COPII Subunit Interactions in the Assembly of the Vesicle Coat

Abstract

In vitro analysis of COPII vesicle formation in the yeast Saccharomyces cerevisiae has demonstrated the requirement for three cytosolic factors: Sec31p-Sec13p, Sec23p-Sec24p, and Sar1p. Convergent evidence suggests that the peripheral endoplasmic reticulum (ER) membrane protein Sec16p also represents an important component of the vesicle assembly apparatus: SEC16 interacts genetically with all five COPII genes; Sec16p binds to Sec23p and Sec24p, is found on ER-derived transport vesicles, and is required in vitro for the efficient release of ER-derived vesicle cargo. In this report, we demonstrate an important functional interaction between Sec16p and Sec31p. First, we map onto Sec31p binding regions for Sec16p, Sec23p, Sec24p, and Sec13p. Second, we show that a truncation mutant of Sec31p specifically defective for Sec16p binding is unable to complement a sec31Δ mutant and cannot rescue the secretion defect of a temperature-sensitive sec31 mutant at nonpermissive temperatures. We propose that Sec16p organizes the assembly of a coat that is stabilized both by the interaction of Sec31p with Sec23p and Sec24p and by the interaction of these three components with Sec16p. In vitro analysis of COPII vesicle formation in the yeast Saccharomyces cerevisiae has demonstrated the requirement for three cytosolic factors: Sec31p-Sec13p, Sec23p-Sec24p, and Sar1p. Convergent evidence suggests that the peripheral endoplasmic reticulum (ER) membrane protein Sec16p also represents an important component of the vesicle assembly apparatus: SEC16 interacts genetically with all five COPII genes; Sec16p binds to Sec23p and Sec24p, is found on ER-derived transport vesicles, and is required in vitro for the efficient release of ER-derived vesicle cargo. In this report, we demonstrate an important functional interaction between Sec16p and Sec31p. First, we map onto Sec31p binding regions for Sec16p, Sec23p, Sec24p, and Sec13p. Second, we show that a truncation mutant of Sec31p specifically defective for Sec16p binding is unable to complement a sec31Δ mutant and cannot rescue the secretion defect of a temperature-sensitive sec31 mutant at nonpermissive temperatures. We propose that Sec16p organizes the assembly of a coat that is stabilized both by the interaction of Sec31p with Sec23p and Sec24p and by the interaction of these three components with Sec16p. In eukaryotic cells, proteins in the secretory pathway are transported from the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; GST, glutathione S-transferase; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; CPY, carboxypeptidase Y; 5-FOA, 5-fluoroorotic acid. to the Golgi apparatus via membrane-bounded vesicles (1Palade G. Science. 1975; 189: 347-358Crossref PubMed Scopus (2341) Google Scholar) that are formed by the recruitment and assembly of cytosolic coat components upon the ER membrane (1Palade G. Science. 1975; 189: 347-358Crossref PubMed Scopus (2341) Google Scholar, 2Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2007) Google Scholar, 3Schekman R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (818) Google Scholar). In the yeast Saccharomyces cerevisiae, three cytoplasmic factors, collectively termed COPII, have been shown to be required for vesicle formation: the Sec31p-Sec13p protein complex, the Sec23p-Sec24p protein complex, and the small GTP-binding protein Sar1p (4Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1048) Google Scholar, 5Salama N. Yeung R. Schekman R.W. EMBO J. 1993; 12: 4073-4082Crossref PubMed Scopus (176) Google Scholar). The addition of these three factors to urea-washed ER membranes stimulates the formation of coated, fusion-competent vesicles (4Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1048) Google Scholar). Although in vitro analysis has defined soluble components required for vesicle assembly, membrane-associated factors have remained less accessible to biochemical study. Thus, the mechanism of COPII recruitment and assembly onto the membrane is still unknown. However, converging evidence suggests that Sec16p, an essential 240-kDa multidomain protein, may be involved intimately in this process.SEC16 is required for transport vesicle formation (6Espenshade P. Gimeno R.E. Holzmacher E. Teung P. Kaiser C.A. J. Cell Biol. 1995; 131: 311-324Crossref PubMed Scopus (149) Google Scholar, 7Kaiser C. Schekman R. Cell. 1990; 61: 723-733Abstract Full Text PDF PubMed Scopus (545) Google Scholar) and exhibits genetic interactions with all five COPII genes (7Kaiser C. Schekman R. Cell. 1990; 61: 723-733Abstract Full Text PDF PubMed Scopus (545) Google Scholar, 8Gimeno R.E. Espenshade P. Kaiser C.A. J. Cell Biol. 1995; 131: 325-338Crossref PubMed Scopus (66) Google Scholar, 9Nakano A. Muramatsu M. J. Cell Biol. 1989; 109: 2677-2691Crossref PubMed Scopus (338) Google Scholar, 10Salama N.R. Chuang J.S. Schekman R.W. Mol. Biol. Cell. 1997; 8: 205-217Crossref PubMed Scopus (127) Google Scholar). Sec16p is tightly associated with the periphery of the ER and is also found on ER-derived transport vesicles (6Espenshade P. Gimeno R.E. Holzmacher E. Teung P. Kaiser C.A. J. Cell Biol. 1995; 131: 311-324Crossref PubMed Scopus (149) Google Scholar); Sec16p cannot be extracted from membranes by urea (6Espenshade P. Gimeno R.E. Holzmacher E. Teung P. Kaiser C.A. J. Cell Biol. 1995; 131: 311-324Crossref PubMed Scopus (149) Google Scholar). Membranes prepared from sec16 mutant strains exhibit a marked deficit in the release of vesicle cargo molecules (11Campbell J.L. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 837-842Crossref PubMed Scopus (72) Google Scholar). Finally, Sec16p directly binds the COPII subunits Sec23p and Sec24p (6Espenshade P. Gimeno R.E. Holzmacher E. Teung P. Kaiser C.A. J. Cell Biol. 1995; 131: 311-324Crossref PubMed Scopus (149) Google Scholar, 12Gimeno R.E. Espenshade P. Kaiser C.A. Mol. Biol. Cell. 1996; 7: 1815-1823Crossref PubMed Scopus (99) Google Scholar). We now report that Sec16p also binds the COPII subunit Sec31p; we show this interaction is required for ER to Golgi transport. Yeast manipulations were performed by standard methods (13Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994Google Scholar). Western blotting was performed using the following antibodies: anti-HA (12CA5; 1/1000; BAbCO); anti-Sec23 (1/250) (14Hicke L. Yoshihisa T. Schekman R. Mol. Biol. Cell. 1992; 3: 667-676Crossref PubMed Scopus (90) Google Scholar); anti-Sec24p (1/250) (14Hicke L. Yoshihisa T. Schekman R. Mol. Biol. Cell. 1992; 3: 667-676Crossref PubMed Scopus (90) Google Scholar); anti-Sec13p (1/250) (15Pryer N.K. Salama N.R. Schekman R.W. Kaiser C.A. J. Cell Biol. 1993; 120: 867-875Crossref Scopus (120) Google Scholar); and anti-Sec31p (1/10,000) (5Salama N. Yeung R. Schekman R.W. EMBO J. 1993; 12: 4073-4082Crossref PubMed Scopus (176) Google Scholar). The antibodies against Sec23p, Sec24p, and Sec31p were generously provided by R. Schekman. A two-hybrid screen was performed in the strain CKY554, which is the indicator strain L40 (16Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar) with the plasmid pDS99, carrying amino acids 447–1235 of Sec16p fused to LexA in the pBTM116 vector (19Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (303) Google Scholar). CKY554 was transformed with an activation domain fusion library (generously provided by M. White) in the pGADGH vector (20Hannon G.J. Demetrik D. Beach D. Genes Dev. 1993; 7: 2378-2391Crossref PubMed Scopus (405) Google Scholar). Interactions were tested as described (18Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1322) Google Scholar, 21Fields S. Song O.-K. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4863) Google Scholar). Sec31p truncation constructs in pGADGH were subsequently constructed using the cloned SEC31 locus (10Salama N.R. Chuang J.S. Schekman R.W. Mol. Biol. Cell. 1997; 8: 205-217Crossref PubMed Scopus (127) Google Scholar), generously provided by R. Schekman. LexA fusion constructs were made in pBTM116 and represent the entire coding sequence of yeast Sec13p (pDS138) (15Pryer N.K. Salama N.R. Schekman R.W. Kaiser C.A. J. Cell Biol. 1993; 120: 867-875Crossref Scopus (120) Google Scholar) and human Sec13Rp (pDS168) (22Swaroop A. Yang-Feng T.L. Liu W. Gieser L. Barrow L. Chen K.-C. Agarwal N. Meisler M.H. Smith D.I. Hum. Mol. Genet. 1994; 3: 1281-1286Crossref PubMed Scopus (31) Google Scholar, 23Shaywitz D.A. Orci L. Ravazzola M. Swaroop A. Kaiser C.A. J. Cell Biol. 1995; 128: 679-777Crossref Scopus (85) Google Scholar) and amino acids 666–926 of Sec24p (pDS272) (8Gimeno R.E. Espenshade P. Kaiser C.A. J. Cell Biol. 1995; 131: 325-338Crossref PubMed Scopus (66) Google Scholar) and amino acids 447–1043 of Sec16p (pDS116) (6Espenshade P. Gimeno R.E. Holzmacher E. Teung P. Kaiser C.A. J. Cell Biol. 1995; 131: 311-324Crossref PubMed Scopus (149) Google Scholar). The entire coding sequence of yeast Sec23p (14Hicke L. Yoshihisa T. Schekman R. Mol. Biol. Cell. 1992; 3: 667-676Crossref PubMed Scopus (90) Google Scholar) was inserted into pGilda to generate pDS72. pGilda represents a derivative of the pEG202 lexA fusion vector (18Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1322) Google Scholar) that retains the multiple cloning sites of pEG202 but that utilizes the GAL1 promoter instead of the ADH1 promoter; the vector backbone of pGilda is from pRS313 (24Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). The strain CKY556, which is EGY40 (17Golemis E.A. Brent R. Mol. Cell Biol. 1992; 12: 3006-3014Crossref PubMed Scopus (148) Google Scholar) transformed with the indicator plasmid pSH18-24 (18Gyuris J. Golemis E. Chertkov H. Brent R. Cell. 1993; 75: 791-803Abstract Full Text PDF PubMed Scopus (1322) Google Scholar), was co-transformed with LexA and activation domain fusion plasmids. Transformants containing a pBTM116-derived LexA fusion plasmid were grown to exponential phase in selective medium containing glucose. Transformants containing a pGilda-derived LexA fusion plasmid were grown to exponential phase in selective medium containing 2% raffinose and then galactose was added to 2%, and growth continued for another 4 h. At least three independent transformants were assayed for β-galactosidase activity (13Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994Google Scholar). The mean activity of the transformants is given and expressed in Miller units (1000 × A 420/(reaction time × A 600 units assayed)) (25Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 352-355Google Scholar). All values above background were within 35% of the mean value. Recombinant Sec23p and Sec24p were were expressed as GST fusion proteins in Escherichia coli, purified by affinity chromotography, and then released by thrombin cleavage of the GST moiety (12Gimeno R.E. Espenshade P. Kaiser C.A. Mol. Biol. Cell. 1996; 7: 1815-1823Crossref PubMed Scopus (99) Google Scholar). Recombinant Sec13p (kindly provided by K. Saxena and E. Neer) was purified as a His6 fusion protein from E. coli (26Saxena K. Gaitatzes C. Walsh M.T. Eck M. Neer E.J. Smith T.F. Biochemistry. 1996; 35: 15215-15221Crossref PubMed Scopus (37) Google Scholar). The HA epitope-tagged Sec16 protein was prepared by insertion of a fragment encoding amino acids 447–1043 of SEC16 into the pGAL10-HA expression vector pRH165 2R. Gimeno, unpublished observations. to generate pDS216. This plasmid was transformed into the S. cerevisiae strain CKY557 (MATα ura3-52 trp1::hisG GAL +). Cells were grown to exponential phase in selective medium containing 2% raffinose and then supplemented with 2% galactose for 4–6 h to induce expression of the epitope-tagged protein. These cells were then washed in LBB-100 (20 mmHepes-KOH, pH 6.8, 80 mm KOAc, 5 mm MgOAc, 0.02% Triton X-100, 0.1 m NaCl) supplemented with protease inhibitors phenylmethylsulfonyl fluoride (1 mm), leupeptin (0.5 μg/ml), pepstatin (0.7 μg/ml) as well as EDTA (0.5 mm) and then frozen by drops in liquid nitrogen. Frozen cell pellets were lysed using a mortar and pestle and resuspended in LBB-100. The lysate was cleared by centrifugation at 3,000 ×g for 5 min, followed by centrifugation at 100,000 ×g for 40 min. DNA regions encoding the entire Sec31p protein, a fragment of Sec31p lacking the initial 490 amino acids, or a fragment of Sec31p lacking the final 98 amino acids were fused to the 3′ end of the GST coding sequence expressed from pGAL1 promoter in pPE127, a vector identical to pRD56 but in a different reading frame (6Espenshade P. Gimeno R.E. Holzmacher E. Teung P. Kaiser C.A. J. Cell Biol. 1995; 131: 311-324Crossref PubMed Scopus (149) Google Scholar). Clarified cytosolic extracts were prepared as described above, except that the concentration of NaCl used was 0.6 m; this buffer is referred to as LBB-600. Glutathione-Sepharose 4B beads (Pharmacia Biotech Inc.) were incubated with the extracts for 30 min at 25 °C and then washed three times with LBB-600. Beads prepared in this fashion were decorated with 2–5 pmol of the fusion protein. For the binding reactions involving Sec23p, Sec24p, and Sec13p, the decorated beads were washed twice with binding buffer (25 mm K-Hepes, pH 6.8, 0.1% Triton X-100, 1 mm MgCl2, 0.25 mg/ml bovine serum albumin). The beads were then resuspended in 45 μl of salt-supplemented binding buffer, and 5 μl of the relevant recombinant protein was then added. Binding of Sec23p (2 pmol) and Sec24p (1 pmol) was carried out in 50 mm NaCl, whereas binding of Sec13p (3 pmol) was carried out in 150 mm NaCl. After incubation for 1 h at 25 °C, the beads were washed two or three times with binding buffer (no additional salt) and then resuspended in extract sample buffer (12Gimeno R.E. Espenshade P. Kaiser C.A. Mol. Biol. Cell. 1996; 7: 1815-1823Crossref PubMed Scopus (99) Google Scholar). For the reactions involving Sec16p, the beads were washed twice with LBB-100 and then incubated with a yeast cytosolic extract prepared as described. These binding reactions were carried out in a volume of 100 μl and utilized extract containing 126 μg of total protein; NaCl was added to a final concentration of 0.2 m. Following a 1-h incubation at 25 °C, the beads were washed twice with LBB-100 and then resuspended in extract sample buffer. All proteins were subjected to SDS-PAGE and Western blotting. Both the SEC31 deletion strain RSY1109 (MATa ade2-1 ura3-1 leu2-3, 112 his3-11, 15 trp1-Δ1 s31::TRP1[pNS3111-SEC31-URA3-CEN]), kindly provided by R. Schekman, and the temperature-sensitive sec31-2 strain CKY555 (MATa sec31-2 ura3-52 leu2-3, 112), kindly provided by A. Frand, were transformed with pDS321, or sec31-2 was by A. in a screen for temperature-sensitive for ER to Golgi the in sec31-2 was by rescue of to a to amino acids the SEC31 locus inserted into the and both a SEC31 which represents a truncation that the coding sequence for the 98 amino acids of the protein. is a plasmid from and is a plasmid from R.S. M. J.H. P. 1992; PubMed Scopus Google Scholar). For the strains were grown to exponential phase at and then to nonpermissive for min. of cells and of were performed as described D.A. Orci L. Ravazzola M. Swaroop A. Kaiser C.A. J. Cell Biol. 1995; 128: 679-777Crossref Scopus (85) Google Scholar). of the SEC16 coding sequence were for regions that by as fused to a domain and be for two-hybrid pDS99, the coding sequence for amino acids 447–1235 of SEC16 inserted into the pBTM116 LexA fusion vector (19Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (303) Google Scholar), was of the constructs that this This fragment of Sec16p the to Sec24p amino acids and was different from the to Sec23p amino acids (6Espenshade P. Gimeno R.E. Holzmacher E. Teung P. Kaiser C.A. J. Cell Biol. 1995; 131: 311-324Crossref PubMed Scopus (149) Google Scholar). The L40 strain (16Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar) was transformed with both and a S. cerevisiae library constructed in the activation domain fusion vector pGADGH (20Hannon G.J. Demetrik D. Beach D. Genes Dev. 1993; 7: 2378-2391Crossref PubMed Scopus (405) Google Scholar). were from strains for expression of both the and A screen of × S. cerevisiae activation of expression upon the of the fusion protein. of the from the 3′ of the SEC31 (10Salama N.R. Chuang J.S. Schekman R.W. Mol. Biol. Cell. 1997; 8: 205-217Crossref PubMed Scopus (127) Google Scholar). The of these a of amino acids, the of the Sec31p In an to the functional of a of SEC31 were constructed in pGADGH and by two-hybrid analysis against a of expressed as LexA fusion proteins in the pBTM116 vector this the to the of the Sec31p protein, a that E.J. Smith T.F. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, E.J. R. Smith T.F. Nature. 1994; PubMed Scopus Google Scholar). at least the 490 amino acids of as with Sec13p, whereas constructs lacking this to Sec13p of (26Saxena K. Gaitatzes C. Walsh M.T. Eck M. Neer E.J. Smith T.F. Biochemistry. 1996; 35: 15215-15221Crossref PubMed Scopus (37) Google Scholar), the interaction of Sec13p with the of Sec31p that these regions of in a We also the interactions of the human Sec13p Sec13Rp (22Swaroop A. Yang-Feng T.L. Liu W. Gieser L. Barrow L. Chen K.-C. Agarwal N. Meisler M.H. Smith D.I. Hum. Mol. Genet. 1994; 3: 1281-1286Crossref PubMed Scopus (31) Google Scholar, 23Shaywitz D.A. Orci L. Ravazzola M. Swaroop A. Kaiser C.A. J. Cell Biol. 1995; 128: 679-777Crossref Scopus (85) Google Scholar). Sec13Rp a two-hybrid interaction identical to that of yeast Sec13p, specifically with These the of between yeast and COPII D.A. Orci L. Ravazzola M. Swaroop A. Kaiser C.A. J. Cell Biol. 1995; 128: 679-777Crossref Scopus (85) Google Scholar, J. G. W. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar, C. Orci L. T. Amherdt M. H. Ravazzola M. J.E. J. Cell Biol. 1994; PubMed Scopus Google Scholar, W. Ravazzola M. Amherdt M. Schekman R. Orci L. Mol. Biol. Cell. 1996; 7: PubMed Scopus Google Scholar) and the of a protein. We Sec31p with Sec23p or a domain of Sec24p that with Sec23p (12Gimeno R.E. Espenshade P. Kaiser C.A. Mol. Biol. Cell. 1996; 7: 1815-1823Crossref PubMed Scopus (99) Google Scholar), we that Sec24p interacts with the of Sec31p a that with Sec13p or Sec16p. The of Sec23p binding to Sec31p required the of an fusion protein, the of to fusion was We constructed the vector which for the expression of fusion proteins expressed from pGilda required different growth and were at expressed from pBTM116 but the that Sec23p specifically with a of Sec31p the interactions by two-hybrid we Sec16p, Sec23p, Sec24p, and Sec13p to different fusion proteins from This has been used to demonstrate the binding of and Sec24p to different regions of Sec16p (12Gimeno R.E. Espenshade P. Kaiser C.A. Mol. Biol. Cell. 1996; 7: 1815-1823Crossref PubMed Scopus (99) Google Scholar). We three different GST fusion Sec31p lacking the 490 amino acids and Sec31p lacking the 98 amino acids For the binding purified recombinant Sec23p, Sec24p, and Sec13p were The of Sec16p for this was extracts of yeast the of Sec16p acids with a The from these binding were in with the two-hybrid Sec31p was to Sec13p, Sec23p, Sec24p, and Sec16p. However, was specifically defective for Sec13p whereas was specifically defective for Sec16p Sec23p and Sec24p to both of the but to GST these binding reactions were performed in buffer containing 50 mm NaCl binding of Sec24p and binding of Sec23p were the mm NaCl) used for the Sec16p and Sec13p binding The of which is specifically defective for Sec16p to the in of this interaction First, we for A strain SEC31 on a plasmid was transformed with carrying SEC31 or Transformants were grown with for the plasmid and then on medium containing 5-fluoroorotic yeast of in the of the plasmid be to these The strains carrying the plasmid with that on that the protein an essential of Sec31p. a for protein expression the yeast strain (6Espenshade P. Gimeno R.E. Holzmacher E. Teung P. Kaiser C.A. J. Cell Biol. 1995; 131: 311-324Crossref PubMed Scopus (149) Google Scholar) was transformed with a plasmid carrying or with vector from both strains were by using anti-Sec31p (5Salama N. Yeung R. Schekman R.W. EMBO J. 1993; 12: 4073-4082Crossref PubMed Scopus (176) Google Scholar). was in to the of the protein. directly the of the of Sec31p in we utilized a temperature-sensitive of which was in a screen for defective for ER to Golgi transport. rescue the secretion defect of sec31-2 at nonpermissive the of to the of Sec31p in ER to Golgi transport be The sec31-2 mutant was transformed with encoding Sec31p or growth of the transformants at the nonpermissive of °C was then Although transformed with the SEC31 plasmid at °C, transformed with the plasmid remained temperature-sensitive for growth these were in transformed with a or a The of ER to Golgi transport of the cargo protein was followed by analysis of the transformants at The plasmid rescue the transport defect the binding that the defect of is in binding to Sec16p, the transport defect by the of SEC31 that the binding of Sec31p to Sec16p is required for ER to Golgi transport. However, we cannot the that the of Sec31p an additional that has been defined that is for in both yeast and cells demonstrate that vesicle coat formation be by the addition of a defined of cytosolic factors to washed membranes (4Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1048) Google Scholar, 5Salama N. Yeung R. Schekman R.W. EMBO J. 1993; 12: 4073-4082Crossref PubMed Scopus (176) Google Scholar, J. Orci L. K. Amherdt M. Ravazzola M. J.E. Cell. 1993; 75: Full Text PDF PubMed Scopus Google Scholar). For transport between Golgi in cells, these factors are the small GTP-binding protein and the complex, of subunits that in the and to the membrane S. Orci L. Amherdt M. Ravazzola M. J.E. J. Cell Biol. 1994; PubMed Scopus Google Scholar). For transport between yeast ER and two different cytosolic protein in addition to the small GTP-binding protein Sar1p are to the COPII vesicle coat R. Orci L. Science. 1996; 271: 1526-1533Crossref PubMed Scopus (818) Google Scholar, L. Yoshihisa T. Schekman R. Mol. Biol. Cell. 1992; 3: 667-676Crossref PubMed Scopus (90) Google Scholar, N.K. Salama N.R. Schekman R.W. Kaiser C.A. J. Cell Biol. 1993; 120: 867-875Crossref Scopus (120) Google Scholar). The interaction that we between purified components of the Sec31p-Sec13p and the Sec23p-Sec24p that these two in the this we expressed a fusion protein in yeast and Sec23p or Sec24p be found associated with this fusion protein in a cytosolic extract prepared of the in vitro transport (4Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1048) Google Scholar, N.K. Salama N.R. Schekman R.W. Kaiser C.A. J. Cell Biol. 1993; 120: 867-875Crossref Scopus (120) Google Scholar). We were unable to of these proteins in the This was with of the of the interactions between binding of to recombinant Sec23p and Sec24p was demonstrated at 50 mm NaCl but was at 150 mm NaCl, a concentration to that used for the in vitro Thus, is that the between the two COPII the of the ER that SEC16 interacts genetically with all five COPII genes and a peripheral ER membrane protein that is on ER-derived transport vesicles, required for vesicle and binds directly to Sec23p, Sec24p, and we propose that Sec16p as a for the of the COPII coat from soluble protein the that Sec31p binds directly to both Sec23p and Sec24p suggests that the COPII subunits are stabilized by interactions with Sec16p but also by interactions with We R. Schekman, M. E. R. A. Frand, K. and E. Neer for generously and J. D. P. C. and A. and the of the Kaiser for and