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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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Expression of Kar5p  in pheromone-treated cells.  Equal amounts of cellular proteins were loaded and run on a  10% polyacrylamide gel, transferred, and probed with affinity  purified anti-Kar5p antibodies.  (A) The kar5-Δ2 strain MS3986  treated with α-factor. (B)  MS3987 without pheromone  treatment, (C) MS3987 after 2.5  h pheromone treatment. The  band corresponding to Kar5p is  indicated by an arrow. Uninduced levels of Kar5p were not  detectable by Western blot.  Kar5p corresponds to a 58-kD  protein that was expressed at  substantially greater levels in  pheromone-treated cells. A contaminating band, indicated with  an asterisk (*), corresponded to a cell membrane protein (as determined by fractionation), which could be removed with successive preadsorption in other experiments (see Materials and  Methods). This cross-reacting protein did not contribute to staining in immunofluorescence experiments (Fig. 9).
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Figure 5: Expression of Kar5p in pheromone-treated cells. Equal amounts of cellular proteins were loaded and run on a 10% polyacrylamide gel, transferred, and probed with affinity purified anti-Kar5p antibodies. (A) The kar5-Δ2 strain MS3986 treated with α-factor. (B) MS3987 without pheromone treatment, (C) MS3987 after 2.5 h pheromone treatment. The band corresponding to Kar5p is indicated by an arrow. Uninduced levels of Kar5p were not detectable by Western blot. Kar5p corresponds to a 58-kD protein that was expressed at substantially greater levels in pheromone-treated cells. A contaminating band, indicated with an asterisk (*), corresponded to a cell membrane protein (as determined by fractionation), which could be removed with successive preadsorption in other experiments (see Materials and Methods). This cross-reacting protein did not contribute to staining in immunofluorescence experiments (Fig. 9).

Mentions: To characterize the Kar5 protein, we used GST–Kar5p expressed in E. coli to produce polyclonal antibodies that were then affinity-purified (see Materials and Methods). To detect Kar5p, total protein was extracted from cells overexpressing KAR5 from a multicopy plasmid, with and without pheromone induction. On protein blots, the Kar5p antibodies recognized a 58-kD protein after pheromone induction, corresponding to the predicted size of unmodified Kar5p (58,396 D). In contrast, the 58-kD protein was not detectable after pheromone induction in a strain bearing a deletion of the KAR5 gene (MS3986) (Fig. 5). Taken together, these data demonstrate that the 58-kD protein was Kar5p. In some experiments (e.g., Fig. 7), the Kar5p band was resolved into a doublet. The molecular nature of the two bands has not been determined. Overexpression of KAR5 had no detectable adverse phenotype (data not shown). In addition, both a low molecular mass protein (Fig. 5) and a slightly higher molecular mass protein (Fig. 7) were sometimes detected with the antibody. Since these proteins were observed in the kar5-Δ strain, they are not derived from Kar5p.


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

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

Expression of Kar5p  in pheromone-treated cells.  Equal amounts of cellular proteins were loaded and run on a  10% polyacrylamide gel, transferred, and probed with affinity  purified anti-Kar5p antibodies.  (A) The kar5-Δ2 strain MS3986  treated with α-factor. (B)  MS3987 without pheromone  treatment, (C) MS3987 after 2.5  h pheromone treatment. The  band corresponding to Kar5p is  indicated by an arrow. Uninduced levels of Kar5p were not  detectable by Western blot.  Kar5p corresponds to a 58-kD  protein that was expressed at  substantially greater levels in  pheromone-treated cells. A contaminating band, indicated with  an asterisk (*), corresponded to a cell membrane protein (as determined by fractionation), which could be removed with successive preadsorption in other experiments (see Materials and  Methods). This cross-reacting protein did not contribute to staining in immunofluorescence experiments (Fig. 9).
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Figure 5: Expression of Kar5p in pheromone-treated cells. Equal amounts of cellular proteins were loaded and run on a 10% polyacrylamide gel, transferred, and probed with affinity purified anti-Kar5p antibodies. (A) The kar5-Δ2 strain MS3986 treated with α-factor. (B) MS3987 without pheromone treatment, (C) MS3987 after 2.5 h pheromone treatment. The band corresponding to Kar5p is indicated by an arrow. Uninduced levels of Kar5p were not detectable by Western blot. Kar5p corresponds to a 58-kD protein that was expressed at substantially greater levels in pheromone-treated cells. A contaminating band, indicated with an asterisk (*), corresponded to a cell membrane protein (as determined by fractionation), which could be removed with successive preadsorption in other experiments (see Materials and Methods). This cross-reacting protein did not contribute to staining in immunofluorescence experiments (Fig. 9).
Mentions: To characterize the Kar5 protein, we used GST–Kar5p expressed in E. coli to produce polyclonal antibodies that were then affinity-purified (see Materials and Methods). To detect Kar5p, total protein was extracted from cells overexpressing KAR5 from a multicopy plasmid, with and without pheromone induction. On protein blots, the Kar5p antibodies recognized a 58-kD protein after pheromone induction, corresponding to the predicted size of unmodified Kar5p (58,396 D). In contrast, the 58-kD protein was not detectable after pheromone induction in a strain bearing a deletion of the KAR5 gene (MS3986) (Fig. 5). Taken together, these data demonstrate that the 58-kD protein was Kar5p. In some experiments (e.g., Fig. 7), the Kar5p band was resolved into a doublet. The molecular nature of the two bands has not been determined. Overexpression of KAR5 had no detectable adverse phenotype (data not shown). In addition, both a low molecular mass protein (Fig. 5) and a slightly higher molecular mass protein (Fig. 7) were sometimes detected with the antibody. Since these proteins were observed in the kar5-Δ strain, they are not derived from Kar5p.

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