title: Yeast Rsp5 ubiquitin ligase affects the actin cytoskeleton in vivo and in vitro.
abstract: Yeast Rsp5 ubiquitin ligase is involved in several cellular processes, including endocytosis. Actin patches are sites of endocytosis, a process involving actin assembly and disassembly. Here we show Rsp5 localization in cortical patches and demonstrate its involvement in actin cytoskeleton organization and dynamics. We found that the Rsp5-F1-GFP2 N-terminal fragment and full length GFP-Rsp5 were recruited to peripheral patches that temporarily co-localized with Abp1-mCherry, a marker of actin patches. Actin cytoskeleton organization was defective in a strain lacking RSP5 or overexpressing RSP5, and this phenotype was accompanied by morphological abnormalities. Overexpression of RSP5 caused hypersensitivity of cells to Latrunculin A, an actin-depolymerizing drug and was toxic to cells lacking Las17, an activator of actin nucleation. Moreover, Rsp5 was required for efficient actin polymerization in a whole cell extract based in vitro system. Rsp5 interacted with Las17 and Las17-binding proteins, Lsb1 and Lsb2, in a GST-Rsp5-WW2/3 pull down assay. Rsp5 ubiquitinated Lsb1-HA and Lsb2-HA without directing them for degradation. Overexpression of RSP5 increased the cellular level of HA-Las17 in wild type and in lsb1Δ lsb2Δ strains in which the basal level of Las17 was already elevated. This increase was prevented in a strain devoid of Las17-binding protein Sla1 which is also a target of Rsp5 ubiquitination. Thus, Rsp5 together with Lsb1, Lsb2 and Sla1 regulate the level of Las17, an important activator of actin polymerization.
authors: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.
authors: Joanna
authors: J
authors: Kaminska
authors: Matthias
authors: M
authors: Spiess
authors: Marta
authors: M
authors: Stawiecka-Mirota
authors: Rasa
authors: R
authors: Monkaityte
authors: Rosine
authors: R
authors: Haguenauer-Tsapis
authors: Daniele
authors: D
authors: Urban-Grimal
authors: Barbara
authors: B
authors: Winsor
authors: Teresa
authors: T
authors: Zoladek
mesh_terms: D008841:Actin Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000201:enzymology / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*; D051316:Wiskott-Aldrich Syndrome Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; C480361:LAS17 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D051316:Wiskott-Aldrich Syndrome Protein; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae