Figure 7.

Slx5-K proteins undergo auto-ubiquitination in cis, and are unique to the yeast STUbL. (A) Strain PSY3884 (slx5∆) was transformed with LEU2/CEN/ARS plasmids that contained either SLX5 (WT), no insert (∆), the indicated slx5-K mutant, or a compound allele also containing the ring⁻ mutation. All inserts express N-terminal His6-tagged Slx5 protein. Cell extracts were incubated with Ni-NTA agarose beads, and bound proteins were immunoblotted as in Figure 6A. (B) Wild-type strain JMY3107 (SLX5) was treated as in (A). (C) Strain PSY3884 (slx5∆) was transformed with plasmids containing the indicated SLX5 alleles and treated as in (A), except that extracts were directly immunoblotted for Slx5. A nonspecific band (*) on the blot is used as an internal loading control. (D) Mammalian cell extracts were prepared from wt MEFs (lane 1), Rnf4^−/− MEFs (lane 2), or from Rnf4^−/− MEFs that were stably transduced with empty vector or the indicated RNF4 alleles (lanes 4–9). Cell extracts (25 µg) were immunoblotted with anti-RNF4 antibodies (upper), and the blot was reprobed with antibody to α-tubulin as control (lower). Extracts were also prepared from wt RNF4 transductants that had been treated for 4 hr with 20 µM MG132 in DMSO (+) or with DMSO alone (−) (lanes 10 and 11). Purified His6-RNF4 (80 pg) was included as positive control (lane 3).