Fig. 1. Kinase activity is essential. (A) Alignment of the catalytic domain of Sid1p with the catalytic domains of the most closely related PAK/GC family members as determined by the BLAST and MULTALIN programs. Relatives include severin kinase from Dictyostelium discoideum (AAC24522), Caenorhabditis elegans contig yk19h11.5 (AAC9038), BnMAPK4 from Brassica napus (CAA08757), human Ste20-like kinase Mst-3 (4505261) and human oxidant stress-activated Ste20-like kinase SOK-1 (5454174). Roman numerals indicate the 11 subdomains characteristic ofSer/Thr protein kinases (Hanks et al., 1988). Boxes enclose conserved residues. (B) Immune complex kinase assays were performed using Myc or HA antibodies as appropriate (see Materials and methods) on lysates prepared from wild-type cells, or cells expressing 13Myc-Sid1p, HA-Sid1p or HA-Sid1-K38R. Assays were carried out with (+) or without (–) antibody or the artificial substrate MBP. A control assay was also carried out in the absence of cell lysate (protein G beads). (C) sid1-125 cells (YDM435) transformed with vector alone or with plasmids expressing either HA-Sid1p or HA-Sid1-K38R were plated at 25 or 36°C.