Identification of novel membrane-binding domains in multiple yeast Cdc42 effectors

Mol Biol Cell. 2007 Dec;18(12):4945-56. doi: 10.1091/mbc.e07-07-0676. Epub 2007 Oct 3.

Abstract

The Rho-type GTPase Cdc42 is a central regulator of eukaryotic cell polarity and signal transduction. In budding yeast, Cdc42 regulates polarity and mitogen-activated protein (MAP) kinase signaling in part through the PAK-family kinase Ste20. Activation of Ste20 requires a Cdc42/Rac interactive binding (CRIB) domain, which mediates its recruitment to membrane-associated Cdc42. Here, we identify a separate domain in Ste20 that interacts directly with membrane phospholipids and is critical for its function. This short region, termed the basic-rich (BR) domain, can target green fluorescent protein to the plasma membrane in vivo and binds PIP(2)-containing liposomes in vitro. Mutation of basic or hydrophobic residues in the BR domain abolishes polarized localization of Ste20 and its function in both MAP kinase-dependent and independent pathways. Thus, Cdc42 binding is required but is insufficient; instead, direct membrane binding by Ste20 is also required. Nevertheless, phospholipid specificity is not essential in vivo, because the BR domain can be replaced with several heterologous lipid-binding domains of varying lipid preferences. We also identify functionally important BR domains in two other yeast Cdc42 effectors, Gic1 and Gic2, suggesting that cooperation between protein-protein and protein-membrane interactions is a prevalent mechanism during Cdc42-regulated signaling and perhaps for other dynamic localization events at the cell cortex.

Publication types

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

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Binding Sites
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Membrane / metabolism*
  • Hydrophobic and Hydrophilic Interactions
  • Intracellular Signaling Peptides and Proteins
  • MAP Kinase Kinase Kinases
  • Molecular Sequence Data
  • Protein Binding
  • Protein Serine-Threonine Kinases / chemistry
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • cdc42 GTP-Binding Protein / genetics
  • cdc42 GTP-Binding Protein / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • GIC1 protein, S cerevisiae
  • GIC2 protein, S cerevisiae
  • Intracellular Signaling Peptides and Proteins
  • Saccharomyces cerevisiae Proteins
  • Protein Serine-Threonine Kinases
  • MAP Kinase Kinase Kinases
  • STE20 protein, S cerevisiae
  • cdc42 GTP-Binding Protein