Fig 1. Biotinylated ubiquitin is incorporated into conjugates in the Drosophila adult eye.

(A) Schematic illustration of the in vivo biotinylation of ubiquitin [21]. The construct is expressed as a polyubiquitin chain fused to the E.coli BirA enzyme, which is digested by the endogenous deubiquitinating enzymes (DUBs). While this clearly indicates that DUB enzymes are capable of deconstructing linear ^bioUb chains, we have no evidence to support that E2/E3 enzymes are actually capable of constructing those linear ^bioUb chains the same way they would do with the more abundant endogenous ubiquitin. Afterwards, the BirA enzyme recognizes the short motif incorporated at the N-terminus of each ubiquitin (sequence is indicated) and attaches a biotin molecule to its lysine residue (red). The biotinylated ubiquitins are then conjugated to the target proteins. (B) Western blot with anti-BirA antibody using total Drosophila head extracts confirmed the full digestion of the ^bioUb precursor by endogenous DUBs. No undigested forms of the precursor were found above the expected molecular size of BirA (35 kDa). (C) Anti-biotin Western blot on the same total extracts confirmed the biotinylation and conjugation of the GMR^GAL4–driven expressed ubiquitin. An endogenous protein known to be biotinylated (CG1516) appeared in all the samples (arrowhead). The expected smear corresponding to biotinylated ubiquitin conjugates was only present in the ^bioUb sample. WT: Oregon R; BirA: GMR ^GAL4 /CyO;^UAS BirA/TM6; ^bioUb: GMR ^GAL4,^UAS ( ^bio Ub) ₆ -BirA/CyO.