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. Author manuscript; available in PMC: 2013 Jan 19.
Published in final edited form as: Nature. 2011 Dec 21;481(7381):371–375. doi: 10.1038/nature10693

Figure 2. CBF-β is a stoichiometric component of the Vif E3 ubiquitin ligase.

Figure 2

a, Size exclusion chromatography of recombinant purifiedCUL5/RBX2 (blue) overlaid with CUL5/RBX2 mixed with 1.5 equivalents of purified Vif substrate adaptor containing Vif, ELOBC and CBF-β (red). b, Coomassie-stained SDS–PAGE of fractions labelled 1–3 in a indicating the Vif substrate adaptor and a six-protein assembly (CRL5–Vif–CBF-β) co-purify as stable monodisperse species. c, A3G, but not A3A, directly binds the tetrameric Vif substrate adaptor in pull-down experiments in vitro. d, CRL5–Vif–CBF-β is an E3 ligase that promotes polyubiquitination of A3G, but not A3A (detected using an anti-c-Myc antibody to the C-terminal tag on the deaminases). Ub, ubiquitin. e, CRL5–Vif–CBF-β and UBE2R1 catalyse formation of K48-linked chains on A3G. Immunoblots showing substrate in ubiquitination reactions containing UBE2R1 as E2, no ubiquitin, Me-ubiquitin, K48R-ubiquitin, K48-only ubiquitin or wild-type ubiquitin. Reactions with Me-ubiquitin indicate at least two distinct sites are modified on A3G; K48R recapitulates the pattern observed with Me-ubiquitin, whereas both wild type and K48R-only ubiquitin result in extensive polyubiquitin chains.