Figure 1.

Using the split-Ub technique to identify the binding sites of Sec62p for the Sec-complex by an in vivo competition assay. N_ug-Sec62p, Sec63-C_ub-RUra3p, and a fragment of Sec62p (X, red bar) are expressed in one cell. Pathway 1: when X does not or only weakly interacts with Sec63p, the N_ug-labeled Sec62p is allowed to bind to the C_ub-labeled Sec63p (both Ub-halves are in green). The induced proximity between N_ug and C_ub leads to their efficient reassociation. The assembled Ub is recognized by the UBPs, and the RUra3p reporter (yellow) is cleaved and subsequently degraded. The cells are phenotypically ura−. Pathway 2: X binds to Sec63p and displaces N_ug-Sec62p from its complex. The increased distance between the N_ug and C_ub inhibits their reassociation. The RUra3p reporter remains linked to C_ub and is not degraded. The cells are phenotypically ura+.