FIGURE 3:

Pkh2 phosphorylates directly Vps27 on residue S613 in vitro. Anti-HA immunoprecipitation was performed from total lysate of pkh1∆ cells transformed with empty plasmid or wild-type or kinase-inactive (K208R) Pkh2-HA–encoding plasmid. Beads were used in an in vitro phosphorylation assay with 25 μg of recombinant Vps27-6xHis (A, B) or vps27^S613A-6xHis (B) in the presence of [γ-³²P]ATP. Samples were analyzed by SDS–PAGE, and the gels were dried and exposed for 24 h on a phosphoscreen. A representative experiment is shown; the experiment was repeated at least three times.