Components of multiprotein complexes are routinely determined by using proteomic approaches. However, this information lacks functional content except when new complex members are identified. To analyze quantitatively the abundance of proteins in human Mediator we used normalized spectral abundance factors generated from shotgun proteomics data sets. With this approach we define a common core of mammalian Mediator subunits shared by alternative forms that variably associate with the kinase module and RNA polymerase (pol) II. Although each version of affinitypurified Mediator contained some kinase module and RNA pol II, Mediator purified through F-Med26 contained the most RNA pol II and the least kinase module as demonstrated by the normalized spectral abundance factor approach. The distinct forms of Mediator were functionally characterized by using a transcriptional activity assay, where F-Med26 Mediator/RNA pol II was the most active. This method of protein complex visualization has important implications for the analysis of multiprotein complexes and assembly of protein interaction networks. multidimensional protein identification technology ͉ proteomics ͉ spectrum counting ͉ mass spectrometry S ince its discovery in yeast (1, 2) and subsequent isolation and initial characterizations in human cells (3), the transcriptional coactivator complex Mediator has been the subject of numerous studies to characterize both its composition and function. Researchers have used a combination of immunoprecipitation (3-14), ion exchange/size-exclusion chromatography (8, 10, 15-19), glycerol gradient (6, 7, 12, 13, 19, 20), SDS/PAGE/ silver stain/Western blot analysis (6,7,9,10,15,18,21), MS (9,16,19,21,22), and ChIP (21,23) to determine the composition of Mediator complexes. Varying in size and subunit composition, the larger complexes (thyroid hormone receptor-associated protein complex, SRB/Med-containing cofactor complex, negative regulator of activated transcription, vitamin D receptor interacting protein complex, and activator recruited cofactor) ranged from 1 to 2 MDa and were composed of Ϸ30 subunits (3, 9-16, 21, 22, 24), whereas the smaller complexes (cofactor required for Sp1, positive cofactor 2, and positive cofactor 4) ranged from Ϸ500 to 700 kDa and were composed of Ϸ9-17 subunits (5-7, 17-20, 25, 26). The main shared difference between large and small complexes appears to be the presence or absence of the kinase module. Furthermore, different complexes possessed different levels of basal transcription, which could be accounted for by several possibilities, including purification method (high ionic strength may have washed away necessary proteins), the adding back of RNA polymerase (pol) II and basal transcription factors, and testing only for activated transcription.Although these studies form the foundation for our present understanding of Mediator, the relationship between different forms of Mediator was unclear and the composition of each form was unclear. The first attempt at standardizing Mediator complex...

The Elongin BC complex was identified initially as a positive regulator of RNA polymerase II (Pol II) elongation factor Elongin A and subsequently as a component of the multiprotein von Hippel-Lindau (VHL) tumor suppressor complex, in which it participates in both tumor suppression and negative regulation of hypoxia-inducible genes. Elongin B is a ubiquitin-like protein, and Elongin C is a Skp1-like protein that binds to a BC-box motif that is present in both Elongin A and VHL and is distinct from the conserved F-box motif recognized by Skp1. In this report, we demonstrate that the Elongin BC complex also binds to a functional BC box present in the SOCS box, a sequence motif identified recently in the suppressor of cytokine signaling-1 (SOCS-1) protein, as well as in a collection of additional proteins belonging to the SOCS, ras, WD-40 repeat, SPRY domain, and ankyrin repeat families. In addition, we present evidence (1) that the Elongin BC complex is a component of a multiprotein SOCS-1 complex that attenuates Jak/STAT signaling by binding to Jak2 and inhibiting Jak2 kinase, and (2) that by interacting with the SOCS box, the Elongin BC complex can increase expression of the SOCS-1 protein by inhibiting its degradation. These results suggest that Elongin BC is a multifunctional regulatory complex capable of controlling multiple pathways in the cell through interaction with a short degenerate sequence motif found in many different proteins.

Mutations in the VHL tumor suppressor gene result in constitutive expression of many hypoxia-inducible genes, at least in part because of increases in the cellular level of hypoxia-inducible transcription factor HIF1␣, which in normal cells is rapidly ubiquitinated and degraded by the proteasome under normoxic conditions. The recent observation that the VHL protein is a subunit of an Skp1-Cul1͞Cdc53-F-box (SCF)-like E3 ubiquitin ligase raised the possibility that VHL may be directly responsible for regulating cellular levels of HIF1␣ by targeting it for ubiquitination and proteolysis. In this report, we test this hypothesis directly. We report development of methods for production of the purified recombinant VHL complex and present direct biochemical evidence that it can function with an E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme to activate HIF1␣ ubiquitination in vitro. Our findings provide new insight into the function of the VHL tumor suppressor protein, and they provide a foundation for future investigations of the mechanisms underlying VHL regulation of oxygen-dependent gene expression.