Figure 1

Disruption, but not replacement, of the S73/S97/loop motif conserved among the Cdc34/Ubc7 E2 family is lethal for yeast. (A). Partial sequence alignment. Asterisks represent residues identical to Cdc34 and dashes represent gaps. Sc - S. cerevisiae; Oc - O. cuniculus; Dm - D. melanogaster; Ce - C. elegans; At - A. thaliana; Hs - H. sapiens; ASFV1 - African swine fever virus (GI:9628248); ASFV2 -African swine fever virus (GI:450743). (B). Structural models of the E2 core (a.a. 1-170) of Cdc34 and ^tmCdc34. Residues corresponding to K73 and D97 in ^tmCdc34, and S73 and S97 in Cdc34, are color coded as in A and shown in the context of structures of a scRad6 and scUbc7 fragment (Methods); navy blue: the acidic loop formed by scUbc7 residues corresponding to amino acids 103-114 in Cdc34; red: the residue corresponding to the catalytic site C95 of Cdc34 and ^tmCdc34. (C). Model of ubiquitin-charged Cdc34 (a.a. 1-170) bound to the RING domain of Rbx1. See Methods. (D). Scheme of Cdc34 domains of interest. (E). Rescue of cdc34-2ts yeast growth with Cdc34 E2 core mutant constructs. Cultures of cdc34-2ts strain carrying the indicated constructs under the GAL10 promoter on a 2μ YEp51 plasmid were grown overnight at 27°C in SD-Leu, adjusted to a density of 1 × 10⁸ cells/ml, serially diluted, spotted onto SD-Leu plates and incubated at permissive (27°C) or non-permissive (37°C) temperature for 4 days. Note that dextrose allows only low expression of the GAL10 controlled constructs.