Nnf1p, Dsn1p, Mtw1p, and Nsl1p: a new group of proteins important for chromosome segregation in Saccharomyces cerevisiae

Abstract

Previously, antibodies were raised against a nuclear envelope-enriched fraction of yeast, and the essential gene NNF1 was cloned by reverse genetics. Here it is shown that the conditional nnf1-17 mutant has decreased stability of a minichromosome in addition to mitotic spindle defects. I have identified the novel essential genes DSN1, DSN3, and NSL1 through genetic interactions with nnf1-17. Dsn3p was found to be equivalent to the kinetochore protein Mtw1p. By indirect immunofluorescence, all four proteins, Nnf1p, Mtw1p, Dsn1p, and Nsl1p, colocalize and are found in the region of the spindle poles. Based on the colocalization of these four proteins, the minichromosome instability and the spindle defects seen in nnf1 mutants, I propose that Nnf1p is part of a new group of proteins necessary for chromosome segregation.

FIG. 1.

nnf1-17 cells have a mitotic defect. (A) Nuclear distribution (percent occurrence) in nnf1-17 cells. Wild-type (GEY145) or nnf1-17 (GEY146) cells were shifted from 25 to 37°C at time zero. After 3 h at 37°C, cells (n > 200 for each time point) were fixed and stained with DAPI. Anucleate cells were not observed, perhaps due to either cell lysis or difficulty in detection. (B) The DNA content of logarithmically growing NNF1 (GEY145) and nnf1-17 (GEY146) cells was analyzed by flow cytometry. (C) MT morphologies in large-budded nnf1-17 cells (percent occurrence) after 3 h at 37°C. Wild-type (GEY122) or nnf1-17 (GEY138) cells were shifted from 25 to 37°C at time zero. MTs were visualized by indirect immunofluorescence with antitubulin antibody. Cells were scored as follows: a, short spindle at or inserted in the bud neck; b, short, misoriented spindle; c, short spindle with missing cytoplasmic MTs; d, short spindle with elongated cytoplasmic MTs; e, extended spindle with divided nucleus; f, anaphase occurring in the mother cell; g. anaphase occurring in the mother cell with missing cytoplasmic MTs; h. anaphase occurring in the mother cell with elongated cytoplasmic MTs; and i, binucleated mother cell.

FIG. 2.

nnf1-17 cells show unstable transmission of a centromeric plasmid. Assays were performed as described in Materials and Methods. (A) The nnf1-17 mutant (GEY165) rapidly loses a centromeric ade3-2p plasmid (pGE102) when grown under nonselective conditions. The arrow points to a half-sectored colony. A wild-type strain (GEY160) carrying pGE102 is shown for comparison. (B) Percentages of each colony type were scored for >800 colonies of the following strains carrying pGE102: NNF1, GEY160; nnf1-17, GEY165; NNF1 + vector, GEY160 + pRS314; nnf1-17 + CEN/NNF1, GEY165 + pGE99; and nnf1-17 + vector, GEY165 + pRS314. Small colonies were ∼1 mm in diameter with irregular margins and thus could not be scored for half-sectoring. (C) The frequency of half-sectored colonies was calculated for each transformant as the number of half-sectored colonies divided by the total number of colonies with one copy of pCEN/ade3-2p (pGE102) upon plating. Errors are standard deviations of the means for the four or five transformants. Approximately 200 colonies were scored for each transformant.

FIG. 3.

Growth of an nnf1-17 mutant with various suppressors at 30, 35, and 37°C. The nnf1-17 strain GEY138 was transformed to Ura⁺ at 25°C with pGE101 (CEN/NNF1), pRS316 (empty vector), pGE164 (2μm/DSN1), pGE190 (2μm/SLG1), or pGE151 (2μm/DSN3). The transformed strains were streaked on the same minimal SC-Ura plates and incubated for 2 days at the indicated temperatures.

FIG. 4.

Complementation of strain YSL68 by NSL1 and of strain YSL29 by DSN3(MTW1). The synthetic lethal mutant YSL68 carrying plasmid pGE100 (CEN/TRP1/ADE3/NNF1) was transformed with either pGE84 (CEN/URA3/nsl1-68) or pGE81 (CEN/URA3/NSL1). The synthetic lethal mutant YSL29 carrying pGE100 was transformed with either pGE90 (CEN/URA3/dsn3-29) or pGE89 (CEN/URA3/DSN3). Transformants were selected on SC-Ura low-Ade plates at 30°C for 5 days. Wild-type NSL1 and DSN3(MTW1) restore the sectoring phenotype to strains YSL68 and YSL29, respectively. Comparable results were obtained with plasmid pGE82 in strain YSL6 and plasmid pGE83 in strain YSL8.

FIG. 5.

Localization of Nnf1p, Mtw1p, Dsn1p, and Nsl1p to the region of the spindle poles. First row, localization of Nnf1p and the SPB component Cnm67p in strain GEY111. For colocalization of Nnf1p with Mtw1p, Dsn1p, and Nsl1p, strain GEY110 was transformed with pGE74, pGE36, or pGE55, respectively. NNF1-myc₆ and CNM67-GFP are integrated in place of wild-type NNF1 or CNM67 in a haploid strain and are expressed from the endogenous promoters. The MTW1-GFP, DSN1-GFP, and NSL1-GFP fusions are expressed from their own promoters in CEN plasmids. For the indirect double-label immunofluorescence, cells were short fixed first in formaldehyde and then in methanol and acetone. The Myc₆ epitope was detected with the monoclonal antibody 9E10 and a Cy3-conjugated goat anti-mouse secondary antibody. GFP fluorescence was enhanced with a rabbit anti-GFP antibody and an FITC-labeled goat anti-rabbit secondary antibody. Bar, 5 μm.

FIG. 6.

The localization of Nnf1p, Dsn1p, Mtw1p, and Nsl1p does not depend on MTs. Cultures of haploid yeast expressing either NNF1-myc₆ (strain GEY110), DSN1-GFP (strain GEY176), MTW1-GFP (strain GEY216), or NSL1-GFP (strain GEY206) were split and with treated either 17 μg of nocodazole per ml or mock treated with DMSO for 2.5 h. Cells were fixed and prepared for indirect immunofluorescence using either antitubulin antibodies (not shown) or anti-Myc or anti-GFP antibodies. Since MT arrays were lost in nocodazole-treated cells, Nnf1p, Dsn1p, Mtw1p, and Nsl1p localizations are independent of MTs (n > 90 for each strain). Each fusion was expressed from its endogenous promoter, and localizations were not dependent on untagged versions of these proteins as only tagged copies were present in each strain. Bar, 5 μm.

FIG. 7.

Summary of interactions among NNF1, DSN1, MTW1, and NSL1. DSN1 and MTW1(DSN3) were isolated as high-copy suppressors of the nnf1-17 growth defect at 35°C. Mutations in NSL1 or MTW1(NSL2) are synthetically lethal with nnf1-17. Finally, Dsn1p and Nsl1p interact in the two-hybrid system (G. Euskirchen, unpublished data).