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PMID: 8175881 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus.

The Journal of cell biology ·Vol. 125 ·No. 3 ·1994-05-00 ·Pages 557-71

Wilson BS, Nuoffer C, Meinkoth JL, McCaffery M, Feramisco JR, Balch WE, Farquhar MG

Abstract

The Golgi apparatus is a dynamic organelle whose structure is sensitive to vesicular traffic and to cell cycle control. We have examined the potential role for rab1a, a GTPase previously associated with ER to Golgi and intra-Golgi transport, in the formation and maintenance of Golgi structure. Bacterially expressed, recombinant rab1a protein was microinjected into rat embryonic fibroblasts, followed by analysis of Golgi morphology by fluorescence and electron microscopy. Three recombinant proteins were tested: wild-type rab, mutant rab1a(S25N), a constitutively GDP-bound form (Nuoffer, C., H. W. Davidson, J. Matteson, J. Meinkoth, and W. E. Balch, 1994. J. Cell Biol. 125: 225-237), and mutant rab1a(N124I) defective in guanine nucleotide binding. Microinjection of wild-type rab1a protein or a variety of negative controls (injection buffer alone or activated ras protein) did not affect the appearance of the Golgi, as visualized by immunofluorescence of alpha-mannosidase II (Man II), used as a Golgi marker. In contrast, microinjection of the mutant forms promoted the disassembly of the Golgi stacks into dispersed vesicular structures visualized by immunofluorescence. When S25N-injected cells were analyzed by EM after immunoperoxidase labeling, Man II was found in isolated ministacks and large vesicular elements that were often surrounded by numerous smaller unlabeled vesicles resembling carrier vesicles. Golgi disassembly caused by rab1a mutants differs from BFA-induced disruption, since beta-COP remains membrane associated, and Man II does not redistribute to the ER. BFA can still cause these residual Golgi elements to fuse and disperse, albeit at a slower rate. Moreover, BFA recovery is incomplete in the presence of rab1 mutants or GTP gamma S. We conclude that GTP exchange and hydrolysis by GTPases, specifically rab1a, are required to form and maintain normal Golgi stacks. The similarity of Golgi disassembly seen with rab1a mutants to that occurring during mitosis, may point to a molecular basis involving rab1a for fragmentation of the Golgi apparatus during cell division.

MeSH Terms
Animals Brefeldin A Cells, Cultured Coatomer Protein Cyclopentanes/pharmacology GTP Phosphohydrolases/metabolism,pharmacology GTP-Binding Proteins/genetics,metabolism Golgi Apparatus/drug effects,metabolism,ultrastructure In Vitro Techniques Mannosidases/metabolism Microinjections Microscopy, Electron Microtubule-Associated Proteins/metabolism Mutation Rats Recombinant Proteins rab1 GTP-Binding Proteins
Chemicals
Coatomer Protein Cyclopentanes Microtubule-Associated Proteins Recombinant Proteins Brefeldin A Mannosidases mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase GTP Phosphohydrolases GTP-Binding Proteins rab1 GTP-Binding Proteins
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Wilson B S
Division of Cellular and Molecular Medicine, University of California, San Diego 92093-0651.
Nuoffer C
Meinkoth J L
McCaffery M
Feramisco J R
Balch W E
Farquhar M G
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.125.3.557
Published
1994-05-00
Pages
557-71
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2119990
Subset
IM
Grants
NCI NIH HHS · CA46128 · United States
NCI NIH HHS · CA58689 · United States
NIDDK NIH HHS · DK17780 · United States
External Links
PubMed source DOI: 10.1083/jcb.125.3.557 PMC Full Text View journal details
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