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Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly

Nature Cell Biology volume 3, pages 384–391 (2001)Cite this article

Abstract

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.

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Figure 1: Identification of proteins associated with Cdc53 and Skp1.
Figure 2: Rav1, Rav2 and Skp1 form RAVE, which binds to V-ATPase.
Figure 3: Topology of the RAVE complex.
Figure 4: RAVE mutants exhibit V-ATPase deficiency.
Figure 5: Cytoplasmic RAVE promotes glucose-regulated assembly of V1 with V0 to form the V-ATPase holoenzyme.
Figure 6: Network of protein interactions involving Cdc53 and Skp1.

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Acknowledgements

We thank K. Hoffman for pointing out the F-box homologies in Ybr280 and Ymr258. We also thank D. Klionsky for antibodies against Vma1, Vma2, Vma4 and Vma8, P. Kane for monoclonal anti-Vph1 antibodies, T. Stevens for vph1Δ (KHY31) strains and polyclonal anti-Vph1 antibodies, T-M. Yi for constructing the GFP-tagging cassette, and members of the Deshaies laboratory for comments on the manuscript. This work was supported by grants from the National Institutes of Health and the W. M. Keck Foundation. J. H. S. was supported by a fellowship from the Leukemia and Lymphoma Society and by the Howard Hughes Medical Institute.

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  1. Division of Biology and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, 91125, California, USA

    Jae Hong Seol & Raymond J. Deshaies

  2. Protein and Peptide Group, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, 69012, Germany

    Anna Shevchenko & Andrej Shevchenko

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Correspondence to Andrej Shevchenko or Raymond J. Deshaies.

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Seol, J., Shevchenko, A., Shevchenko, A. et al. Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly. Nat Cell Biol 3, 384–391 (2001). https://doi.org/10.1038/35070067

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