Verprolin function in endocytosis and actin organization. Roles of the Las17p (yeast WASP)-binding domain and a novel C-terminal actin-binding domain.
Vrp1p (verprolin, End5p) is the yeast ortholog of human Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Vrp1p localizes to the cortical actin cytoskeleton, is necessary for its polarization to sites of growth and is also essential for endocytosis. At elevated temperature, Vrp1p becomes essential for growth. A C-terminal Vrp1p fragment (C-Vrp1p) retains the ability to localize to the cortical actin cytoskeleton and function in actin-cytoskeleton polarization, endocytosis and growth. Here, we demonstrate that two submodules in C-Vrp1p are required for actin-cytoskeleton polarization: a novel C-terminal actin-binding submodule (CABS) that contains a novel G-actin-binding domain, which we call a verprolin homology 2 C-terminal (VH2-C) domain; and a second submodule comprising the Las17p-binding domain (LBD) that binds Las17p (yeast WASP). The LBD localizes C-Vrp1p to membranes and the cortical actin cytoskeleton. Intriguingly, the LBD is sufficient to restore endocytosis and growth at elevated temperature to Vrp1p-deficient cells. The CABS also restores these functions, but only if modified by a lipid anchor to provide membrane association. Our findings highlight the role of Las17p binding for Vrp1p membrane association, suggest general membrane association may be more important than specific targeting to the cortical actin cytoskeleton for Vrp1p function in endocytosis and cell growth, and suggest that Vrp1p binding to individual effectors may alter their physiological activity.
Thanabalu
Thirumaran
T
Institute of Molecular and Cell Biology, A*STAR Biomedical Science Institutes, Singapore.
Rajmohan
Rajamuthiah
R
Meng
Lei
L
Ren
Gang
G
Vajjhala
Parimala R
PR
Munn
Alan L
AL
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D004705:Endocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013696:Temperature; D051316:Wiskott-Aldrich Syndrome Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D000199:Actins; C480361:LAS17 protein, S cerevisiae; D008840:Microfilament Proteins; D029701:Saccharomyces cerevisiae Proteins; C090350:VRP1 protein, S cerevisiae; D051316:Wiskott-Aldrich Syndrome Protein; D049452:Green Fluorescent Proteins; D001120:Arginine; D008239:Lysine