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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 extraction from nuclei. To determine whether  Kar5p was a lumenal, integral, or peripheral membrane protein, a  purified membrane fraction (derived from MS3987 after 2.5-h  α-factor induction) was subjected to various extraction conditions  (see Materials and Methods): 1 M NaCl; 0.1 M Na2CO3, pH 11.0;  2 M urea; 1% Triton X-100; 1% Triton X-100, 0.5 M NaCl; and  buffer alone. As a control, an equivalent membrane fraction from  the kar5-Δ strain, MS3986, was used to confirm the identity of the  protein species. After incubation for 1 h at 4°C, the samples were  centrifuged at 128,000 g. The supernatant was collected, and the  pellet was resuspended in the original volume. Equal volumes of  supernatant (S) and pellet (P) were analyzed by Western blot.  Blots were probed with anti-Sec61p, anti-Kar2p, anti-Sec72p, and  anti-Kar5p antibodies.
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Figure 7: Kar5p extraction from nuclei. To determine whether Kar5p was a lumenal, integral, or peripheral membrane protein, a purified membrane fraction (derived from MS3987 after 2.5-h α-factor induction) was subjected to various extraction conditions (see Materials and Methods): 1 M NaCl; 0.1 M Na2CO3, pH 11.0; 2 M urea; 1% Triton X-100; 1% Triton X-100, 0.5 M NaCl; and buffer alone. As a control, an equivalent membrane fraction from the kar5-Δ strain, MS3986, was used to confirm the identity of the protein species. After incubation for 1 h at 4°C, the samples were centrifuged at 128,000 g. The supernatant was collected, and the pellet was resuspended in the original volume. Equal volumes of supernatant (S) and pellet (P) were analyzed by Western blot. Blots were probed with anti-Sec61p, anti-Kar2p, anti-Sec72p, and anti-Kar5p antibodies.

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)

Kar5p extraction from nuclei. To determine whether  Kar5p was a lumenal, integral, or peripheral membrane protein, a  purified membrane fraction (derived from MS3987 after 2.5-h  α-factor induction) was subjected to various extraction conditions  (see Materials and Methods): 1 M NaCl; 0.1 M Na2CO3, pH 11.0;  2 M urea; 1% Triton X-100; 1% Triton X-100, 0.5 M NaCl; and  buffer alone. As a control, an equivalent membrane fraction from  the kar5-Δ strain, MS3986, was used to confirm the identity of the  protein species. After incubation for 1 h at 4°C, the samples were  centrifuged at 128,000 g. The supernatant was collected, and the  pellet was resuspended in the original volume. Equal volumes of  supernatant (S) and pellet (P) were analyzed by Western blot.  Blots were probed with anti-Sec61p, anti-Kar2p, anti-Sec72p, and  anti-Kar5p antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140214&req=5

Figure 7: Kar5p extraction from nuclei. To determine whether Kar5p was a lumenal, integral, or peripheral membrane protein, a purified membrane fraction (derived from MS3987 after 2.5-h α-factor induction) was subjected to various extraction conditions (see Materials and Methods): 1 M NaCl; 0.1 M Na2CO3, pH 11.0; 2 M urea; 1% Triton X-100; 1% Triton X-100, 0.5 M NaCl; and buffer alone. As a control, an equivalent membrane fraction from the kar5-Δ strain, MS3986, was used to confirm the identity of the protein species. After incubation for 1 h at 4°C, the samples were centrifuged at 128,000 g. The supernatant was collected, and the pellet was resuspended in the original volume. Equal volumes of supernatant (S) and pellet (P) were analyzed by Western blot. Blots were probed with anti-Sec61p, anti-Kar2p, anti-Sec72p, and anti-Kar5p antibodies.
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