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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 is enriched  in isolated nuclei. Equal  amounts of protein (20 μg),  isolated from whole cells (A  and B) or purified nuclei (C  and D) were loaded onto 10% SDS polyacrylamide gels, transferred, and probed with anti-Kar5p and anti-Kar2p antibodies  (1/50 of the protein was loaded for the Kar2p blot). In lanes A  and C, protein extracts were derived from MS3987 after 2.5 h of  α-factor induction. In lanes B and D, protein extracts were derived from the kar5-Δ2 strain MS3986, which had also been  treated with α-factor for 2.5 h. Both Kar2p and Kar5p were  highly enriched in the nuclear fractions.
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Figure 6: Kar5p is enriched in isolated nuclei. Equal amounts of protein (20 μg), isolated from whole cells (A and B) or purified nuclei (C and D) were loaded onto 10% SDS polyacrylamide gels, transferred, and probed with anti-Kar5p and anti-Kar2p antibodies (1/50 of the protein was loaded for the Kar2p blot). In lanes A and C, protein extracts were derived from MS3987 after 2.5 h of α-factor induction. In lanes B and D, protein extracts were derived from the kar5-Δ2 strain MS3986, which had also been treated with α-factor for 2.5 h. Both Kar2p and Kar5p were highly enriched in the nuclear fractions.

Mentions: To determine if Kar5p was enriched in nuclear membranes, nuclei were isolated by the method of Aris and Blobel (1991). The enriched nuclear proteins were electrophoretically separated by SDS-PAGE and analyzed by protein blot. Kar5p was found to be highly enriched in the purified nuclei (Fig. 6). As a control, we also examined the ER/nuclear envelope protein Kar2p and found it to be enriched in the nuclear fraction.


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

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

Kar5p is enriched  in isolated nuclei. Equal  amounts of protein (20 μg),  isolated from whole cells (A  and B) or purified nuclei (C  and D) were loaded onto 10% SDS polyacrylamide gels, transferred, and probed with anti-Kar5p and anti-Kar2p antibodies  (1/50 of the protein was loaded for the Kar2p blot). In lanes A  and C, protein extracts were derived from MS3987 after 2.5 h of  α-factor induction. In lanes B and D, protein extracts were derived from the kar5-Δ2 strain MS3986, which had also been  treated with α-factor for 2.5 h. Both Kar2p and Kar5p were  highly enriched in the nuclear fractions.
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Related In: Results  -  Collection

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Figure 6: Kar5p is enriched in isolated nuclei. Equal amounts of protein (20 μg), isolated from whole cells (A and B) or purified nuclei (C and D) were loaded onto 10% SDS polyacrylamide gels, transferred, and probed with anti-Kar5p and anti-Kar2p antibodies (1/50 of the protein was loaded for the Kar2p blot). In lanes A and C, protein extracts were derived from MS3987 after 2.5 h of α-factor induction. In lanes B and D, protein extracts were derived from the kar5-Δ2 strain MS3986, which had also been treated with α-factor for 2.5 h. Both Kar2p and Kar5p were highly enriched in the nuclear fractions.
Mentions: To determine if Kar5p was enriched in nuclear membranes, nuclei were isolated by the method of Aris and Blobel (1991). The enriched nuclear proteins were electrophoretically separated by SDS-PAGE and analyzed by protein blot. Kar5p was found to be highly enriched in the purified nuclei (Fig. 6). As a control, we also examined the ER/nuclear envelope protein Kar2p and found it to be enriched in the nuclear fraction.

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