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KAR5 encodes a novel pheromone-inducible protein required for homotypic nuclear fusion.

Beh CT, Brizzio V, Rose MD - J. Cell Biol. (1997)

Bottom Line: KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion.Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum.In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA.

ABSTRACT
KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion. Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum. In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy. We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex.

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Kar5p was localized at or in the vicinity of the spindle  pole body after pheromone induction. To determine whether  Kar5p was localized to the spindle pole body, the fixation method  of Rout and Kilmartin (1990) was used. Before fixation and staining, cells were induced with pheromone until >80% of the cells  had formed shmoos. Indirect immunofluorescence using antibodies against Kar5p and the 90-kD SPB antigen were performed on  wild-type (A–C), KAR5 overexpressing (D–F), and kar5-Δ2 (G–I)  strains. The nucleus was identified by DAPI staining shown in A,  D, and G. Localization of the 90-kD protein is shown in C, F, and  I and indicates the position of the SPB on the nucleus. Localization of Kar5p is shown in B and E. Kar5p largely colocalized with  the 90-kD protein and was not detected in the kar5-Δ2 control  strain (H, MS3986). Of 48 cells counted, 77% (37/48) exhibited  Kar5p staining and 87% (41/48) exhibited 90-kD staining.
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Figure 9: Kar5p was localized at or in the vicinity of the spindle pole body after pheromone induction. To determine whether Kar5p was localized to the spindle pole body, the fixation method of Rout and Kilmartin (1990) was used. Before fixation and staining, cells were induced with pheromone until >80% of the cells had formed shmoos. Indirect immunofluorescence using antibodies against Kar5p and the 90-kD SPB antigen were performed on wild-type (A–C), KAR5 overexpressing (D–F), and kar5-Δ2 (G–I) strains. The nucleus was identified by DAPI staining shown in A, D, and G. Localization of the 90-kD protein is shown in C, F, and I and indicates the position of the SPB on the nucleus. Localization of Kar5p is shown in B and E. Kar5p largely colocalized with the 90-kD protein and was not detected in the kar5-Δ2 control strain (H, MS3986). Of 48 cells counted, 77% (37/48) exhibited Kar5p staining and 87% (41/48) exhibited 90-kD staining.

Mentions: In wild-type cells, and in cells in which Kar5p was overexpressed, Kar5p localization was confined to a spot on the periphery of the nucleus (Figs. 9 and 10). Most cells had a single large spot of localization, but some cells had one or more additional smaller spots along the nuclear periphery (71% had one spot, 19% had two spots, and 10% had three spots, out of 105 cells counted). The minor spots were largely observed in cells using methanol-acetone fixation and only rarely in cells fixed with paraformaldehyde. The minor spots may therefore result from inadequate fixation.


KAR5 encodes a novel pheromone-inducible protein required for homotypic nuclear fusion.

Beh CT, Brizzio V, Rose MD - J. Cell Biol. (1997)

Kar5p was localized at or in the vicinity of the spindle  pole body after pheromone induction. To determine whether  Kar5p was localized to the spindle pole body, the fixation method  of Rout and Kilmartin (1990) was used. Before fixation and staining, cells were induced with pheromone until >80% of the cells  had formed shmoos. Indirect immunofluorescence using antibodies against Kar5p and the 90-kD SPB antigen were performed on  wild-type (A–C), KAR5 overexpressing (D–F), and kar5-Δ2 (G–I)  strains. The nucleus was identified by DAPI staining shown in A,  D, and G. Localization of the 90-kD protein is shown in C, F, and  I and indicates the position of the SPB on the nucleus. Localization of Kar5p is shown in B and E. Kar5p largely colocalized with  the 90-kD protein and was not detected in the kar5-Δ2 control  strain (H, MS3986). Of 48 cells counted, 77% (37/48) exhibited  Kar5p staining and 87% (41/48) exhibited 90-kD staining.
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Related In: Results  -  Collection

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Figure 9: Kar5p was localized at or in the vicinity of the spindle pole body after pheromone induction. To determine whether Kar5p was localized to the spindle pole body, the fixation method of Rout and Kilmartin (1990) was used. Before fixation and staining, cells were induced with pheromone until >80% of the cells had formed shmoos. Indirect immunofluorescence using antibodies against Kar5p and the 90-kD SPB antigen were performed on wild-type (A–C), KAR5 overexpressing (D–F), and kar5-Δ2 (G–I) strains. The nucleus was identified by DAPI staining shown in A, D, and G. Localization of the 90-kD protein is shown in C, F, and I and indicates the position of the SPB on the nucleus. Localization of Kar5p is shown in B and E. Kar5p largely colocalized with the 90-kD protein and was not detected in the kar5-Δ2 control strain (H, MS3986). Of 48 cells counted, 77% (37/48) exhibited Kar5p staining and 87% (41/48) exhibited 90-kD staining.
Mentions: In wild-type cells, and in cells in which Kar5p was overexpressed, Kar5p localization was confined to a spot on the periphery of the nucleus (Figs. 9 and 10). Most cells had a single large spot of localization, but some cells had one or more additional smaller spots along the nuclear periphery (71% had one spot, 19% had two spots, and 10% had three spots, out of 105 cells counted). The minor spots were largely observed in cells using methanol-acetone fixation and only rarely in cells fixed with paraformaldehyde. The minor spots may therefore result from inadequate fixation.

Bottom Line: KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion.Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum.In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA.

ABSTRACT
KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion. Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum. In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy. We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex.

Show MeSH
Related in: MedlinePlus