Purification of NSP1 reveals complex formation with 'GLFG' nucleoporins and a novel nuclear pore protein NIC96

EMBO J. 1993 Aug;12(8):3061-71.

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Calcium-Binding Proteins*
  • Cell Division
  • Centrifugation
  • Chromatography, Affinity
  • Cloning, Molecular
  • DNA, Fungal
  • Electrophoresis, Polyacrylamide Gel
  • Epitopes
  • Fungal Proteins / genetics
  • Fungal Proteins / isolation & purification*
  • Fungal Proteins / metabolism*
  • Immunoglobulin G / immunology
  • Membrane Glycoproteins / isolation & purification*
  • Membrane Glycoproteins / metabolism
  • Membrane Proteins / isolation & purification*
  • Membrane Proteins / metabolism
  • Molecular Sequence Data
  • Nuclear Pore Complex Proteins
  • Nuclear Proteins / genetics
  • Nuclear Proteins / isolation & purification*
  • Nuclear Proteins / metabolism*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins*

Substances

  • Calcium-Binding Proteins
  • DNA, Fungal
  • Epitopes
  • Fungal Proteins
  • Immunoglobulin G
  • Membrane Glycoproteins
  • Membrane Proteins
  • NIC96 protein, S cerevisiae
  • NSP1 protein, S cerevisiae
  • Nuclear Pore Complex Proteins
  • Nuclear Proteins
  • Saccharomyces cerevisiae Proteins
  • nuclear pore protein p62

Associated data

  • GENBANK/X72923