Vrp1p–Las17p interaction is critical for actin patch polarization but is not essential for growth or fluid phase endocytosis in S. cerevisiae

Vrp1p (yeast WIP) forms a protein complex with Las17p (yeast WASP), however the physiological significance of the interaction has not been fully characterized. Vrp1p residues, 788MPKPR792 are essential for Vrp1p–Las17p interaction. While C-Vrp1p364–817 complements all the defects of the vrp1Δ strain, C-Vrp1p364–8175A (788AAAAA792) does not complement any of the defects, due to its inability to localize to cortical patches. Targeting C-Vrp1p364–8175A to membranes using CAAX motif (C-Vrp1p364–8175A-CAAX) rescued the growth and endocytosis defect but not the actin patch polarization defect of vrp1Δ. Vrp1p can localize to cortical patches, either by binding to Las17p through LBD (Las17 Binding Domain, Vrp1p760–817) or independent of Las17p through residues in N-Vrp1p1–364. Unlike Vrp1p, Vrp1p5A localizes poorly to cortical patches and complements all the defects of vrp1Δ strain except actin patch polarization at elevated temperature. N-Vrp1p1–364 complements all the defects of vrp1Δ strain except the actin patch polarization defect while N-Vrp1p1–364–LBD fusion protein complements all the defects. Thus our results show that while both Vrp1p and Las17p are essential for many cellular processes, the two proteins do not necessarily have to bind to each other to carry out these cellular functions. However, Las17p–Vrp1p interaction is essential for actin patch polarization at elevated temperature.

Research Highlights
► Vrp1p residues 787–797 are essential for Vrp1p–Las17p interaction.
► The motif 788MPKPR792 is essential for the direct interaction between Vrp1p and Las17p.
► Fusion of CAAX motif to C-Vrp1p364–8175A mutant can restore its activity in growth and endocytosis.
► N-Vrp1p1–364 fused to LBD complements the actin patch polarization defect of vrp1Δ strain.