Figure 1.

A system to select for protein interactions in vivo. (A) The split-ubiquitin system. Ubiquitin, fused to the N terminus of Ura3p displaying an arginine as its first amino acid (RUra3p), is recognized by the UBPs (line 1). The cleaved RUra3p is rapidly degraded by the N-end rule pathway of protein degradation (line 4). No cleavage of RUra3p takes place if only the C-terminal half of ubiquitin (C_ub) is fused between Gal4p and RUra3p (line 2). A protein X is attached to the N-terminal half of ubiquitin. If X interacts with Gal4p, the two coupled Ub peptides are forced into close proximity, a ubiquitin-like molecule is reconstituted, and cleavage by the UBPs is observed (line 3). The freed RUra3p reporter is now rapidly degraded by the enzymes of the N-end rule, resulting in uracil auxotrophy and FOA resistance (line 4). (B) Gal4p interacts with Gal80p in vivo. Shown are serial dilutions of cells coexpressing N_ub or a N_ub-Gal80p fusion together with Gal4(1–147 + 768–881)-C_ub-RUra3p on plates lacking tryptophan and leucine (Top), additionally lacking uracil (Middle), or containing FOA (Bottom). All proteins were expressed from single-copy vectors. (C) Tup1p interacts with Ssn6p in vivo. Shown are serial dilutions of cells coexpressing the depicted N_ub and C_ub fusions on plates lacking tryptophan and leucine (Upper) or on plates additionally lacking uracil (Lower). All proteins were expressed from single-copy vectors.