Fig. 2.

Lysineless (K0) Ub penetrates into the conjugated Ub pool increasing mono/poly Ub ratio. A, WT yeast cells were transformed with plasmids for expression of either RGS-His₈ tagged lysineless Ub (K0 Ub) or with tagged WT Ub as control. Rapidly lysed whole cell extracts were resolved by 18% SDS-PAGE, transferred and immunoblotted with anti RGS-His antibodies (left panel) or with anti Ub antibodies (right panel). B–D, Equal amounts of cells expressing WT Ub, K0 Ub, or empty vector (serving as background strain) were lysed and whole-cell extracts resolved by gradient SDS-PAGE. The high MW region was excised and subjected to Ub-AQUA analysis as described in Fig. 1. B, As in Fig. 1, expression of WT Ub from a plasmid did not significantly alter total conjugates relative to background strain (empty vector). Ubiquitin levels in both strains were normalized using common internal peptide standards. K0 Ub expression resulted in a roughly 50% increase in total ubiquitin conjugates. C, Increase in mono/end cap modifications accounts for bulk of ubiquitin conjugates accumulating in K0 Ub expressing cells relative to nontransformed cells. D, The relative abundance of different Ub modifications in cells expressing K0 Ub is displayed as a percentage of total cellular conjugated Ub.