QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 26 February 2008
doi: 10.1242/jcs.019463

This Article Figures Only Full Text Full Text (PDF) Supplementary Material All Versions of this Article: jcs.019463v1 121/6/865    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Dukhovny, A. Articles by Hirschberg, K. Search for Related Content PubMed PubMed Citation Articles by Dukhovny, A. Articles by Hirschberg, K.

Quantitative live-cell analysis of microtubule-uncoupled cargo-protein sorting in the ER

Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel

^* Author for correspondence (e-mail: koty{at}post.tau.ac.il)

Accepted 11 December 2007

The sorting and concentration of cargo proteins within ER exit sites (ERESs) is a fundamental function of the secretory machinery. The mechanism by which peripheral coat complexes and their small GTPase effectors mediate this function with export membrane domains is only partially understood. The secretory-machinery-mediated sorting to ERESs is a process that counters the entropy-driven even distribution of membrane proteins within organellar membranes. Here, for the first time, we quantified the dynamic properties of GFP-VSVG sorting to ERESs in living cells by uncoupling it from later translocation steps using microtubule depolymerization. The dynamics of the ER to ERES redistribution of cargo proteins was quantified in single cells by measuring changes in fluorescence-intensity variance after shift to the permissive temperature. Cargo concentration within ERESs continued in cells overexpressing the GTP-locked ARF1Q71L or in the presence of brefeldin A. In the absence of COPI and microtubules, ERESs transformed from tubulovesicular to spherical membranes that actively accumulated secretory cargo and excluded ER-membrane markers. We found sorting to ERESs to be a slow and diffusion-unlimited process. Our findings exclude COPI, and identify the COPII protein complex to be directly involved in the secretory cargo sorting and redistribution functions of ERESs.

Key words: Endoplasmic reticulum, ER-exit sites, sorting, secretory pathway, membrane, live-cell imaging, COPII, COPI