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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: 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.

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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KAR5 is a pheromone-induced gene. Northern blots of mRNA from (A)  a vegetatively growing wild-type strain (MY3265)—a low  level of KAR5 message could  be detected; (B) MY3265 after addition of α-factor—a dramatic induction of KAR5 transcription was observed; (C) a vegetatively growing ste12-Δ strain  (MY3264); and (D) the ste12-Δ strain after addition of pheromone. In the ste12-Δ strain, the levels of KAR5 transcripts were  the same before and after addition of pheromone.
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Figure 4: KAR5 is a pheromone-induced gene. Northern blots of mRNA from (A) a vegetatively growing wild-type strain (MY3265)—a low level of KAR5 message could be detected; (B) MY3265 after addition of α-factor—a dramatic induction of KAR5 transcription was observed; (C) a vegetatively growing ste12-Δ strain (MY3264); and (D) the ste12-Δ strain after addition of pheromone. In the ste12-Δ strain, the levels of KAR5 transcripts were the same before and after addition of pheromone.

Mentions: The PRE sequence motif found upstream of most pheromone-inducible genes mediates transcriptional induction in response to activation of the pheromone-activated protein kinase pathway by mating factor. Because of the PREs observed upstream of KAR5, we tested directly whether the expression of KAR5 was induced upon pheromone treatment. Total RNA was isolated from strains before and after α-factor induction, and equal amounts were analyzed by RNA-blot hybridization (Fig. 4). The observed size of the KAR5 mRNA, 2.1 kb, was consistent with the predicted length of the KAR5 open reading frame. KAR5 mRNA was present at low levels in vegetatively growing cells (more readily apparent after longer exposures of the autoradiograph) and was markedly induced after α-factor treatment (Fig. 4, lane B). In a ste12-Δ strain, the level of KAR5 mRNA did not increase, indicating that induction required the STE12 transcription factor and the mating-response signal transduction pathway. The vegetative level of KAR5 transcription was not higher in diploids and was not present in kar5-Δ strains (data not shown). Induction by mating pheromone is consistent with a specific role for Kar5p in nuclear fusion during mating.


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

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

KAR5 is a pheromone-induced gene. Northern blots of mRNA from (A)  a vegetatively growing wild-type strain (MY3265)—a low  level of KAR5 message could  be detected; (B) MY3265 after addition of α-factor—a dramatic induction of KAR5 transcription was observed; (C) a vegetatively growing ste12-Δ strain  (MY3264); and (D) the ste12-Δ strain after addition of pheromone. In the ste12-Δ strain, the levels of KAR5 transcripts were  the same before and after addition of pheromone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140214&req=5

Figure 4: KAR5 is a pheromone-induced gene. Northern blots of mRNA from (A) a vegetatively growing wild-type strain (MY3265)—a low level of KAR5 message could be detected; (B) MY3265 after addition of α-factor—a dramatic induction of KAR5 transcription was observed; (C) a vegetatively growing ste12-Δ strain (MY3264); and (D) the ste12-Δ strain after addition of pheromone. In the ste12-Δ strain, the levels of KAR5 transcripts were the same before and after addition of pheromone.
Mentions: The PRE sequence motif found upstream of most pheromone-inducible genes mediates transcriptional induction in response to activation of the pheromone-activated protein kinase pathway by mating factor. Because of the PREs observed upstream of KAR5, we tested directly whether the expression of KAR5 was induced upon pheromone treatment. Total RNA was isolated from strains before and after α-factor induction, and equal amounts were analyzed by RNA-blot hybridization (Fig. 4). The observed size of the KAR5 mRNA, 2.1 kb, was consistent with the predicted length of the KAR5 open reading frame. KAR5 mRNA was present at low levels in vegetatively growing cells (more readily apparent after longer exposures of the autoradiograph) and was markedly induced after α-factor treatment (Fig. 4, lane B). In a ste12-Δ strain, the level of KAR5 mRNA did not increase, indicating that induction required the STE12 transcription factor and the mating-response signal transduction pathway. The vegetative level of KAR5 transcription was not higher in diploids and was not present in kar5-Δ strains (data not shown). Induction by mating pheromone is consistent with a specific role for Kar5p in nuclear fusion during mating.

Bottom Line: 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.

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