A short linear motif, conserved from yeast to human, binds to members of the Spa2/GIT1 family of cortical scaffold proteins

Abstract

Tip growth is closely tied to fungal pathogenicity. Spa2, a multi-domain protein and member of the polarisome, orchestrates tip growth in yeast and other fungi. We identified a conserved short linear motif in the RabGAPs Msb3 and Msb4, and the MAP kinase kinase Ste7 and Mkk1, which mediates their interaction with Spa2. AlphaFold predictions suggest that these initially unstructured motifs adopt an alpha-helical conformation upon binding to the hydrophobic cleft of Spa2s N-terminal domain. Altering the predicted key contact residues in either Spa2 or the motif reduces complex stability. Such mutations also cause mis-localization of Msb3, Msb4, and Ste7 within the cell. Deleting the motif in Msb3 or Msb4 abolishes tip-directed growth of the yeast bud. Protein assemblies that spatially confine secretion to specific membrane regions are a common feature of eukaryotic cells. Accordingly, Spa2-motif complexes were predicted in orthologs and paralogs across selected Opisthokonta, including pathogenic fungi and humans. A search for functional motifs in conformationally flexible regions of all yeast proteins identified Dse3 as a novel Spa2-binding partner.