Purification of NSP1 reveals complex formation with 'GLFG' nucleoporins and a novel nuclear pore protein NIC96.
The essential C-terminal domain of NSP1 mediates assembly into the nuclear pore complex (NPC). To identify components which interact physically with this yeast nucleoporin, the tagged C-terminal domain of NSP1 (ProtA-NSP1) was isolated by affinity chromatography under non-denaturing conditions. The purified complex contains ProtA-NSP1, two previously identified 'GLFG' nucleoporins, NUP49 (NSP49) and p54 and a novel protein designated NIC96 (for Nucleoporin-Interacting Component of 96 kDa). Conversely, affinity purification of tagged NSP49 enriches for NSP1, the p54 and the NIC96 component. The NIC96 gene was cloned; it encodes a novel 839 amino acid protein essential for cell growth. By immunofluorescence, protein A-tagged NIC96 exhibits a punctate nuclear membrane staining indicative of nuclear pore location. Therefore, affinity purification of tagged nucleoporins has allowed the definition of a subcomplex of the NPC and analysis of physical interactions between nuclear pore proteins.
Grandi
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P
EMBL, Heidelberg, Germany.
Doye
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V
Hurt
E C
EC
D000595:Amino Acid Sequence; D001483:Base Sequence; D002135:Calcium-Binding Proteins; D002455:Cell Division; D002498:Centrifugation; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007074:Immunoglobulin G / Q000276:immunology; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D002135:Calcium-Binding Proteins; D004271:DNA, Fungal; D000939:Epitopes; D005656:Fungal Proteins; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C082533:NIC96 protein, S cerevisiae; C062748:NSP1 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C049312:nuclear pore protein p62