Stimulation of actin polymerization by vacuoles via Cdc42p-dependent signaling.
We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction.
Isgandarova
Sabina
S
Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Jones
Lynden
L
Forsberg
Daniel
D
Loncar
Ana
A
Dawson
John
J
Tedrick
Kelly
K
Eitzen
Gary
G
D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D021961:Cell Membrane Structures / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000494:pharmacology; D017353:Gene Deletion; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011721:Pyrenes / Q000494:pharmacology; D050683:R-SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014617:Vacuoles / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D005456:Fluorescent Dyes; D008840:Microfilament Proteins; D011721:Pyrenes; D050683:R-SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; C090350:VRP1 protein, S cerevisiae; C107127:YKT6 protein, S cerevisiae; D000255:Adenosine Triphosphate; C030984:pyrene; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae