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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 contained within the ER/nuclear envelope  lumen. Protease protection was used to determined if Kar5p was  exposed to the cytoplasm or sequestered within the ER between  the inner and outer nuclear envelopes. Membrane fractions from  pheromone-induced cells (MS3987) were either treated with proteinase K or proteinase K with Triton X-100 for 0, 1, 2, 5, 10, and  20 min. After protease treatment, proteins were separated by  SDS gel electrophoresis, transferred to nitrocellulose, and probed  with anti-Kar5p, anti-Kar2p, or anti-Sec72p antibodies. Sec72p is  an ER protein exposed to the cytoplasm (Feldheim and Schekman, 1994), and Kar2p is an ER lumenal protein (Rose et al.,  1989). Sec72p was nearly entirely digested by protease after 20  min regardless of whether Triton X-100 was added. Kar2p and  Kar5p were protease protected in the absence of detergent for  >20 min. Kar2p and Kar5p were not inherently protease resistant  since neither was resistant to proteinase K when Triton X-100  was added; the solubilization of the nuclear membrane allowed  the protease access to lumen proteins. Thus, both Kar2p and  Kar5p are ER/nuclear envelope lumenal proteins.
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Figure 8: Kar5p was contained within the ER/nuclear envelope lumen. Protease protection was used to determined if Kar5p was exposed to the cytoplasm or sequestered within the ER between the inner and outer nuclear envelopes. Membrane fractions from pheromone-induced cells (MS3987) were either treated with proteinase K or proteinase K with Triton X-100 for 0, 1, 2, 5, 10, and 20 min. After protease treatment, proteins were separated by SDS gel electrophoresis, transferred to nitrocellulose, and probed with anti-Kar5p, anti-Kar2p, or anti-Sec72p antibodies. Sec72p is an ER protein exposed to the cytoplasm (Feldheim and Schekman, 1994), and Kar2p is an ER lumenal protein (Rose et al., 1989). Sec72p was nearly entirely digested by protease after 20 min regardless of whether Triton X-100 was added. Kar2p and Kar5p were protease protected in the absence of detergent for >20 min. Kar2p and Kar5p were not inherently protease resistant since neither was resistant to proteinase K when Triton X-100 was added; the solubilization of the nuclear membrane allowed the protease access to lumen proteins. Thus, both Kar2p and Kar5p are ER/nuclear envelope lumenal proteins.

Mentions: To determine the orientation of Kar5p with respect to the ER membrane/nuclear envelope, the sensitivity of Kar5p to protease was examined. Proteins exposed on the surface of the membrane are sensitive to exogenous proteases. In contrast, proteins contained within the lumen between the inner and outer nuclear membranes are resistant to the addition of proteases. Lumenal proteins would become sensitive to proteases upon addition of detergent to disrupt the membrane. Sec72p, a peripheral ER/nuclear envelope protein (Feldheim and Schekman, 1994), was sensitive to proteinase K (Fig. 8) regardless of the presence of detergent. In contrast, Kar2p, an ER-lumenal protein (Normington et al., 1989; Rose et al., 1989), was resistant to proteinase K in the absence of detergent but sensitive in the presence of detergent. Like Kar2p, Kar5p was resistant to protease in the absence of detergent but sensitive in its presence. The detergent-dependent protease sensitivity of Kar5p indicates that the protein was not inherently protease resistant. Furthermore, the fact that Kar5p was not significantly trimmed by protease in the absence of detergent suggests that very little if any of the protein is exposed on the cytoplasmic side of the membrane. This is consistent with the observation that the putative transmembrane domains are present at the extreme carboxyl terminus of the protein. Taken together, these results demonstrate that Kar5p is a nuclear envelope/ER membrane protein, anchored in the membrane by one or more hydrophobic transmembrane domains and topologically oriented such that most or all of the hydrophilic portion of the protein is situated within the ER/nuclear envelope lumen.


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

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

Kar5p was contained within the ER/nuclear envelope  lumen. Protease protection was used to determined if Kar5p was  exposed to the cytoplasm or sequestered within the ER between  the inner and outer nuclear envelopes. Membrane fractions from  pheromone-induced cells (MS3987) were either treated with proteinase K or proteinase K with Triton X-100 for 0, 1, 2, 5, 10, and  20 min. After protease treatment, proteins were separated by  SDS gel electrophoresis, transferred to nitrocellulose, and probed  with anti-Kar5p, anti-Kar2p, or anti-Sec72p antibodies. Sec72p is  an ER protein exposed to the cytoplasm (Feldheim and Schekman, 1994), and Kar2p is an ER lumenal protein (Rose et al.,  1989). Sec72p was nearly entirely digested by protease after 20  min regardless of whether Triton X-100 was added. Kar2p and  Kar5p were protease protected in the absence of detergent for  >20 min. Kar2p and Kar5p were not inherently protease resistant  since neither was resistant to proteinase K when Triton X-100  was added; the solubilization of the nuclear membrane allowed  the protease access to lumen proteins. Thus, both Kar2p and  Kar5p are ER/nuclear envelope lumenal proteins.
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Figure 8: Kar5p was contained within the ER/nuclear envelope lumen. Protease protection was used to determined if Kar5p was exposed to the cytoplasm or sequestered within the ER between the inner and outer nuclear envelopes. Membrane fractions from pheromone-induced cells (MS3987) were either treated with proteinase K or proteinase K with Triton X-100 for 0, 1, 2, 5, 10, and 20 min. After protease treatment, proteins were separated by SDS gel electrophoresis, transferred to nitrocellulose, and probed with anti-Kar5p, anti-Kar2p, or anti-Sec72p antibodies. Sec72p is an ER protein exposed to the cytoplasm (Feldheim and Schekman, 1994), and Kar2p is an ER lumenal protein (Rose et al., 1989). Sec72p was nearly entirely digested by protease after 20 min regardless of whether Triton X-100 was added. Kar2p and Kar5p were protease protected in the absence of detergent for >20 min. Kar2p and Kar5p were not inherently protease resistant since neither was resistant to proteinase K when Triton X-100 was added; the solubilization of the nuclear membrane allowed the protease access to lumen proteins. Thus, both Kar2p and Kar5p are ER/nuclear envelope lumenal proteins.
Mentions: To determine the orientation of Kar5p with respect to the ER membrane/nuclear envelope, the sensitivity of Kar5p to protease was examined. Proteins exposed on the surface of the membrane are sensitive to exogenous proteases. In contrast, proteins contained within the lumen between the inner and outer nuclear membranes are resistant to the addition of proteases. Lumenal proteins would become sensitive to proteases upon addition of detergent to disrupt the membrane. Sec72p, a peripheral ER/nuclear envelope protein (Feldheim and Schekman, 1994), was sensitive to proteinase K (Fig. 8) regardless of the presence of detergent. In contrast, Kar2p, an ER-lumenal protein (Normington et al., 1989; Rose et al., 1989), was resistant to proteinase K in the absence of detergent but sensitive in the presence of detergent. Like Kar2p, Kar5p was resistant to protease in the absence of detergent but sensitive in its presence. The detergent-dependent protease sensitivity of Kar5p indicates that the protein was not inherently protease resistant. Furthermore, the fact that Kar5p was not significantly trimmed by protease in the absence of detergent suggests that very little if any of the protein is exposed on the cytoplasmic side of the membrane. This is consistent with the observation that the putative transmembrane domains are present at the extreme carboxyl terminus of the protein. Taken together, these results demonstrate that Kar5p is a nuclear envelope/ER membrane protein, anchored in the membrane by one or more hydrophobic transmembrane domains and topologically oriented such that most or all of the hydrophilic portion of the protein is situated within the ER/nuclear envelope lumen.

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