Opposite roles of the F-box protein Rcy1p and the GTPase-activating protein Gyp2p during recycling of internalized proteins in yeast.
The F-box protein Rcy1p is part of a non-SCF (Skp1p-cullin-F-box protein) complex involved in recycling of internalized material. Like rcy1Delta, cells lacking the Rab-GTPase Ypt6p or its heterodimeric GEFs Rgp1p and Ric1p are unable to recycle the v-SNARE Snc1p. Here we provide genetic evidence suggesting that Rcy1p is a positive regulator of Ypt6p. Deletion of the GAP Gyp2p restores recycling in rcy1Delta, while overexpression of an active form of Ypt6p partially suppresses the recycling defect of rcy1Delta cells. Conversely, overexpression of Gyp2p in wild-type cells interferes with recycling of GFP-Snc1p, and the cells accumulate membrane structures as evidenced by electron microscopy. Gyp2p-GFP is distributed throughout the cytoplasm and accumulates in punctate structures, which concentrate in an actin-dependent manner at sites of polarized growth. Taken together, our results suggest that the F-box protein Rcy1p may activate the Ypt6p GTPase module during recycling.
Lafourcade
Céline
C
Swiss Federal Institute of Technology Zurich (ETH), Institute of Biochemistry, Switzerland.
Galan
Jean-Marc
JM
Peter
Matthias
M
D000199:Actins / Q000378:metabolism; D001692:Biological Transport; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004251:DNA Transposable Elements; D019281:Dimerization; D044783:F-Box Proteins; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000502:physiology*; D017353:Gene Deletion; D005838:Genotype; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008854:Microscopy, Electron; D008957:Models, Genetic; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D050683:R-SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology; D013696:Temperature; D013997:Time Factors; D014157:Transcription Factors / Q000502:physiology; D033921:Vesicular Transport Proteins
D000199:Actins; D004251:DNA Transposable Elements; D044783:F-Box Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; C482449:Gyp2 protein, S cerevisiae; D008164:Luminescent Proteins; D008565:Membrane Proteins; D050683:R-SNARE Proteins; C407703:RCY1 protein, S cerevisiae; C421763:RGP1 protein, S cerevisiae; C105592:RIC1 protein, S cerevisiae; C083241:SNC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D020559:Monomeric GTP-Binding Proteins