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PMID: 12654904 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

The Ran GTPase cycle is required for yeast nuclear pore complex assembly.

The Journal of cell biology ·Vol. 160 ·No. 7 ·2003-03-31 ·Pages 1041-53

Ryan KJ, McCaffery JM, Wente SR

Abstract

Here, we report the first evidence that the Ran GTPase cycle is required for nuclear pore complex (NPC) assembly. Using a genetic approach, factors required for NPC assembly were identified in Saccharomyces cerevisiae. Four mutant complementation groups were characterized that correspond to respective mutations in genes encoding Ran (gsp1), and essential Ran regulatory factors Ran GTPase-activating protein (rna1), Ran guanine nucleotide exchange factor (prp20), and the RanGDP import factor (ntf2). All the mutants showed temperature-dependent mislocalization of green fluorescence protein (GFP)-tagged nucleoporins (nups) and the pore-membrane protein Pom152. A decrease in GFP fluorescence associated with the nuclear envelope was observed along with an increase in the diffuse, cytoplasmic signal with GFP foci. The defects did not affect the stability of existing NPCs, and nup mislocalization was dependent on de novo protein synthesis and continued cell growth. Electron microscopy analysis revealed striking membrane perturbations and the accumulation of vesicles in arrested mutants. Using both biochemical fractionation and immunoelectron microscopy methods, these vesicles were shown to contain nups. We propose a model wherein a Ran-mediated vesicular fusion step is required for NPC assembly into intact nuclear envelopes.

MeSH Terms
Alleles Cycloheximide/pharmacology Cytoplasmic Vesicles/metabolism,ultrastructure Genes, Fungal Green Fluorescent Proteins Luminescent Proteins/metabolism Membrane Proteins/metabolism Mutation Nuclear Envelope/metabolism,ultrastructure Nuclear Pore/drug effects,genetics,metabolism,ultrastructure Nuclear Pore Complex Proteins/metabolism Protein Synthesis Inhibitors/pharmacology Recombinant Fusion Proteins/metabolism Saccharomyces cerevisiae/genetics,metabolism Saccharomyces cerevisiae Proteins/drug effects,genetics,metabolism Temperature ran GTP-Binding Protein/genetics,metabolism
Chemicals
Luminescent Proteins Membrane Proteins Nuclear Pore Complex Proteins Protein Synthesis Inhibitors Recombinant Fusion Proteins Saccharomyces cerevisiae Proteins Green Fluorescent Proteins Cycloheximide ran GTP-Binding Protein
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Ryan Kathryn J
Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
McCaffery J Michael
Wente Susan R
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.200209116
Published
2003-03-31
Epub
2003-00-24
Pages
1041-53
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2172763
Subset
IM
Grants
NIGMS NIH HHS · F32 GM020308 · United States
NIGMS NIH HHS · R01 GM057438 · United States
NIGMS NIH HHS · GM20308 · United States
NIGMS NIH HHS · R01 GM57438 · United States
External Links
PubMed source DOI: 10.1083/jcb.200209116 PMC Full Text View journal details
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