articles NATURE CELL BIOLOGY VOL 3 APRIL 2001 http://cellbio.nature.com 384 Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly Jae Hong Seol*, Anna Shevchenko†, Andrej Shevchenko†‡ and Raymond J. Deshaies*§ *Division of Biology and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA †Protein and Peptide Group, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69012 Heidelberg, Germany ‡e-mail: shevchenko@embl-heidelberg.de §e-mail: deshaies@its.caltech.edu 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, affini- ty 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 V 1 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 glu- cose-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. S CF ubiquitin ligases regulate a wide range of cellular process- es, including immunity, signalling, cell division and transcrip- tion 1 . SCF contains four subunits — Skp1, Cdc53 (also known as cullin 1 or Cul1), an F-box protein, and the newly discovered RING-H2 protein Hrt1 (also known as Roc1 or Rbx1; refs 2–5). A remarkable property of SCF ubiquitin ligases is that their specifici- ty can be programmed through an array of distinct substrate recep- tors 6,7 . These receptors are recruited to SCF by the conserved F-box Control Cdc53–Myc 9 Skp1–Myc 9 S4 S6 M r (K) M r (K) a b Control RCY1–Myc 9 YBR280–Myc 9 RAV1–Myc 9 RAV2–Myc 9 Control RCY1–Myc 9 YBR280–Myc 9 RAV1–Myc 9 RAV2–Myc 9 Control RCY1–Myc 9 YBR280–Myc 9 RAV1–Myc 9 RAV2–Myc 9 Anti-Skp1 Anti-Cdc53 Anti-Myc 250 98 64 50 36 30 C1 C2 C3 C4 C5 C6 250 98 64 50 36 30 16 6 S1 S2 S3 S5 S7 S8 S9 S10 S11 S12 S13 Figure 1 Identification of proteins associated with Cdc53 and Skp1. a, Proteins immunoprecipitated with an anti-Myc antibody (9E10) from 35 mg of wild- type, CDC53–Myc 9 and SKP1–Myc 9 cell extracts were eluted with SDS, separated in an 8–16% gradient SDS–polyacrylamide gel and visualized by silver staining. Specific bands present only in Cdc53–Myc 9 or Skp1–Myc 9 immunoprecipitates were identified by mass spectrometry. b, Extracts (6 mg) from untagged control, RCY1–Myc 9 , YBR280–Myc 9 , RAV1–Myc 9 and RAV2–Myc 9 cells were immunoprecipi- tated with 9E10, fractionated by SDS–PAGE, and immunoblotted with antibodies against Myc, Cdc53 or Skp1 as indicated. © 2001 Macmillan Magazines Ltd