Abstract
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.
Highlights
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
We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A
These results highlight that small primary sequence differences between the two mammalian Secretion-associated Rasrelated GTPase 1 (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
Summary
There are only 20 of 198 divergent amino acids that distinguish human SAR1A and SAR1B. We found that constructs in which the amino acids of the divergent apical ␣-helix (SAR1AϾB-helix and SAR1BϾA-helix) were swapped did not reverse the kinetics (Fig. 2F) These data suggest that the GTP-adjacent divergent residues are necessary and sufficient for the increased kinetics of SAR1 GTP exchange. Addition of SEC23/24 heterodimer greatly increased the size of particles and reduced their number (Fig. 5B), as expected, because SEC23 binds directly to SAR1A and SAR1B, and the heterodimers bind each other To test whether this effect was due to either of the divergent peptide clusters, we repeated this assay with the WT (Fig. 5C), helix (Fig. 5D), and GTP-adjacent cluster (Fig. 5E) SAR1 constructs. Overexpression of the GTP-adjacent swap SAR1BϾA produced an ϳ2-fold increase in ApoB secretion, similar to the effect of WT SAR1B, suggesting that the GTP-adjacent amino acid cluster is not relevant to the SAR1B-specific function. The apical ␣-helix cluster causes SAR1B to have a higher affinity for SEC23A and has an important role in lipoprotein secretion
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