Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI.
ABSTRACT Exocytic transport from the endoplasmic reticulum (ER) to the Golgi complex has been visualized in living cells using a chimera of the temperature-sensitive glycoprotein of vesicular stomatitis virus and green fluorescent protein (ts-G-GFP[ct]). Upon shifting to permissive temperature, ts-G-GFP(ct) concentrates into COPII-positive structures close to the ER, which then build up to form an intermediate compartment or transport complex, containing ERGIC-53 and the KDEL receptor, where COPII is replaced by COPI. These structures appear heterogenous and move in a microtubule-dependent manner toward the Golgi complex. Our results suggest a sequential mode of COPII and COPI action and indicate that the transport complexes are ER-to-Golgi transport intermediates from which COPI may be involved in recycling material to the ER.
Full-textDOI: · Available from: Suzie J Scales, Apr 27, 2015
SourceAvailable from: Mattia Gazzola01/1994, Degree: PhD, Supervisor: Petros Koumoutsakos
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ABSTRACT: Tethering factors regulate the targeting of membrane-enclosed vesicles under the control of Rab GTPases. P115, a golgin family tether, has been shown to participate in multiple stages of ER/Golgi transport. Despite extensive study, p115's mechanism of action is poorly understood. SNARE proteins make up the machinery for membrane fusion, and strong evidence shows that p115's function is directly linked to its interaction with SNAREs. Using a gel filtration binding assay, we have demonstrated that in solution p115 stably interacts with ER/Golgi SNAREs rbet1 and sec22b, but not membrin and syntaxin 5. These binding preferences stemmed from selectivity of p115 for monomeric SNARE motifs as opposed to SNARE oligomers. Soluble monomeric rbet1 can compete off p115 from COPII vesicles. Furthermore, excess p115 inhibits p115 function in trafficking. We conclude that monomeric SNAREs are a major binding site for p115 on COPII vesicles, and that p115 dissociates from its SNARE partners upon SNAREpin assembly. Our results suggest a model in which p115 forms a mixed p115/SNARE helix bundle with a monomeric SNARE, facilitates the binding activity and/or concentration of the SNARE at pre-fusion sites, and is subsequently ejected as SNARE complex formation and fusion proceed.Traffic 11/2014; DOI:10.1111/tra.12242 · 4.71 Impact Factor
The Journal of Cell Biology 01/2001; 154(2):317-330. DOI:10.1083/jcb.200104073 · 9.69 Impact Factor