Coated vesicles transport proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The formation of transport vesicles in vitro requires the incubation of an ER-membrane fraction with three protein fractions collectively known as coat protein II (COPII; Sar1p, Sec23p/Sec24p, and Sec13p/Sec31p). We used this assay to investigate how targeting [v-SNARE, vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor], putative adapter (e.g., Emp24p), and cargo molecules are captured into ER-derived COPII vesicles. Analysis of fusion proteins strongly suggests that the cytoplasmic domain of the v-SNARE protein Sec22p is required for its packaging into ER-derived COPII vesicles. We examined the packaging requirements for various molecules by individually titrating each of the COPII components. More Sar1p (the GTP-binding protein that initiates vesicles budding) is needed to package the membrane-associated v-SNAREs and Emp24p than is needed to package the soluble secretory protein glycosylated pro-alpha-factor (gp alphaF). Microsomes prepared from a strain overproducing Sec12p (the nucleotide exchange protein that recruits Sar1p to the ER) produce vesicles containing gp alphaF without the addition of exogenous Sar1p, whereas the v-SNAREs and Emp24p are not efficiently packaged under these conditions. Addition of Sar1p to these microsomes leads to increased packaging of v-SNAREs and Emp24p with no increase in the packaging of gp alphaF. Finally, we show that membranes prepared from strains with mutations in the SEC16 gene are more defective for the packaging of v-SNARE molecules and Emp24p than they are for the packaging of gp alphaF. These results point to the possibility that diverse signals or adapters participate in the capture of secretory and membrane cargo molecules into COPII transport vesicles.

• PMID: 9023343
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Campbell JL, Schekman R

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