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Abstract
ABSTRACTSecretory cargo is recognized, concentrated and trafficked from endoplasmic reticulum (ER) exit sites (ERES) to the Golgi. Cargo export from the ER begins when a series of highly conserved COPII coat proteins accumulate at the ER and regulate the formation of cargo-loaded COPII vesicles. In animal cells, capturing live de novo cargo trafficking past this point is challenging; it has been difficult to discriminate whether cargo is trafficked to the Golgi in a COPII-coated vesicle. Here, we describe a recently developed live-cell cargo export system that can be synchronously released from ERES to illustrate de novo trafficking in animal cells. We found that components of the COPII coat remain associated with the ERES while cargo is extruded into COPII-uncoated, non-ER associated, Rab1 (herein referring to Rab1a or Rab1b)-dependent carriers. Our data suggest that, in animal cells, COPII coat components remain stably associated with the ER at exit sites to generate a specialized compartment, but once cargo is sorted and organized, Rab1 labels these export carriers and facilitates efficient forward trafficking.This article has an associated First Person interview with the first author of the paper.
Highlights
The ER serves as the entry point for the secretory pathway
COPII components localize to stable domains on peripheral ER tubules We used live confocal fluorescence microscopy to visualize the distribution and dynamics of four major COPII components at ER exit sites (ERES) in three different animal cell lines: COS-7, HeLa and U2OS
COPII components were scored as remaining associated with the ER if they remained tethered to the ER throughout the two-minute movie
Summary
The ER serves as the entry point for the secretory pathway. Following successful translation and translocation, secretory proteins synthesized in the ER are concentrated by COPII proteins at ER exit sites (ERES) and trafficked to the Golgi within vesicular carriers (D’Arcangelo et al, 2013). The inner coat recruits the heterotetramer “outer coat” complex composed of Sec13/Sec resulting in the formation of a fully formed COPII-coated transport vesicle that will deliver incorporated cargo to the Golgi complex via anterograde trafficking from the ER (Kirk and Ward, 2007; Lederkremer et al, 2001; Lee et al, 2004; Rossanese et al, 1999; Stagg et al, 2006) Many of these studies have been performed in yeast cells because of the advantages of yeast genetics to identify COPII components combined with temperature sensitive (ts) mutants that can stall cargo as it is trafficked
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