ABSTRACT
Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. Here we show that p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the p97-Ufd1-Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a p97-p47 complex. This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation.
Subject(s)
Adenosine Triphosphatases/metabolism , Membrane Fusion/physiology , Nuclear Envelope/enzymology , Nuclear Pore Complex Proteins , Nuclear Proteins/metabolism , Animals , Carrier Proteins/metabolism , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Nucleocytoplasmic Transport Proteins , Oocytes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Vesicular Transport Proteins , XenopusABSTRACT
Nuclear formation in Xenopus egg extracts requires cytosol and is inhibited by GTP gamma S, indicating a requirement for GTPase activity. Nuclear envelope (NE) vesicle fusion is extensively inhibited by GTP gamma S and two mutant forms of the Ran GTPase, Q69L and T24N. Depletion of either Ran or RCC1, the exchange factor for Ran, from the assembly reaction also inhibits this step of NE formation. Ran depletion can be complemented by the addition of Ran loaded with either GTP or GDP but not with GTP gamma S. RCC1 depletion is only complemented by RCC1 itself or by RanGTP. Thus, generation of RanGTP by RCC1 and GTP hydrolysis by Ran are both required for the extensive membrane fusion events that lead to NE formation.
Subject(s)
Cell Cycle Proteins , Guanine Nucleotide Exchange Factors , Guanosine Triphosphate/metabolism , Nuclear Envelope/metabolism , Nuclear Proteins , Xenopus laevis , ran GTP-Binding Protein/metabolism , Amino Acid Substitution , Animals , Chromatin/chemistry , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Female , Fluorescent Antibody Technique , Fluorescent Dyes , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/pharmacology , Hydrolysis/drug effects , Male , Membrane Fusion/drug effects , Mutation , Nuclear Envelope/drug effects , Oocytes/cytology , Oocytes/metabolism , Solubility , Sperm Head/metabolism , Xenopus Proteins , ran GTP-Binding Protein/geneticsABSTRACT
The characterization of a novel yeast-splicing factor, Luc7p, is presented. The LUC7 gene was identified by a mutation that causes lethality in a yeast strain lacking the nuclear cap-binding complex (CBC). Luc7p is similar in sequence to metazoan proteins that have arginine-serine and arginine-glutamic acid repeat sequences characteristic of a family of splicing factors. We show that Luc7p is a component of yeast U1 snRNP and is essential for vegetative growth. The composition of yeast U1 snRNP is altered in luc7 mutant strains. Extracts of these strains are unable to support any of the defined steps of splicing unless recombinant Luc7p is added. Although the in vivo defect in splicing wild-type reporter introns in a luc7 mutant strain is comparatively mild, splicing of introns with nonconsensus 5' splice site or branchpoint sequences is more defective in the mutant strain than in wild-type strains. By use of reporters that have two competing 5' splice sites, a loss of efficient splicing to the cap proximal splice site is observed in luc7 cells, analogous to the defect seen in strains lacking CBC. CBC can be coprecipitated with U1 snRNP from wild-type, but not from luc7, yeast strains. These data suggest that the loss of Luc7p disrupts U1 snRNP-CBC interaction, and that this interaction contributes to normal 5' splice site recognition.