Figure 1: Bacterial genetics approach for selection of ubiquitylated proteins.

(a) A genetic selection system for discovering ubiquitylated proteins in bacteria. The ubiquitylation apparatus is expressed from two compatible plasmids. Each plasmid harbors different antibiotic resistance and origin of replication, facilitating cotransformation and selection of the vectors regardless of ubiquitylation. pND–Ub denotes the N-terminal fragment of DHFR fused to Ub. pCD–Sub denotes the C-terminal fragment of DHFR fused to a ubiquitylation substrate. A complete system (consists of pND–Ub, pCD–Sub and a cognate set of ubiquitylating enzymes) confers antibiotic resistance and bacterial growth in the presence of trimethoprim (TRIM). Red cross represents bacteria that express an incomplete system and therefore are not resistant to the selective media. K, lysine residue. (b) Vps9 ubiquitylation. 2.5 μl of E. coli W3110 (at OD₆₀₀ of 0.2), expressing a complete (cDHFR–Vps9, nDHFR–Ub, yeast Ubc4 and Rsp5) or incomplete (ΔE1,ΔE2; ΔUb or ΔE3) ubiquitylation apparatus, were seeded as spots on selective (10 μg/ml TRIM) or nonselective minimal agar plates. Bacterial spots were visualized by a UV video camera. (c) Same as in b, except the Vps9 cognate E3 ligase (Rsp5) was replaced by a noncognate E3 ligase (SIAH2). (d) As shown in b, but the trimethoprim concentration is varied. Binding, bacteria expressing the pND–Ub and pCD–Vps9 without a complete ubiquitylation apparatus (ΔE1,ΔE2). Ubiquitylation, bacteria expressing the pND–Ub and pCD–Vps9 along with a cognate ubiquitylation apparatus.