Figure 6.—

The α1 protein is ubiquitinated and this modification requires multiple ubiquitin ligases. Ubiquitinated proteins from lysates of cells of the indicated genotypes expressing His₆-ubiquitin (His₆-Ub) or untagged ubiquitin (Ub) and HA-tagged or untagged α1 were purified by binding to Ni-NTA beads. The affinity-purified material was analyzed for the presence of α1^3HA by α-HA immunoblotting (top). The unpurified lysates were analyzed by immunoblotting with α-Pgk1 (bottom) in parallel. The bracket (top) indicates ubiquitinated α1^3HA and the positions of molecular weight markers are given on the left side of the gel. Note the enrichment of ubiquitinated α1^3HA in cells expressing the tagged forms of both α1 and Ub (compare lane 3 to lanes 1 and 2) and the reduced levels of ubiquitinated α1^3HA in strains lacking the ubiquitin ligases Ufd4, Dma1, Dma2, and Slx5–Slx8 (compare lanes 4–6 to lane 3). Equal amounts of protein were loaded in each lane and proteins were equivalently transferred, as judged by the levels of Pgk1 in the unpurified lysates. Numbers indicate the relative amount of ubiquitinated α1^3HA after normalizing to the levels of Pgk1.