A rapid and quantitative coat protein complex II vesicle formation assay using luciferase reporters

The cytosolic coat protein complex II (COPII) mediates vesicle formation from the endoplasmic reticulum (ER) and is essential for ER-to-Golgi trafficking. The minimal machinery for COPII assembly is well established. However, additional factors may regulate the process in mammalian cells. Here, a morphological COPII assembly assay using purified COPII proteins and digitonin-permeabilized cells has been applied to demonstrate a role for a novel component of the COPII assembly pathway. The factor was purified and identified by mass spectrometry as Nm23H2, one of eight isoforms of nucleoside diphosphate kinase in mammalian cells. Importantly, recombinant Nm23H2, as well as a catalytically inactive version, promoted COPII assembly in vitro, suggesting a noncatalytic role for Nm23H2. Consistent with a function for Nm23H2 in ER export, Nm23H2 localized to a reticular network that also stained for the ER marker calnexin. Finally, an in vivo role for Nm23H2 in COPII assembly was confirmed by isoform-specific knockdown of Nm23H2 by using short interfering RNA. Knockdown of Nm23H2, but not its most closely related isoform Nm23H1, resulted in diminished COPII assembly at steady state and reduced kinetics of ER export. These results strongly suggest a previously unappreciated role for Nm23H2 in mammalian ER export.

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Specific interaction of the yeast cis-Golgi syntaxin Sed5p and the coat protein complex II component Sec24p of endoplasmic reticulum-derived transport vesicles

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.96.7.3751 ◽

1999 ◽

Vol 96 (7) ◽

pp. 3751-3756 ◽

Cited By ~ 48

Author(s):

R. Peng ◽

R. Grabowski ◽

A. De Antoni ◽

D. Gallwitz

Keyword(s):

Endoplasmic Reticulum ◽

Coat Protein ◽

Protein Complex ◽

Specific Interaction ◽

Complex Ii ◽

Transport Vesicles ◽

Coat Protein Complex

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Faculty Opinions recommendation of Uncoupled packaging of amyloid precursor protein and presenilin 1 into coat protein complex II vesicles.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1023422.270727 ◽

2005 ◽

Author(s):

Sebastian Springer

Keyword(s):

Coat Protein ◽

Amyloid Precursor Protein ◽

Protein Complex ◽

Precursor Protein ◽

Presenilin 1 ◽

Complex Ii ◽

Coat Protein Complex

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Small sequence variations between two mammalian paralogs of the small GTPase SAR1 underlie functional differences in coat protein complex II assembly

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012964 ◽

2020 ◽

Vol 295 (25) ◽

pp. 8401-8412 ◽

Cited By ~ 2

Author(s):

David B. Melville ◽

Sean Studer ◽

Randy Schekman

Keyword(s):

Endoplasmic Reticulum ◽

Coat Protein ◽

Binding Site ◽

Protein Complex ◽

Small Gtpase ◽

The Other ◽

Complex Ii ◽

Gtp Binding ◽

Lipoprotein Secretion ◽

Coat Protein Complex

Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog–specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases.

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