Abstract
Accurate maintenance of organelle identity in the secretory pathway relies on retention and retrieval of resident proteins. In the endoplasmic reticulum (ER), secretory proteins are packaged into COPII vesicles that largely exclude ER residents and misfolded proteins by mechanisms that remain unresolved. Here we combined biochemistry and genetics with correlative light and electron microscopy (CLEM) to explore how selectivity is achieved. Our data suggest that vesicle occupancy contributes to ER retention: in the absence of abundant cargo, nonspecific bulk flow increases. We demonstrate that ER leakage is influenced by vesicle size and cargo occupancy: overexpressing an inert cargo protein or reducing vesicle size restores sorting stringency. We propose that cargo recruitment into vesicles creates a crowded lumen that drives selectivity. Retention of ER residents thus derives in part from the biophysical process of cargo enrichment into a constrained spherical membrane-bound carrier.
Highlights
Protein trafficking within the eukaryotic secretory pathway occurs via cargobearing vesicles that shuttle proteins and lipids from one compartment to another
To understand the basis for vesicle formation in the absence of Sec13, we first sought to visualize the ultrastructure of ER exit sites (ERES) with high spatial resolution
Membrane morphology falls below the diffraction limit such that detailed structural information can only be obtained by electron microscopy (Orci et al, 1991; Zeuschner et al, 2006)
Summary
Protein trafficking within the eukaryotic secretory pathway occurs via cargobearing vesicles that shuttle proteins and lipids from one compartment to another. The inner cargo- and lipid-bound layer comprises the small GTPase, Sar, and the cargo adaptor complex, Sec23/Sec. The inner cargo- and lipid-bound layer comprises the small GTPase, Sar, and the cargo adaptor complex, Sec23/Sec24 This inner coat in turn recruits an outer coat of heterotetrameric Sec13/Sec, which forms rod-like structures that can selfassemble into a polyhedral cage that is thought to contribute to vesicle architecture (Fath et al, 2007; Noble et al, 2013; Zanetti et al, 2013). Sec is one example of an accessory protein that is thought to define sites for COPII recruitment and assist in coat assembly (Supek et al, 2002; Kung et al, 2012)
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