chem. 1996; 271: 26939-26946Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 4Marzioch M. Henthorn D.C. Herrmann J.M. Wilson R. Thomas D.Y. Bergeron J.J. Solari R.C. Rowley A. Mol. Biol. Cell. 1999; 10: 1923-1938Crossref PubMed Scopus (154) Google Scholar, 13Gommel D. Orci L. Emig E.M. Hannah M.J. Ravazzola M. Nickel W. Helms J.B. Wieland F.T. Sohn K. FEBS Lett. 1999; 447: 179-185Crossref PubMed Scopus (66) Google Scholar, 16Füllekrug J. Suganuma T. Tang B.L. Hong W. Storrie B. Nilsson T. Mol. Biol. Cell. 1999; 10: 1939-1955Crossref PubMed Scopus (117) Google Scholar). Moreover, overexpression of a single p24 protein leads to a mislocalization of all p24 proteins in ER-derived structures (13Gommel D. Orci L. Emig E.M. Hannah M.J. Ravazzola M. Nickel W. Helms J.B. Wieland F.T. Sohn K. FEBS Lett. 1999; 447: 179-185Crossref PubMed Scopus (66) Google Scholar, 15Emery G. Rojo M. Grünberg J. J. Cell Sci. 2000; 113: 2507-2516Crossref PubMed Google Scholar,16Füllekrug J. Suganuma T. Tang B.L. Hong W. Storrie B. Nilsson T. Mol. Biol. Cell. 1999; 10: 1939-1955Crossref PubMed Scopus (117) Google Scholar), and only the simultaneous overexpression of p24 proteins of all subfamilies results in a convincing perinuclear Golgi localization (15Emery G. Rojo M. Grünberg J. J. Cell Sci. 2000; 113: 2507-2516Crossref PubMed Google Scholar,16Füllekrug J. Suganuma T. Tang B.L. Hong W. Storrie B. Nilsson T. Mol. Biol. Cell. 1999; 10: 1939-1955Crossref PubMed Scopus (117) Google Scholar). Furthermore, in yeast cells lacking one p24 protein (4Marzioch M. Henthorn D.C. Herrmann J.M. Wilson R. Thomas D.Y. Bergeron J.J. Solari R.C. Rowley A. Mol. Biol. Cell. 1999; 10: 1923-1938Crossref PubMed Scopus (154) Google Scholar) and in cells from mice lacking one allele of p23 (18Denzel A. Otto F. Girod A. Pepperkok R. Watson R. Rosewell I. Bergeron J.J. Solari R.C. Owen M.J. Curr. Biol. 2000; 10: 55-58Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) other family members are degraded. Thus, there is a strict dependence among p24 proteins in terms of stability, transport, and/or localization. Consequently, the formation of hetero-oligomers seems to be a prerequisite for their correct function. To set a basis for understanding the molecular functionality of p24 proteins, we have undertaken a detailed study of their oligomeric behavior, their stoichiometric relation, and cycling in the membranes of the secretory pathway. We demonstrate that p24 proteins, in contrast to earlier results (1Dominguez M. Dejgaard K. Füllekrug J. Dahan S. Fazel A. Paccaud J.P. Thomas D.Y. Bergeron J.J. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (292) Google Scholar, 4Marzioch M. Henthorn D.C. Herrmann J.M. Wilson R. Thomas D.Y. Bergeron J.J. Solari R.C. Rowley A. Mol. Biol. Cell. 1999; 10: 1923-1938Crossref PubMed Scopus (154) Google Scholar), exist either as dimers or monomers with no higher oligomers observed. Moreover, we find different ratios between dimers and monomers depending both on a given p24 protein and on its subcellular localization. p23 and p27 build up concentration gradients, and p24 and p25 are distributed equally in the early secretory pathway, with differential cycling between these compartments. Therefore, we assume highly dynamic and complex interactions of these four p24 proteins. In contrast, p26 and tp24 do not seem to interact with the other four members as they occur exclusively either as monomer or as dimer, respectively, and their concentrations are vastly different from other p24 proteins. Antibodies directed against p23, p24, p25, p26, tp24, and p27 were raised in rabbits, chicken, and guinea pigs with the peptides or recombinant proteins indicated in parentheses coupled to keyhole limpet hemocyanin (Sigma): Henriette (p23 recombinant luminal domain), HAC344 (p23-tail, CLRRFFKAKKLIE), Thelma (p23, KITDSAGHILYSK), #1327chicken (p23, KITDSAGHILYSK), Elfriede (p24 tail, (13Gommel D. Orci L. Emig E.M. Hannah M.J. Ravazzola M. Nickel W. Helms J.B. Wieland F.T. Sohn K. FEBS Lett. 1999; 447: 179-185Crossref PubMed Scopus (66) Google Scholar)), Frieda (p24 tail, CYLKRFFEVRRVV), #1593chicken (p24, CYLKRFFEVRRVV), #2469R1 (p25-tail, CYLKSFFEAKKLV), #2088M2 (p26-tail, CLLKSFFTEKRPISRHVHS), #2087R2 (p27-tail, CLLKSFFSDKRTTTTRVGS), #2501R2 (tp24 tail, CTLKRFFQDKRPVPT). Immunization and affinity purification was performed according to standard protocols (19Harlowe E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Rabbit polyclonal antibodies against cytochrome b5 were a kind gift of N. Borgese (University of Milan, Italy), monoclonal antibodies against ERGIC-53 were from H.-P. Hauri (Biocenter, Basel, Switzerland) (20Schweizer A. Fransen J.A. Bachi T. Ginsel L. Hauri H.P. J. Cell Biol. 1988; 107: 1643-1653Crossref PubMed Scopus (377) Google Scholar), and rabbit polyclonal antibodies against p30 (mitochondrial protein) and PMP69 (peroxisomal membrane protein) were provided by W. Just (Biochemie-Zentrum Heidelberg, University of Heidelberg, Germany). Further antibodies used were: anti-protein disulfide isomerase (PDI, BD Transduction Laboratories), anti-KDEL-receptor (Stressgen Biotechnologies), anti-transferrin receptor (Zymed Laboratories Inc.), anti-rabbit-peroxidase (POD, BioRad Laboratories), anti-mouse-POD (Dianova), anti-guinea pig-POD (Dianova), anti-chicken-POD (Dianova), monoclonal anti-rabbit-POD (Sigma), anti-mouse-Alexa-488 (Molecular Probes), and anti-guinea pig-Alexa-456 (Molecular Probes). HeLa cells (ATTC: CCL-2.2) were grown in Spinner culture according to standard conditions up to 4–6 × 105 cells/ml. Homogenization and centrifugation were performed according to Ref. 16Füllekrug J. Suganuma T. Tang B.L. Hong W. Storrie B. Nilsson T. Mol. Biol. Cell. 1999; 10: 1939-1955Crossref PubMed Scopus (117) Google Scholar. After centrifugation, 18 instead of 9 fractions were taken from the top of the gradient and were diluted 1:1 with buffer and centrifuged for 1 h at 100,000 × g at 4 °C. Membrane pellets were used for further analysis. Galactosyltransferase activity was measured according to Ref. 21Brew K. Shaper J.H. Olsen K.W. Trayer I.P. Hill R.L. J. Biol. Chem. 1975; 250: 1434-1444Abstract Full Text PDF PubMed Google Scholar, and alkaline phosphodiesterase corresponding to Ref. 22Warnock D.E. Roberts C. Lutz M.S. Blackburn W.A. Young Jr., W.W. Baenziger J.U. J. Biol. Chem. 1993; 268: 10145-10153Abstract Full Text PDF PubMed Google Scholar. The content of other marker proteins was determined by Western blot analysis with the appropriate antibodies using the ECL detection system (AmershamBiosciences). The signals were quantified with the QuantityOne® software from BioRad Laboratories, and the relative protein amounts were calculated. Solubilization conditions were tested with 50 μg of rabbit liver Golgi membranes (23Tabas I. Kornfeld S. J. Biol. Chem. 1979; 254: 11655-11663Abstract Full Text PDF PubMed Google Scholar). After pelleting, the membranes were solubilized in 50 μl of the indicated buffers (containing 3% 8-n-octyl-poly-oxyethylene (POE), Alexis Corporation; 2% cholic acid, Sigma; or 4% octylglucoside (n-octyl-β-d-glucopyranoside), Calbiochem), incubated for 30 min on ice and subjected to 100,000 × gcentrifugation for 30 min. Pellets and the trichloroacetic acid-precipitated supernatants were then analyzed by Western blotting with an antibody against p23 (Henriette). Membrane pellets (equivalent to 50 nmol of phospholipid as determined by phosphate measurement) (24Rouser G. Fleischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2880) Google Scholar) from subcellular fractionations were solubilized for 30 min on ice in 4% octylglucoside, 20 mmHepes, pH 6.8, 50 mm NaCl, unless indicated differently. After centrifugation (1 h, 100,000 × g at 4 °C), the supernatant was fractionated on a Superdex 200 column on the SMART system (Amersham Biosciences) using a flow rate of 50 μl/min and 1% octylglucoside, 20 mm Hepes, pH 6.8, 50 mm NaCl as running buffer. All fractions were trichloroacetic acid-precipitated, applied to SDS-PAGE (25Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar), and further analyzed by Western blotting with several p24 protein antibodies (Henriette, Elfriede, #2469R1, #2088M, #2087R2, #2501R2). The column was calibrated with gel filtration standards from BioRad Laboratories (thyroglobin (670 kDa), bovine-γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), vitamin B12 (1.35 kDa)) for calibration with membrane proteins. Golgi membranes from rabbit liver (23Tabas I. Kornfeld S. J. Biol. Chem. 1979; 254: 11655-11663Abstract Full Text PDF PubMed Google Scholar) and from Chinese hamster ovary cells containing a CD8-Myc-construct (CD8-LT) (26Nickel W. Malsam J. Gorgas K. Ravazzola M. Jenne N. Helms J.B. Wieland F.T. J. Cell Sci. 1998; 111: 3081-3090Crossref PubMed Google Scholar), were mixed, pelleted, solubilized, and analyzed by gel filtration as described before. Thereafter, the fractions were subjected to Western blot analysis with antibodies against transferrin receptor (190 kDa), Calnexin (90 kDa), Myc (CD8-LT, 60 kDa) and cytochrome b5 (15 kDa). ER, intermediate compartment (IC), and Golgi membranes according to a phospholipid content of 6.25 nmol were diluted in phosphate-buffered saline and pelleted for 1 h at 100,000 × g at 4 °C. Pellets were resuspended in phosphate-buffered saline and treated either with DSG (final concentration 0.6 mm) in Me2SO or Me2SO alone for 30 min on ice. Tris-HCl, pH7.5, was added to stop the reaction, and the samples were further analyzed by Western blotting using an anti-p23 antibody (Thelma). Immunoprecipitations were carried out according to Ref. 13Gommel D. Orci L. Emig E.M. Hannah M.J. Ravazzola M. Nickel W. Helms J.B. Wieland F.T. Sohn K. FEBS Lett. 1999; 447: 179-185Crossref PubMed Scopus (66) Google Scholar. In contrast to this study, the antibodies were covalently coupled to the protein A-Sepharose (19Harlowe E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Sense and antisense oligonucleotides were synthesized corresponding to the cytoplasmic tail sequences of p24, p25, p26, p27, and tp24 with overhanging restriction sites for EcoRI andBamHI. After annealing of the oligonucleotides they were cloned into the EcoRI and BamHI site of the pGEX-2T vector (Amersham Biosciences). The GST-p24 tail constructs were overexpressed and purified according to manufacturers' specifications. The sequence for the luminal domain of p23 was cloned into theBamHI and HindIII site of the pQE-30 vector (Qiagen), overexpressed, and purified by nickel-nitrilotriacetic acid-agarose. The purity of the overexpressed proteins was tested by Coomassie staining, and protein concentration was determined by the Bradford method (BioRad Laboratories). For immunofluorescence studies, Vero cells (ATTC CCL-81) were cultured on coverslips, incubated for 2 h at 15 °C in carbonate-free Hepes-buffered standard medium and afterward transferred to 37 °C for various time periods. Cells were then fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100, and labeled with p24 protein antibodies. They were affinity purified and anti-p23 (HAC344), -p24 (Elfriede), and -p25 (#2469R1) directly labeled with cy3, anti-p27 (#2087R2) with cy5 according to the manufacturers specifications (Amersham Biosciences). Additional antibodies used were detected with Alexa-488/456-coupled secondary antibodies. For confocal analysis a Leica TCS-SP with the Leica confocal software was used. For Western blot analysis s-HeLa cells were grown in Spinner culture, transferred in 15 °C cold, carbonate-free, Hepes-buffered standard medium, and incubated at 15 °C for 2.5 h. Cells were harvested at various time points, and subcellular fractionation was performed as described before. For each condition the distribution of marker enzymes was tested, and fractions were pooled accordingly. The amount of membrane in each pool was determined by nano-ESI-MS/MS (electrospray ionization-tandem mass spectrometry) (27Brügger B. Sandhoff R. Wegehingel S. Gorgas K. Malsam J. Helms J.B. Lehmann W.D. Nickel W. Wieland F.T. J. Cell Biol. 2000; 151: 507-518Crossref PubMed Scopus (186) Google Scholar), and identical amounts based on phosphatidylcholine content were subjected to Western blot analysis with p24 protein antibodies (Henriette, Elfriede, #2469R1, #2088M2, #2087R2, #2501R2), using the ECL detection system. The signals obtained were quantified with QuantityOne software, and the signals for each compartment were used to calculate relative protein amounts. GST-p24 tail constructs (p24, p25, p26, p27, and tp24) or the luminal domain of p23 were used as p24 standard proteins. Increasing amounts of standard protein (in ng) and membranes (in nmol of phospholipid) were subjected to gel electrophoresis (28Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and Western blotting with antibodies against p24 proteins (Henriette, Frieda, #2469R1, #2088M, #2087R2, #2501R2), and developed with the ECL detection system. The antibodies had been tested before to be specific for each p24 protein and not to cross-react with GST. The signals were quantified with the QuantityOne software, and standard curves with the data of the p24 standards were established. Only values of the membrane samples within these linear curves were taken into account to calculate the concentration of each p24 protein in ng of protein/nmol of phospholipid (see supplemental data Figure I at http://www.jbc.org ). For each protein, at least three independent estimations were made, each time with both the standards and the samples on the same blot. Independent experiments with different p24 protein antibodies in a second quantitative Western blot analysis were performed as an additional control, and comparable results were obtained (supplemental data Figure II). To fractionate ER, IC, and Golgi, HeLa cell membranes were subjected to density gradient centrifugation. As shown in Fig.1, these membranes of the early secretory pathway are well separated. Golgi membranes were found in light fractions (fractions 3 + 4), characterized by galactosyltransferase activity and the KDEL receptor (KDEL-R.), and are separated from plasma membrane in fraction 2 as shown by alkaline phosphodiesterase (AP) activity. The second peak of the KDEL receptor in the intermediate density fractions represents the membranes of the IC, as this protein localizes to the cis-Golgi and the IC, respectively (29Tang B.L. Wong S.H. Qi X.L. Low S.H. Hong W. J. Cell Biol. 1993; 120: 325-328Crossref PubMed Scopus (117) Google Scholar, 30Griffiths G. Ericsson M. Krijnse-Locker J. Nilsson T. Goud B. Söling H.D. Tang B.L. Wong S.H. Hong W. J. Cell Biol. 1994; 127: 1557-1574Crossref PubMed Scopus (170) Google Scholar). In contrast, ER membranes move to the high density region of the gradient (fractions 15–17), as shown by the distribution of PDI. The ER pool also contains mitochondria and peroxisomes as indicated by the distribution of p30 and PMP69. However, the amount of membrane according to peroxisomes in ER fractions can be disregarded as these membranes only constitute 1% of total membranes (31Griffiths G. Back R. Marsh M. J. Cell Biol. 1989; 109: 2703-2720Crossref PubMed Scopus (239) Google Scholar). In contrast, mitochondria represent about 32% of total cell membranes (estimated for baby hamster kidney cells, (31Griffiths G. Back R. Marsh M. J. Cell Biol. 1989; 109: 2703-2720Crossref PubMed Scopus (239) Google Scholar)) and thus only part of the membranes in the ER pool represents ER membranes. This was quantified and taken into account. Three different detergents were tested under the same buffer and salt conditions described before for the solubilization of p24 proteins (1Dominguez M. Dejgaard K. Füllekrug J. Dahan S. Fazel A. Paccaud J.P. Thomas D.Y. Bergeron J.J. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (292) Google Scholar, 4Marzioch M. Henthorn D.C. Herrmann J.M. Wilson R. Thomas D.Y. Bergeron J.J. Solari R.C. Rowley A. Mol. Biol. Cell. 1999; 10: 1923-1938Crossref PubMed Scopus (154) Google Scholar, 32Rojo M. Emery G. Marjomaki V. McDowall A.W. Parton R.G. Grünberg J. J. Cell Sci. 2000; 113: 1043-1057Crossref PubMed Google Scholar). As shown in Fig.2, only octylglucoside and 8POE were able to solubilize p23 almost quantitatively. Cholic acid, used previously to determine an oligomeric size of mammalian p24 proteins of 35S (1Dominguez M. Dejgaard K. Füllekrug J. Dahan S. Fazel A. Paccaud J.P. Thomas D.Y. Bergeron J.J. Nilsson T. J. Cell Biol. 1998; 140: 751-765Crossref PubMed Scopus (292) Google Scholar) in our hands emerged to be unsuited for solubilization. Since an extensive study of p24 proteins in yeast was performed with octylglucoside, and this detergent solubilizes p23 nearly quantitatively (Fig. 2) (4Marzioch M. Henthorn D.C. Herrmann J.M. Wilson R. Thomas D.Y. Bergeron J.J. Solari R.C. Rowley A. Mol. Biol. Cell. 1999; 10: 1923-1938Crossref PubMed Scopus (154) Google Scholar), it was used for solubilization in further experiments. Equal amounts of ER, IC, and Golgi with regard to their phospholipid content were solubilized in 4% octylglucoside, and the p24 oligomers were separated by gel filtration. The fractions were subsequently analyzed by Western blotting with antibodies against all p24 proteins. As shown in Fig. 3, p24 proteins elute at two defined volumes with peaks in fractions 18 and 21. To determine the size of the p24 oligomers in these two peaks, we calibrated the gel filtration column with membrane proteins rather than soluble proteins, which, like p24 proteins, need a detergent micelle to stay in solution (Fig. 4 A). With reference to this calibration, p24 proteins appear as dimers and monomers in the ER, IC, and Golgi pools. To assess the oligomeric state of p23 independently, i.e. without the use of detergent, (i) soluble recombinant luminal domain of p23 was analyzed by gel filtration in the absence of detergent and (ii) ER, IC, and Golgi membranes were treated with a cross-linker (DSG) and afterward analyzed by Western blotting with an anti-p23 antibody (supplemental data Figs. III and IV). In both detergent-independent experiments a dimer is the highest oligomer observed. This clearly shows that the dimer observed in the gel filtration analysis is physiological and is not a result of the disruption of higher oligomers by the use of detergent.Figure 3Gel filtration analysis of p24 proteins. A, 50 nmol of ER, IC, and Golgi membrane according to phospholipid content were solubilized in 4% octylglucoside and separated on a Superdex 200 column. All fractions were trichloroacetic acid-precipitated and applied to Western blot analysis with p24 antibodies. B, D and M illustrate ratios of dimer to monomer. Bold letters indicate the prevailing form.View Large Image Figure ViewerDownload (PPT)Figure 4Calibration of the gel filtration column. The gel filtration column was either calibrated using membrane proteins (A) or soluble proteins (B), as indicated under “Experimental Procedures.” Note that the presence of detergent leads to an overestimation of the size of membrane proteins.View Large Image Figure ViewerDownload (PPT) In addition, we observed a different ratio between dimer and monomer, depending on the organelle investigated. As depicted in Fig.3 B, p23 is predominantly found as a dimer in the ER, but in the IC and the Golgi there are about equal amounts of dimer and monomer. In contrast, p24 shows a similar ratio between monomer and dimer in all membrane fractions. p25 and p27 exist predominantly as monomers, with p25 showing the highest amount of dimer in the ER and p27 in the Golgi. However, p26 and tp24, which coimmunoprecipitate to a significantly lower extent with other p24 members (16Füllekrug J. Suganuma T. Tang B.L. Hong W. Storrie B. Nilsson T. Mol. Biol. Cell. 1999; 10: 1939-1955Crossref PubMed Scopus (117) Google Scholar), are present only as either monomers or dimers, respectively. As shown in Fig. 5 an anti-p24 antibody coimmunoprecipitates p23 and p25, and an anti-p27 antibody p23 and p24 from all compartments of the early secretory pathway. Therefore, a part of p24 proteins always exists as heterodimers, but the amount
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