Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly.
SCF ubiquitin ligases are composed of Skp1, Cdc53, Hrt1 and one member of a large family of substrate receptors known as F-box proteins (FBPs). Here we report the identification, using sequential rounds of epitope tagging, affinity purification and mass spectrometry, of 16 Skp1 and Cdc53-associated proteins in budding yeast, including all components of SCF, 9 FBPs, Yjr033 (Rav1) and Ydr202 (Rav2). Rav1, Rav2 and Skp1 form a complex that we have named 'regulator of the (H+)-ATPase of the vacuolar and endosomal membranes' (RAVE), which associates with the V1 domain of the vacuolar membrane (H+)-ATPase (V-ATPase). V-ATPases are conserved throughout eukaryotes, and have been implicated in tumour metastasis and multidrug resistance, and here we show that RAVE promotes glucose-triggered assembly of the V-ATPase holoenzyme. Previous systematic genome-wide two-hybrid screens yielded 17 proteins that interact with Skp1 and Cdc53, only 3 of which overlap with those reported here. Thus, our results provide a distinct view of the interactions that link proteins into a comprehensive cellular network.
Division of Biology and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
J H
JH
Seol
A
A
Shevchenko
A
A
Shevchenko
R J
RJ
Deshaies
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D044842:Cullin Proteins; D003593:Cytoplasm / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D020035:Holoenzymes / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D025262:Vacuolar Proton-Translocating ATPases
C100988:Cdc53 protein, S cerevisiae; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D005656:Fungal Proteins; D020035:Holoenzymes; D011993:Recombinant Fusion Proteins; D044786:S-Phase Kinase-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D005947:Glucose