Interdependence of the Ypt/RabGAP Gyp5p and Gyl1p for recruitment to the sites of polarized growth.
Gyp5p and Gyl1p are two members of the Ypt/Rab guanosine triphosphatases-activating proteins involved in the control of polarized exocytosis in Saccharomyces cerevisiae. We had previously shown that Gyp5p and Gyl1p colocalize at the sites of polarized growth and belong to the same complex in subcellular fractions enriched in plasma membrane or secretory vesicles. Here, we investigate the interaction between Gyp5p and Gyl1p as well as the mechanism of their localization to the sites of polarized growth. We show that purified recombinant Gyp5p and Gyl1p interact directly in vitro. In vivo, both Gyp5p and Gyl1p are mutually required to concentrate at the sites of polarized growth. Moreover, the localization of Gyp5p and Gyl1p to the sites of polarized growth requires the formins Bni1p and Bnr1p and depends on actin cables. We show that, in a sec6-4 mutant, blocking secretion leads to coaccumulation of Gyp5p and Gyl1p, together with Sec4p. Electron microscopy experiments demonstrate that Gyp5p is associated with secretory vesicles. Altogether, our results indicate that both Gyp5p and Gyl1p access the sites of polarized growth by transport on secretory vesicles. Two polarisome components, Spa2p and Bud6p, are involved in maintaining Gyp5p and Gyl1p colocalized at the sites of polarized growth.
Chesneau
Laurent
L
Institut de Génétique et Microbiologie, Université Paris Sud 11, CNRS UMR8621, Bâtiment 400, F-91405 Orsay Cedex, France.
Prigent
Magali
M
Boy-Marcotte
Emmanuelle
E
Daraspe
Jean
J
Fortier
Guillaume
G
Jacquet
Michel
M
Verbavatz
Jean-Marc
JM
Cuif
Marie-Hélène
MH
D000199:Actins / Q000378:metabolism; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D048708:Cell Growth Processes; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism / Q000648:ultrastructure; D053778:Thiazolidines / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; C106463:BNR1 protein, S cerevisiae; C107727:BUD6 protein, S cerevisiae; C105191:Bni1 protein, S cerevisiae; D019086:Bridged Bicyclo Compounds, Heterocyclic; C099502:CDC11 protein, S cerevisiae; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D020690:GTPase-Activating Proteins; C504621:Gyl1 protein, S cerevisiae; C504620:Gyp5 protein, S cerevisiae; D008840:Microfilament Proteins; D011994:Recombinant Proteins; C065889:SPA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D053778:Thiazolidines; C064320:SEC4 protein, S cerevisiae; D020691:rab GTP-Binding Proteins; C037067:latrunculin A