Role of NCD in Cdc17 function. (A) Expression of Cdc17-NΔ390 from nmt41 promoter fails to rescue haploid cdc17::kanMX6. (B) Immunoblot of protein extracts prepared from wild-type cells (grown in the absence of thiamine) expressing Cdc17, Cdc17-NΔ125, Cdc17-NΔ390 and Cdc17-K416A from the nmt41 promoter in plasmid pREP41X (lanes 2–5, respectively) or Cdc17-NΔ390 from the full-strength nmt1 promoter in pREP3X (lane 6). Lane 1 contains protein prepared from cells carrying the empty vector pREP41X. Note that under these conditions, detection of the endogenous Cdc17 protein was not possible. (C) Location of mutations in temperature-sensitive Cdc17 proteins, together with protein sequence alignments showing context of mutations. Sp, S.pombe. Sc, S.cerevisiae. Hs, human. Xl, Xenopus. See legend to Figure 1 for Swiss-Prot accession numbers.