The Ran GTPase cycle is required for yeast nuclear pore complex assembly.
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.
Ryan
Kathryn J
KJ
Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
McCaffery
J Michael
JM
Wente
Susan R
SR
D000483:Alleles; D003513:Cycloheximide / Q000494:pharmacology; D022162:Cytoplasmic Vesicles / Q000378:metabolism / Q000648:ultrastructure; D005800:Genes, Fungal; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism / Q000648:ultrastructure; D022022:Nuclear Pore / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D008164:Luminescent Proteins; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D003513:Cycloheximide; D020931:ran GTP-Binding Protein