Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae

Abstract

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.

Figure 1

(A and B) Phenotype of class II Kar⁻ zygotes. Shown are examples of wild-type (A) and class II Kar⁻ (B) zygotes, respectively. Zygotes from filter matings between wild-type strains (MS1554 × MS23) or between kar7-1039 strains (MS3259 × MS3539) were analyzed by microscopy. Each image shows the nucleus by DAPI fluorescence and the zygote morphology by DIC. (C) Temperature-sensitive defect of kar7-1039. Streaks of wild-type (MS1554), kar7-1039 (MS3259), and sec71Δ (MS3910) strains were incubated at 30°C (left panel) or 37°C (right panel).

Figure 2

(A) Restriction map of KAR7/SEC71 and surrounding region on chromosome II. Shown are several subclones generated to further define KAR7. Bars represent the DNA fragments present in the different plasmids. The ability (+) or inability (−) of the different plasmids to suppress the mating defect is indicated to the right. Shown at the bottom is the structure of plasmids pMR3056 and pMR3057 used in the linkage analysis and the generation of sec71Δ by one-step gene replacement respectively. (B) Restriction map of KAR8/JEM1 and surrounding region on chromosome X. One of the original plasmids (pMR2935) able to rescue the kar8-1333 mating defect is depicted. Several subclones were generated and tested for rescuing activity. Bars represent the DNA fragments present in the different plasmids. The ability (+) or inability (−) of the different plasmids to suppress the mating defect is indicated to the right. Shown at the bottom is the structure of plasmid pMR3133 used in the linkage analysis of JEM1 and the structure of the jem1/kar8Δ allele generated by one-step gene replacement.

Figure 3

sec71Δ and sec72Δ, but not sec63-1, membranes show a defect in the in vitro ER–nuclear membrane fusion assay at 37°C. (A) Donor and acceptor membranes (75 μg protein each) prepared from wild-type strains (MLY1601 and MLY1600), strains deleted for the SEC71 gene (MLY1889 and MLY1890), or strains deleted for the SEC72 gene (MLY1891 and MLY1892) were combined in the presence of an ATP regeneration system in a total volume of 50 μl and held on ice. Reactions were incubated for 60 min at 24 or 37°C. (B) In a separate experiment, donor and acceptor membranes prepared from wild-type strains MLY1600 and MLY1601 and sec63-1 strains MLY1651 and MLY1652) were combined and incubated as above. In all cases, the experiments were repeated three times, and the mean values and SDs are shown. All strains were grown at 24°C before membrane isolation. The amount of glucose trimming, indicative of the successful fusion of membranes, was assessed as described previously (Latterich and Schekman, 1994).

Figure 4

Kar5p is mislocalized in kar7-1039 but not in other karyogamy mutants. In each series (A–F), the left panel shows the morphology of the cell (shmoo) by DIC. The middle panel shows the Kar5p immunofluorescence, and the right panel shows the nucleus stained with DAPI. (A) Kar5 shmoos (MS3987); (B) kar5Δ2 shmoos (MS3986); (C) kar1-1 shmoos (MS4021); (D) kar2-1 shmoos (MS4020); (E) kar7-1039 shmoos (MS3991); (F) kar8-1333 shmoos (MS3989). Cells were treated with α-factor for 2–2.5 hr before preparation for immunofluorescence. A and B are reproduced from Beh et al. (1997) J. Cell. Biol. 139, 1063–1076, by copyright permission of The Rockefeller University Press.

Figure 5

Kar5p was not detected in the kar7-1039 mutant. (A) Western blot analysis. The strains kar5Δ2 and kar7-1039 transformed with 2μ KAR5 plasmid (MS3987 and MS3991) were treated (+) or left untreated (−) with α-factor for 2 hr. Total protein extracts were analyzed by Western blot using affinity-purified anti-Kar5p antibodies. (B) Pulse labeling. MS3986 and MS3987, kar5Δ2 strains transformed with either vector (lane 1) or 2μ KAR5 plasmid (lanes 2 and 3), and MS3991, a kar7-1039 strain transformed with 2μ KAR5 plasmid (lane 4), were ³⁵S pulsed for 5 min at 23°C after treatment (+) or no treatment (−) with α-factor. Pulse-labeled extracts were immunoprecipitated with crude anti-Kar5p antibodies, run on a polyacrylamide gel, and visualized by PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Figure 6

Electron micrographs of serial section of two kar8-1333 zygotes. The kar8-1333 mating partners used in this study were MS2705 and MS2706. (A–C) Micrographs of three consecutive serial sections (of seven) through a kar8-1333 mutant zygote shown at two different magnifications. (D–F) Micrographs of sections 1, 3, and 5 (of five) of another kar8-1333 mutant zygote also shown at two different magnifications. Each section is 70 nm thick. n, nuclei; *, nuclear pores; arrows, kar8-1333 bridges between the two nuclei.

Figure 7

Electron micrographs of serial sections through a kar5Δ kar8Δ double mutant zygote. The kar5Δ kar8Δ mating partners used in this study were MS4359 and MS4360. (A–C) Micrographs of three consecutive 90-nm-thick serial sections through a kar5Δ kar8Δ mutant zygote. n, nuclei; arrows, morphology of the bridges observed in kar5Δ kar8Δ zygotes.