Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Base Sequence
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Calcium-Binding Proteins*
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Cell Division
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Centrifugation
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Chromatography, Affinity
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Cloning, Molecular
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DNA, Fungal
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Electrophoresis, Polyacrylamide Gel
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Epitopes
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Fungal Proteins / genetics
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Fungal Proteins / isolation & purification*
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Fungal Proteins / metabolism*
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Immunoglobulin G / immunology
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Membrane Glycoproteins / isolation & purification*
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Membrane Glycoproteins / metabolism
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Membrane Proteins / isolation & purification*
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Membrane Proteins / metabolism
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Molecular Sequence Data
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Nuclear Pore Complex Proteins
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Nuclear Proteins / genetics
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Nuclear Proteins / isolation & purification*
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Nuclear Proteins / metabolism*
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins*
Substances
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Calcium-Binding Proteins
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DNA, Fungal
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Epitopes
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Fungal Proteins
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Immunoglobulin G
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Membrane Glycoproteins
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Membrane Proteins
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NIC96 protein, S cerevisiae
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NSP1 protein, S cerevisiae
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Nuclear Pore Complex Proteins
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Nuclear Proteins
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Saccharomyces cerevisiae Proteins
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nuclear pore protein p62