Abstract
The COPII coat complex found on endoplasmic reticulum (ER)-derived vesicles plays a critical role in cargo selection. We now address the potential role of biosynthetic cargo in modulating COPII coat assembly and vesicle budding. The ER accumulation of vesicular stomatitis glycoprotein (VSV-G), a transmembrane protein, or the soluble PiZ variant of alpha1-antitrypsin, reduced levels of general COPII vesicle formation in vivo. Consistent with this result, conditions that prevent the export of VSV-G from the ER led to a significant inhibition of general COPII vesicle budding from ER microsomes and the export of an endogenous recycling protein p58 in vitro. In contrast, synchronized export of VSV-G stimulated COPII vesicle budding both in vivo and in vitro. Under conditions where VSV-G is retained in the ER, we find that it can to be recovered in pre-budding complexes containing COPII components. These results suggest that the export of biosynthetic cargo is integrated with ER functions involved in protein folding and oligomerization. The ability of biosynthetic cargo to prevent or enhance ER export suggests that interactions of cargo with the COPII machinery contribute to the formation of vesicles budding from the ER.
Highlights
This paper is dedicated to the memory of Thomas Kreis. ‡ Recipient of a fellowship grant from the European Molecular Biology Organization and the Human Frontier Sciences Program. § Recipient of a Cystic Fibrosis Foundation Post-doctoral fellowship. ¶ Recipient of a fellowship from the Human Frontier Sciences Program and the Muscular Dystrophy Association. ʈ To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, The Scripps Research Institute, 10666 N
To assess the effect of cargo on endoplasmic reticulum (ER) export in vivo, we examined whether the number of buds found on the ER surface can be modulated by biosynthetic cargo using electron microscopy and quantitative stereology
Biosynthetic cargo exiting the ER includes a wide collection of newly synthesized molecules, protein and lipids, that are mobilized from the ER to distinct cellular and extracellular destinations
Summary
(Received for publication, June 17, 1998, and in revised form, November 5, 1998). Meir Aridor‡, Sergei I. Biosynthetic cargo is sorted from resident ER proteins and selected for incorporation into vesicles by interacting with the Sar and Sec23/24 components of the COPII coat prior to vesicle formation [4, 5]. We have applied a variety of techniques to monitor the effect of biosynthetic cargo on vesicle budding from secretory compartments These include the following: (i) quantitative stereology to follow membrane flow at the ultra-structural level in vivo [24], (ii) biochemical assays to monitor ER vesicle budding in vitro [25], (iii) an assay to monitor the interactions of cargo with COPII components in the ER prior to vesicle forma-.
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