FIG. 2.

ndc1-39 is a novel temperature-sensitive (ts) allele of NDC1. (A) Six amino acid changes in the ndc1-39 allele are shown. The black and white bars represent the structure of NDC1 as described for Fig. 1. (B) Suspensions of NDC1 (strain 1264) and ndc1-39 (strain 2710) cells were plated on YPD plates at an optical density at 600 nm of 3.0 in fivefold serial dilutions and were grown at the permissive temperature of 23°C (left) and at the restrictive temperature of 35°C (right) for 3 days. Two isolates of ndc1-39 mutants are shown. (C) Subcellular localization of Ndc1p and Ndc1-39p as visualized by IF microscopy. NDC1 (strain 3451; top two rows) and ndc1-39 (strain 3452; bottom two rows) cells were shifted to 35°C for 3 h in YPD medium. Ndc1p and Ndc1-39p cells tagged with the Myc epitope are shown in red (see Materials and Methods), DNA is shown in blue, autofluorescence of Spc42p tagged with GFP is shown in green, and colocalization of Spc42p-GFP with either Ndc1p-myc or Ndc1-39p-myc is shown in yellow. Each image shown was projected from five consecutive images taken at 0.1-μm intervals along the z axis that have been deconvolved. The ndc1-39 allele used for this experiment did not include the F643L amino acid change that does not contribute to the ts phenotype. Bar, 1 μm.