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Published online 24 March 2003. doi:10.1083/jcb.200209116
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© The Rockefeller University Press, 0021-9525/2003/3/1041 $5.00
The Journal of Cell Biology, Volume 160, Number 7, 1041-1053

Article

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



Kathryn J. Ryan1, J. Michael McCaffery2 and Susan R. Wente1

1 Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232
2 Integrated Imaging Center, Department of Biology, The Johns Hopkins University, Baltimore, MD 21218

Address correspondence to Susan R. Wente, Dept. of Cell and Developmental Biology, Vanderbilt University Medical Center, 3120 A Medical Research Building III, 465 21st Ave. South, Nashville, TN 37232-8240. Tel.: (615) 936-3443. Fax: (615) 936-3439. E-mail: susan.wente{at}vanderbilt.edu

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.

Key Words: Ran; vesicular trafficking; nucleoporin; NPC; nuclear envelope

* Abbreviations used in this paper: GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; NE, nuclear envelope; npa, nuclear pore complex assembly mutant; NPC, nuclear pore complex; nup, nucleoporin; ts, temperature-sensitive.


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