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Proc. Natl. Acad. Sci. USA Vol. 94, pp. 837–842, February 1997 Biochemistry Selective packaging of cargo molecules into endoplasmic reticulum-derived COPII vesicles (Sar1pySec16pysecretory proteinsySNARE proteins) JOSEPH L. CAMPBELL AND RANDY SCHEKMAN Department of Molecular and Cell Biology, and Howard Hughes Medical Institute, Barker Hall, University of California, Berkeley, CA 94720-3202 Contributed by Randy Schekman, November 27, 1996 ABSTRACT Coated vesicles transport proteins from the Despite our knowledge of the cytosolic factors involved in endoplasmic reticulum (ER) to the Golgi apparatus. The vesicle formation, little is known about how cargo is recognized formation of transport vesicles in vitro requires the incubation and packaged into these vesicles. One class of proteins that of an ER-membrane fraction with three protein fractions must be packaged into vesicles is known collectively as collectively known as coat protein II (COPII; Sar1p, Sec23py v-SNARE [vesicle-soluble NSF (N-ethylmaleimide-sensitive Sec24p, and Sec13pySec31p). We used this assay to investigate factor) attachment protein receptor] proteins (for review, see how targeting [v-SNARE, vesicle-soluble NSF (N-ethylmale- ref. 5). v-SNARE proteins are membrane-bound proteins that imide-sensitive factor) attachment protein receptor], putative interact with their cognate t-SNARE (target SNARE) protein adapter (e.g., Emp24p), and cargo molecules are captured into (t-SNARE proteins are located in the membrane of the acceptor ER-derived COPII vesicles. Analysis of fusion proteins strongly organelle) to ensure that vesicles only fuse with the membrane of suggests that the cytoplasmic domain of the v-SNARE protein the appropriate organelle. To examine v-SNARE packaging Sec22p is required for its packaging into ER-derived COPII signals, we employed fusion proteins between Sec22p, a vesicles. We examined the packaging requirements for various v-SNARE protein found in COPII vesicles, and Sec12p, an molecules by individually titrating each of the COPII compo- endoplasmic reticulum (ER) membrane protein required for nents. More Sar1p (the GTP-binding protein that initiates the formation of COPII vesicles but not found in these vesicles. vesicles budding) is needed to package the membrane-associated The results of these and other experiments suggest that there v-SNAREs and Emp24p than is needed to package the soluble is a signal for the packaging of Sec22p on the cytoplasmic secretory protein glycosylated pro-a-factor (gpaF). Microsomes domain of Sec22p. prepared from a strain overproducing Sec12p (the nucleotide We also examined the packaging characteristics of glycosy- a a exchange protein that recruits Sar1p to the ER) produce vesicles lated pro- -factor (gp F), Sec22p, Bos1p, Bet1p, and Emp24p. Bos1p and Bet1p are two additional v-SNARE proteins found containing gpaF without the addition of exogenous Sar1p, in COPII vesicles, and Emp24p is a transmembrane protein whereas the v-SNAREs and Emp24p are not efficiently packaged believed to be involved in the packaging of specific cargo under these conditions. Addition of Sar1p to these microsomes molecules (6). We find differential sensitivity to COPII com- leads to increased packaging of v-SNAREs and Emp24p with no ponent levels and to the presence of functional Sec16p, increase in the packaging of gp F. Finally, we show that mem- a suggesting that all packaging is not handled identically. branes prepared from strains with mutations in the SEC16 gene Preliminary data on the fusion proteins appeared previously are more defective for the packaging of v-SNARE molecules and in the proceedings of a meeting (7). Emp24p than they are for the packaging of gpaF. These results point to the possibility that diverse signals or adapters partici- pate in the capture of secretory and membrane cargo molecules MATERIALS AND METHODS into COPII transport vesicles. Strains. RSY255, MATa ura3-52, leu2-3,112; RSY267, MATa sec16-2 ura3–52 his 4-619; RSY317, MATa sec16-1, leu Palade (1) originally proposed that vesicles carry secretory 2-3,112; RSY658 W303-1B, sec12::LEU2 (pANY1-9); and proteins between organelles. Subsequent genetic and biochem- RSY772, MATa sec16-3 ura3-52, leu2-3,112. pANY1-9 is a ical experiments have identified many of the proteins required 2-mm plasmid from which the expression of Sec12p is driven by for producing these vesicles and fusing them with the appro- its own promoter (8). priate target membrane. These studies have also shown that Reagents. Restriction enzymes, T4 DNA polymerase, and homologous molecules perform the same functions in yeast T4 DNA ligase were purchased from New England BioLabs and mammalian cells (for review, see ref. 2). A set of soluble and Boehringer Mannheim. PFU DNA polymerase was pur- proteins (the Sec13pySec31p complex, the Sec23pySec24p chased from Stratagene. Antibodies for Sec22p (9), Bos1p complex, and Sar1p) that can substitute for cytosol in the (10), Bet1p (10), Emp24p (6), and Sec12p (C. Barlowe, formation of these vesicles were identified in a cell-free assay Dartmouth Medical School) were used in Western blots. (3). This set of soluble proteins forms a coat, COPII, around 125I-labeled protein A was purchased from ICN. Chemicals the vesicles during the budding process (4). The first COP (coat were purchased from Sigma, and media were purchased from protein) complex to be identified, COPI, was discovered in Difco. mammalian cells and mediates the formation of a different Construction of Fusion Proteins. The fusion proteins were class of vesicles (for review, see ref. 2). constructed with a two-step PCR-based method described by Horton et al. (11). For Sec22-12p (a hybrid protein bearing the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: v-SNARE, vesicle-soluble NSF (N-ethylmaleimide- accordance with 18 U.S.C. §1734 solely to indicate this fact. sensitive factor) attachment protein receptor; COP, coat protein; Sec22-12p, hybrid protein bearing the N terminus of Sec22p and the Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA C terminus of Sec12p; Sec12-22p, hybrid protein bearing the N 0027-8424y97y94837-6$2.00y0 terminus of Sec12p and the C terminus of Sec22p; gpaF, glycosylated PNAS is available online at http:yywww.pnas.org. pro-a-factor; ER, endoplasmic reticulum. 837 Downloaded by guest on September 28, 2021 838 Biochemistry: Campbell and Schekman Proc. Natl. Acad. Sci. USA 94 (1997) N terminus of Sec22p and the C terminus of Sec12p), the mgyml. For all reactions except the titrations shown in Fig. 2, following oligonucleotides were used: 59-GCT TCG AGA TCT the COPII components were used at the following concentra- AAA CCC TGA CAG TGA CAC-39,59-CGC AAA AGA tions: Sar1p, 20 mgyml; Sec23pySec24p, 24 mgyml; and TCA ACT TCG ATT TTT TCA CCA ACT TCA TCC T-39, Sec31pySec13p, 44 mgyml. In the titrations shown in Fig. 2, two 59-AGG ATG AAG TTG GTG AAA AAA TCG AAG TTG components were added at the concentrations indicated above, ATC TTT TGC G-39, and 59-GCC GAT TAT CTA GAC GTC and the concentration of the third component was varied. The ACT CTC CTT TTC GC-39. For Sec12-22p (a hybrid protein Sar1p concentration ranged from 0 to 80 mgyml, the Sec23py bearing the N terminus of Sec12p and the C terminus of Sec24p concentration ranged from 0 to 96 mgyml, and the Sec22p), the following oligonucleotides were used: 59-GCC Sec13pySec31p concentration ranged from 0 to 176 mgyml. GAT TAA GAT CTG ATA CGT GAT AGA AAT AC-39, For all of the vesicle budding reactions, after a 30-min incu- 59-TGA AAC AAA AAA TCT CTA AAC TCT TGA TCA bation at the appropriate temperature, the reactions were GTC AAT ATG C-39,59-GCA TAT TGA CTG ATC AAG placed on ice for 5 min, and the vesicles were separated from AGT TTA GAG ATT TTT TGT TTC A-39, and 59-GCT TCG donor membranes by centrifugation at 14,000 3 g for 3 min in TCT AGA CTT GGA CCA AAT TGA TCG-39. The desired a refrigerated microcentrifuge. The release of gpaF into the fragments were gel-purified, and the ends were made blunt vesicle-containing supernatant was quantified as described by with T4 DNA polymerase. Blunt fragments were ligated into Rexach and Schekman (16), and release of the other assayed the EcoRV site of Bluescript II KS1 (Stratagene). The result- molecules into the supernatant was quantified by 125I-labeled ing plasmids were then cut with BglII and XbaI and the protein A immunoblots and a Molecular Dynamics fragment coding for the fusion protein was cloned into PhosphorImager. Immunoblots were performed as described pRH98-1 cut with BamHI and XbaI. pRH98-1, a gift of Randy by Towbin et al. (17). Hampton (University of California, Berkeley, CA), was de- rived as described for pRH98-2 (12) except the parent vector RESULTS was YCplac33 (ARSCEN, URA3) instead of YIp lac211 (in- tegrating, URA3) (13). When genes were cloned into the Sec22p May Possess a Positive Transport Signal. To gain BamHI site of pRH98-1, their expression was driven by the information about how v-SNARE proteins are packaged into glyceraldehyde-3-phosphate dehydrogenase promoter (14). COPII vesicles, we constructed fusions between two type II ER The plasmid bearing Sec22-12p was called pJCY26, and the membrane proteins, Sec22p and Sec12p. A schematic view of plasmid bearing Sec12-22p was called pJCY30. these proteins is shown in Fig. 1. Sec22p is a v-SNARE protein Construction of Sec22p Overproducer. PCR was used to that is packaged efficiently into ER-derived vesicles, whereas generate a fragment containing the entire coding sequence of Sec12p is required for the formation of COPII vesicles but does Sec22p. The following oligonucleotides were used in these not efficiently enter COPII vesicles (4, 9, 16, 18).

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