FIG. 1.

Ubc7p cross-linked to degraded Hmg2p in a mevalonate pathway-regulated manner but not to stable Hmg1p. Interaction of 2HA-Ubc7p with 1myc-HMGR was assessed by the in vivo cross-linking assay. Cells were grown to mid-log phase and removed to amine-free medium. DSP was added at the indicated concentrations to separate aliquots of cells, and cross-linking proceeded at 30°C for 30 min. Lysates were prepared, and 1myc-Hmg2p was immunoprecipitated with antiserum raised against its catalytic domain. Immunoprecipitates were denatured under reducing conditions and analyzed by immunoblotting with either the 9E10 antibody (α-myc) to detect 1myc-Hmg2p or the 12CA5 antibody (α-HA) to detect 2HA-Ubc7p. (a) Ubc7p cross-linked to Hmg2p but did not cross-link to Hmg1p. Cross-linking assay was done with strains expressing either 1myc-Hmg2p or 1myc-Hmg1p and coexpressing either 2HA-Ubc7p or 3HA-Ubc6p. The reduced immunoreactivity of 1myc-HMGR with DSP at 400 μg/ml is due to modification of the lysine residue in the myc epitope sequence, not reduced immunoprecipitation of HMGR (unpublished observations). The small amount of 2HA-Ubc7p that is present with DSP at 0 μg/ml in this and the following figures is similar to that observed when preimmune serum is used instead of HMGR-specific antiserum (unpublished observations), indicating that it is nonspecific immunoprecipitation. (b) Ubc7p cross-linked to a degraded version of Hmg1p; cross-linking assay with strains expressing either 1myc-Hmg2p, 1myc-Hmg1p, or hydrophilic-1myc-Hmg1p and coexpressing 2HA-Ubc7p. (c) Addition of either lovastatin or L-659,699 decreased Ubc7p cross-linking. Regulated Ubc7p cross-linking to Hmg2p was observed by preincubating the cells with either no or 50 μg of lovastatin (Lov) or 10 μg of L-659,599 (L659) per ml for 2 h prior to addition of DSP. Cross-linking assay was performed as in Fig. 1. (d) Cross-linking of Ubc7p to 6myc-Hmg2p was not regulated by the mevalonate pathway. Ubc7p cross-linking to 6myc-Hmg2p was observed by preincubating the cells with either 0 or 50 μg lovastatin or 10 μg of L-659,599 per ml for 2 h prior to addition of DSP.