Utilizing the Dyn2 dimerization-zipper as a tool to probe NPC structure and function

Methods Cell Biol. 2014:122:99-115. doi: 10.1016/B978-0-12-417160-2.00005-9.

Abstract

The discovery of dynein light chain 2 (Dyn2) as a member of the nucleoporins in yeast led to a series of applications to study NPC structure and function. Its intriguing ability to act as a hub for the parallel dimerization of two short amino acid sequence motifs (DID) prompted us to utilize it as a tool for probing nucleocytoplasmic transport in vivo. Further, the distinct structure of the Dyn2-DID rod, which is easily visible in the electron microscope, allowed us to develop a precise structural label on proteins or protein complexes. This label was used to identify the position of subunits in NPC subcomplexes or to derive at pseudo-atomic models of single large Nups. The versatility for various applications of the DID-Dyn2 system makes it an attractive molecular tool beyond the nuclear pore and transport field.

Keywords: DID; Dynein2; Electron microscopy; FG repeats; Label; NPC; Negative stain; Transport channel.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • Dyneins / metabolism*
  • Molecular Sequence Data
  • Nuclear Pore / metabolism
  • Nuclear Pore / ultrastructure*
  • Nuclear Pore Complex Proteins / metabolism
  • Nuclear Pore Complex Proteins / ultrastructure*
  • Protein Binding
  • Protein Multimerization
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Nuclear Pore Complex Proteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • intermediate chain 1 protein, S cerevisiae
  • Dyneins
  • SLC1 protein, S cerevisiae