Yeast cells with the schec1 null mutation can be rescued by human hsHEC1 (A) Schematic diagram of wild-type cell (a. YPH499) and yeast cells rescued by scHEC1 (b. WHL103) or hsHEC1 (c. WHL101). The yeast scHEC1 promoter region is marked by hatched boxes; arrows indicate the primers used in PCR genotyping. (B) Predicted DNA fragment sizes determined by PCR genotyping using the primers shown in panel A. (C) DNA fragments of PCR genotyping analyzed by agarose gel electrophoresis. Lane 1, WHL101; lane 2, WHL 103; lane 3, YPH499. (D) Immunoblot analysis of total cell lysates from YPH499 (lanes 1 and 3) and WHL101 (lanes 2 and 4), using anti-hsHec1p MAb 9G3 and mouse anti-scHec1p sera, respectively. (E) Growth-density curve of the yeast strain carrying the scHEC1 allele (YPH499) and the isogenic yeast strain in which the scHEC1 allele is replaced by hsHEC1 (WHL102) at 25°C. (F) The cell type distributions of yeast grown at 25°C for 4 h in (E) were observed with fluorescence microscopy. The percentages of cells at different phases of the cell cycle were determined according to budding morphologies, nuclear patterns, and spindle formation, and presented as histograms. (G) Immunostaining of Hec1p in yeast cells carrying the scHEC1 (a and b) or hsHEC1 (c and d) allele. scHec1p was detected by mouse polyclonal anti-scHec1p antibodies (b); hsHec1p was detected by purified rabbit polyclonal anti-hsHec1p antibodies (d). Corresponding FITC-conjugated secondary antibodies were used. DNA was stained with DAPI (a and c). (H) Immunostaining of hsHec1p in yeast cells. a, phase-contrast; b, spindle stained by rat antitubulin antibodies and FITC-conjugated secondary antibodies; c, nuclear DNA strained by DAPI; d, hsHec1p stained by rabbit anti-hsHec1p antibodies and Texas red-conjugated secondary antibodies.

Temperature sensitivity of mutated hshec1 alleles. (A) Growth-density curves of yeast strains carrying different mutant alleles or the wild-type allele of hsHEC1 were measured by spectrophotometric methods. Log-phase cells originally growing at 25°C were inoculated and cultured at 25 or 37°C, respectively. After 8 h, half of the cultures grown at 37°C were shifted back to 25°C, as indicated by arrows. (B) Log-phase cells from hsHEC1 or hshec1-113 strains were arrested with α-factor at 5 μg/ml, hydroxyurea at 0.1 M, or nocodazole at 20 μg/ml or were left untreated for 3 h at 25°C. The percentages of arrested cells, determined by DAPI staining and observation by fluorescence microscopy, were between 85 to 95%. While arrested, half of each culture was shifted to 37°C and the remaining was kept at 25°C. After another 3-h incubation, cells were released and plated at 25°C. The ratios between the numbers of colonies formed by the cells exposed to 37°C and those by the cells exposed to 25°C are shown by histograms. Asyn., asynchronous; αf, α-factor; Hu, hydroxyurea; Nz, nocodazole. (C) Log-phase cells from hsHEC1 or hshec1-113 strains were arrested with α-factor at 5 μg/ml for 3 h at 25°C. Immediately after release, half of each culture was shifted to 37°C and the remaining half was kept at 25°C. The numbers of colonies formed by equal aliquots taken from these cultures every 20 min were counted from duplicated plates. Numbers of colonies formed for each sample relative to the 0-min sample were calculated to generate the curves in the upper panel. The lower panel shows the percentages of budding cells in each sample.

Phenotypes of ts hshec1-113 mutants. (A) Characterization of cell cycle progression of hshec1-113 and wild-type alleles. Unsynchronized log-phase cells originally growing at 25°C were shifted to 37°C. At 2, 4, and 8 h, 300 to 500 cells were categorized and placed in different phases of cell cycle according to their budding morphologies, nuclear patterns, and spindle formation under microscopic observation. The percentages of different types of cells are shown by histograms. (B) Morphologies of hshec1-113 cells growing at 25°C (b, e, and h) and 37°C (c, f, and i) for 4 h and of wild-type hsHEC1 cells growing at 37°C (a, d, and g). (C) DNA content analysis of unsynchronized log-phase cells carrying a wild-type hsHEC1 or mutant hshec1-113 allele. Cells were cultured at 25 or 37°C for 6 h. Arrows indicate shoulders representing DNA content of less than 1n or more than 2n. (D) Morphologies of synchronized hshec1-113 (g to l) and wild-type (a to f) cells after release from S phase for 1 and 2 h. The cells were synchronized with 0.1 M hydroxyurea for 5 h at 25°C and shifted to 37°C when released. (E) DNA content analysis of the cells shown in panel D, before release (0 h) and 1, 2, and 3 h after release. Note that 2 h after release, wild-type cells exited from M phase and exhibited a 1n peak, but hshec1-113 cells did not exhibit such a peak (arrowheads). In panels B (a to c) and D (a, d, g, and j), cell morphology is shown by phase-contrast. DNA was stained with DAPI (B, d to f; D, b, e, h, and k). The spindle was stained by antitubulin antibodies and FITC-conjugated secondary antibodies (B, g to i; D, c, f, i, and l). The large-budded cells with unevenly divided nuclei are marked by arrows.

Characterization of the mutant hshec1-113p. (A) Partial nucleotide and protein sequences of wild-type hsHEC1 and mutant hshec1-113 alleles. The nucleotide substitution in hshec1-113p (G to A) and the corresponding amino acid change at position 395 (Trp to a stop codon OPA) are underlined. (B) Identification of hshec1-113p in mutant cells. Cell lysate prepared from wild-type (lanes 1 and 2) and hshec1-113 (lanes 3 and 4) cells growing at 25°C (lanes 1 and 3) or 37°C (lanes 2 and 4) for 4 h were immunoprecipitated by mouse anti-hsHec1p MAbs, followed by SDS-PAGE and Western blotting with the same antibodies. (C) Subcellular localization of the mutant hshec-113p. Log-phase cells of hshec1-113 were grown at 25°C or shifted to 37°C for 2 h. hshec-113p was stained by purified rabbit anti-hsHec1p antibodies and Texas red-conjugated secondary antibodies. Nuclear DNA of the same cells was stained by DAPI; spindle was stained by rat antitubulin antibodies and FITC-conjugated secondary antibodies.

The interaction between the mutated hshec1-113p and Smc1p is ts. (A) Binding between hsHec1-113p and hsHec1p-associated proteins (hsHec1p-Aps) at permissive and nonpermissive temperatures. 15ts113, the cDNA encoding the deletion mutant (aa 251 to 394) of hsHec1p that is truncated at the same position as the hshec1-113p mutant, was generated by in-frame fusion to the Gal4 DNA-binding domain. The same constructs in Table 3 were used to express hsHec1p-associated proteins that were fused with the Gal4 TAD. The interactions at 25 or 37°C were tested in yeast two-hybrid assays. Units represent β-galactosidase activity. (B) Domain mapping of the interaction between Hec1p and SMC1 proteins. The schematic structures of the regions in Hec1p that were fused with the Gal4 DNA-binding domain are shown. Shaded boxes are the three leucine heptad repeat-enriched regions. sb1.8/human SMC1 and Smc1p/yeast SMC1 were expressed as Gal4 TAD fusion proteins and used to test for interaction with Hec1 fusion proteins in yeast two-hybrid assays. (C) Log-phase yeast cells carrying an hshec1-113 or hsHEC1 allele were transferred to fresh medium and grown at either 25 or 37°C. After 3 h, cells were collected and lysed, and equal amounts of protein extracts (about 15 mg) were immunoprecipitated (IP) with mouse preimmune serum (lane 1 to 4) or anti-hsHec1p MAb 9G3 (lane 5 to 8). The immunoprecipitates were then resolved by SDS-PAGE followed by immunoblotting with mouse anti-Smc1p antiserum for Smc1p (a), 9G3 for hsHec1p (b), or hshec1-113p (c). Equal amounts of the same lysates (about 2 mg) were separated by SDS-PAGE followed by immunoblotting with anti-Smc1p antiserum for detecting the expression of Smc1p (d, lanes 9 to 12). Lanes 1, 5, and 9, hsHEC1 at 25°C; lanes 2, 6, and 10, hsHEC1 at 37°C; lanes 3, 7, and 11, hshec1-113 at 25°C; lanes 4, 8, and 12, hshec1-113 at 37°C.

Hec1p interacts with Smc2p. (A) Domain mapping and temperature sensitivity of the interaction between Hec1p and Smc2p. The same constructs of Hec1p as used for Fig. 5 were used in yeast two-hybrid assays to test their binding ability to yeast Smc2p expressed as a Gal4 TAD fusion protein. Binding ability was tested at 25 or 37°C. Gal4-DBD, Gal4 DNA-binding domain; ND, not determined. (B) In vivo interaction between Hec1p and Smc2p. Equal amounts of cell lysates from the log-phase culture of strain Y153 alone (lanes 1 to 3) or Y153 expressing TAD-Smc2p (lanes 4 to 6) were immunoprecipitated (IP) by the antibodies indicated. The resultant immunocomplexes were separated by SDS-PAGE followed by immunoblotting. The upper panel was blotted by polyclonal anti-TAD antibodies, and the lower panel was stained by anti-scHec1p. Ig, immunoglobulin.

Suppression of ts phenotype of smc1 or smc2 by overexpressing hsHEC1 or scHEC1. smc1-2, smc2-6, or wild-type cells were transformed by hsHEC1 or scHEC1 in a GAL1-inducible vector or by the vector alone. Different dilutions of log-phase cells grown in 2% glucose at 25°C were inoculated on the plates with 2% galactose and incubated at 25 or 37°C.