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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| Gene/Complex | Qualifier | Gene Ontology Term | Annotation Extension | Evidence | Source | Assigned On |
|---|---|---|---|---|---|---|
| SAF1 | part of | SCF ubiquitin ligase complex | IDA | SGD | 2013-08-07 | |
| RAV2 | involved in | vacuolar acidification | IMP | SGD | 2013-08-07 | |
| RAV2 | involved in | vacuolar acidification | IGI with RAV1 | SGD | 2013-08-07 | |
| RAV2 | involved in | regulation of protein-containing complex assembly | IPI with SKP1 | SGD | 2013-08-07 | |
| RAV2 | involved in | regulation of protein-containing complex assembly | IPI with RAV1 | SGD | 2013-08-07 | |
| RAV2 | involved in | regulation of protein-containing complex assembly | IGI with RAV1 | SGD | 2013-08-07 | |
| RAV2 | part of | RAVE complex | IPI with SKP1 | SGD | 2013-08-07 | |
| RAV2 | part of | RAVE complex | IPI with RAV1 | SGD | 2013-08-07 | |
| SKP1 | part of | RAVE complex | IPI with RAV1 | SGD | 2013-08-07 | |
| SKP1 | part of | RAVE complex | IPI with RAV2 | SGD | 2013-08-07 |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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| Interactor | Interactor | Assay | Annotation | Action | Modification | |
|---|---|---|---|---|---|---|
| CDC4 | SKP1 | Two-hybrid | manually curated | Hit-Bait | No Modification | |
| CDC53 | HRT1 | Affinity Capture-Western | manually curated | Bait-Hit | No Modification | |
| CDC53 | HRT1 | Affinity Capture-Western | manually curated | Hit-Bait | No Modification | |
| CDC53 | SKP1 | Affinity Capture-Western | manually curated | Hit-Bait | No Modification | |
| CDC53 | ERG27 | Two-hybrid | manually curated | Bait-Hit | No Modification | |
| CDC53 | SKP1 | Two-hybrid | manually curated | Bait-Hit | No Modification | |
| CDC53 | ZIP2 | Two-hybrid | manually curated | Bait-Hit | No Modification | |
| CDC53 | LUG1 | Affinity Capture-MS | manually curated | Bait-Hit | No Modification | |
| CDC53 | DAS1 | Affinity Capture-MS | manually curated | Bait-Hit | No Modification | |
| CDC53 | UFO1 | Affinity Capture-MS | manually curated | Bait-Hit | No Modification |