Yeast pericentrin/Spc110 contains multiple domains required for tethering the γ-tubulin complex to the centrosome

Mol Biol Cell. 2020 Jul 1;31(14):1437-1452. doi: 10.1091/mbc.E20-02-0146. Epub 2020 May 6.

Abstract

The Saccharomyces cerevisiae spindle pole body (SPB) serves as the sole microtubule-organizing center of the cell, nucleating both cytoplasmic and nuclear microtubules. Yeast pericentrin, Spc110, binds to and activates the γ-tubulin complex via its N terminus, allowing nuclear microtubule polymerization to occur. The Spc110 C terminus links the γ-tubulin complex to the central plaque of the SPB by binding to Spc42, Spc29, and calmodulin (Cmd1). Here, we show that overexpression of the C terminus of Spc110 is toxic to cells and correlates with its localization to the SPB. Spc110 domains that are required for SPB localization and toxicity include its Spc42-, Spc29-, and Cmd1-binding sites. Overexpression of the Spc110 C terminus induces SPB defects and disrupts microtubule organization in both cycling and G2/M arrested cells. Notably, the two mitotic SPBs are affected in an asymmetric manner such that one SPB appears to be pulled away from the nucleus toward the cortex but remains attached via a thread of nuclear envelope. This SPB also contains relatively fewer microtubules and less endogenous Spc110. Our data suggest that overexpression of the Spc110 C terminus acts as a dominant-negative mutant that titrates endogenous Spc110 from the SPB causing spindle defects.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antigens / genetics
  • Antigens / metabolism
  • Binding Sites
  • Calmodulin / metabolism
  • Calmodulin-Binding Proteins / genetics
  • Calmodulin-Binding Proteins / metabolism*
  • Cell Nucleus / metabolism
  • Centrosome / metabolism*
  • Centrosome / physiology
  • Cytoskeletal Proteins / genetics
  • Cytoskeletal Proteins / metabolism*
  • Microtubule-Associated Proteins / metabolism
  • Microtubule-Organizing Center / metabolism
  • Microtubules / metabolism
  • Nuclear Proteins / metabolism
  • Phosphoproteins / metabolism
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Spindle Apparatus / metabolism
  • Spindle Pole Bodies / metabolism
  • Tubulin / metabolism*
  • Tubulin / physiology

Substances

  • Antigens
  • CMD1 protein, S cerevisiae
  • Calmodulin
  • Calmodulin-Binding Proteins
  • Cytoskeletal Proteins
  • Microtubule-Associated Proteins
  • Nuclear Proteins
  • Phosphoproteins
  • SPC110 protein, S cerevisiae
  • SPC42 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Spc29 protein, S cerevisiae
  • Tubulin
  • pericentrin