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PMID: 14502225 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Spatiotemporal dynamics of the COPI vesicle machinery.

EMBO reports ·Vol. 4 ·No. 10 ·2003-10-00 ·Pages 1000-4

Elsner M, Hashimoto H, Simpson JC, Cassel D, Nilsson T, Weiss M

Abstract

Assembly of the coat protein I (COPI) vesicle coat is controlled by the small GTPase ADP ribosylation factor 1 (ARF1) and its GTPase-activating protein, ARFGAP1. Here, we investigate the diffusional behaviours of coatomer, the main component of the coat, and also those of ARF1 and ARFGAP1. Using fluorescence-correlation spectroscopy, we found that most ARF1 and ARFGAP1 molecules are highly mobile in the cytosol (diffusion constant D approximately equal to 15 microm(2) s(-1)), whereas coatomer diffuses 5-10 times more slowly than expected (D approximately equal to 1 microm(2) s(-1)). This slow diffusion causes diffusion-limited binding kinetics to Golgi membranes, which, in FRAP (fluorescence recovery after photobleaching) experiments, translates into a twofold slower binding rate. The addition of aluminium fluoride locks coatomer onto Golgi membranes and also decreases the binding kinetics of both ARF1 and ARFGAP1, suggesting that these proteins function in concert to mediate sorting and vesicle formation.

MeSH Terms
ADP-Ribosylation Factor 1/genetics,metabolism ADP-Ribosylation Factors/genetics,metabolism Animals Biological Transport/physiology CHO Cells COP-Coated Vesicles/metabolism Coat Protein Complex I/genetics,metabolism Cricetinae Fluorescence Recovery After Photobleaching GTPase-Activating Proteins/genetics,metabolism Golgi Apparatus/metabolism HeLa Cells Humans Protein Transport RNA Interference Recombinant Fusion Proteins/metabolism Spectrometry, Fluorescence Time Factors
Chemicals
ARFGAP1 protein, human ARFGAP3 protein, human Coat Protein Complex I GTPase-Activating Proteins Recombinant Fusion Proteins ADP-Ribosylation Factor 1 ADP-Ribosylation Factors
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Elsner Markus
Cell Biology and Cell Biophysics Programme, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Hashimoto Hitoshi
Simpson Jeremy C
Cassel Dan
Nilsson Tommy
Weiss Matthias
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22 references, click to expand
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Article Info
Journal
EMBO reports
Abbr.
EMBO Rep
ISSN
1469-221X
DOI
10.1038/sj.embor.embor942
Published
2003-10-00
Epub
2003-00-19
Pages
1000-4
Language
English
Region
England
NLM ID
100963049
PMCID
PMC1326400
Subset
IM
Corrections
CommentIn
PMID: 14528254
External Links
PubMed source DOI: 10.1038/sj.embor.embor942 PMC Full Text View journal details
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