FIG. 6.

Use of zymocin as a diagnostic tool to map a SIR on Sap185. (A) sap190Δ deletion strains coexpressing HA-tagged Sit4 and the indicated c-Myc-tagged variants of Sap185 were subjected to immune precipitation (IP) using the anti-c-Myc antibody. The presence of Sit4, as well as full-length and C-terminal truncations of Sap185 (all indicated by arrows), in these precipitates was monitored by immunoblotting (IB) using anti-HA and anti-c-Myc antibodies. In addition, the content of Sit4 (bottom) and the Sap185 variants (not shown) in the inputs (preIP) was checked by IB using anti-HA and anti-c-Myc antibodies, respectively. (B) Sit4 binding deficits of Sap185 truncations C3 and C4 cause zymocin resistance in a sap190Δ deletion mutant. Strains as in panel A were subjected to killer eclipse assays to score zymocin sensitivity (S) or resistance (R). (C) Assaying the Elp1 phosphobalance involved transformation with pELP1-HA, protein extraction, and Western blot analysis using the anti-HA antibody. Elp1 phosphoforms (circled P) were distinguished from unmodified Elp1 by mobility shifts (27). (D) The SIR of Sap185 maps to a central segment conserved in other members of the yeast Sap family. The sketch summarizes SIR mapping data (gray box) on the basis of C- and N-terminal Sap185 truncation sets, C1 to C4 and N1 to N3 (see Fig. S3 in the supplemental material). The regions with the highest similarity between Sap4, Sap155, Sap185, and Sap190 are highlighted as black boxes (38).