Quantitative live-cell analysis of microtubule-uncoupled cargo-protein sorting in the ER.
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.
Dukhovny
Anna
A
Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel.
Papadopulos
Andreas
A
Hirschberg
Koret
K
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D020755:Coat Protein Complex I / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D036681:Fluorescence Recovery After Photobleaching; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008870:Microtubules / Q000187:drug effects; D009154:Mutation; D015739:Nocodazole / Q000494:pharmacology; D021381:Protein Transport; D050257:Tubulin Modulators / Q000494:pharmacology; D033921:Vesicular Transport Proteins / Q000502:physiology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism
D020755:Coat Protein Complex I; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D050257:Tubulin Modulators; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins; D015739:Nocodazole