FIG. 2.

Physical and functional interactions between VSV M and Rsp5. (A) Full-length VSV M was transcribed and translated in vitro in the presence of [³⁵S]methionoine and incubated with ATP, ubiquitin, E1 enzyme, and E2 protein (UBC8 from Arabidopsis thaliana).+, reactions with wild-type (WT) Rsp5 (lanes 2, 5, 6, 9, and 10); −, reactions without WT Rsp5 (lanes 1, 3, 4, 7, and 8). The positions of unmodified WT VSV M (lanes 3 to 6) and of the unmodified VSV A4 mutant (lanes 7 to 10) are indicated. Multiubiquitinated forms of WT VSV M [M-ub(n)] are shown (lanes 5 and 6). A yeast protein of approximately 52 kDa encoded by the YHL002w gene is shown as a positive control for ubiquitination by Rsp5 (lanes 1 and 2). (B) Far-Western binding assay. Two micrograms of GST-Rsp5 (full-length Rsp5) were immobilized onto nitrocellulose filters and probed with BAP-VSV M (WT) or BAP-VSV M (A4 mutant).