Limits...
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.

Show MeSH

Related in: MedlinePlus

kar5-1162–suppressing subclones. To map the two genomic library isolates containing the KAR5 gene and flanking  DNA, the plasmids were analyzed by restriction mapping (A,  AvrII; B, BamHI; R, EcoRI; H, HindIII; K, KpnI; S, SalI; Sc,  SacI; X, XhoI; Xb, XbaI; designated to scale). To determine the  minimal KAR5 complementing DNA, the indicated subclones  were transformed into a kar5-1162 strain (MS2686). Subclones  that suppressed the kar5-1162 karyogamy defect are indicated by  black bars; open bars indicate no suppression. The minimal complementing DNA subcloned in pMR2710 was used to create the  two disruption constructs pMR2741 and pMR2869. In pMR2741,  the URA3 gene replaces the deleted KAR5 sequence indicated  (kar5-Δ1); in pMR2869, LEU2 replaces deleted KAR5 sequence  (kar5-Δ2).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140214&req=5

Figure 1: kar5-1162–suppressing subclones. To map the two genomic library isolates containing the KAR5 gene and flanking DNA, the plasmids were analyzed by restriction mapping (A, AvrII; B, BamHI; R, EcoRI; H, HindIII; K, KpnI; S, SalI; Sc, SacI; X, XhoI; Xb, XbaI; designated to scale). To determine the minimal KAR5 complementing DNA, the indicated subclones were transformed into a kar5-1162 strain (MS2686). Subclones that suppressed the kar5-1162 karyogamy defect are indicated by black bars; open bars indicate no suppression. The minimal complementing DNA subcloned in pMR2710 was used to create the two disruption constructs pMR2741 and pMR2869. In pMR2741, the URA3 gene replaces the deleted KAR5 sequence indicated (kar5-Δ1); in pMR2869, LEU2 replaces deleted KAR5 sequence (kar5-Δ2).

Mentions: To clone the KAR5 gene, the kar5-1162 mutant strain (MS2686) was transformed with a yeast centromere vector– based genomic library (Rose et al., 1987). A total of 86,000 transformants (57 genomic equivalents) were screened by replica plating onto lawns of kar5-1162 cells of the opposite mating type (MS2685) and allowing them to mate for only 3 h before replica plating to media to select for diploids (interrupted plate matings). Several mating-proficient transformants were isolated; two of these suppressed the kar5-1162 mating defect after isolation of the plasmids in E. coli and retransformation back into kar5-1162 cells. Suppression was therefore plasmid-linked, and the genomic DNA inserts on the plasmids had related restriction maps (Fig. 1).


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

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

kar5-1162–suppressing subclones. To map the two genomic library isolates containing the KAR5 gene and flanking  DNA, the plasmids were analyzed by restriction mapping (A,  AvrII; B, BamHI; R, EcoRI; H, HindIII; K, KpnI; S, SalI; Sc,  SacI; X, XhoI; Xb, XbaI; designated to scale). To determine the  minimal KAR5 complementing DNA, the indicated subclones  were transformed into a kar5-1162 strain (MS2686). Subclones  that suppressed the kar5-1162 karyogamy defect are indicated by  black bars; open bars indicate no suppression. The minimal complementing DNA subcloned in pMR2710 was used to create the  two disruption constructs pMR2741 and pMR2869. In pMR2741,  the URA3 gene replaces the deleted KAR5 sequence indicated  (kar5-Δ1); in pMR2869, LEU2 replaces deleted KAR5 sequence  (kar5-Δ2).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: kar5-1162–suppressing subclones. To map the two genomic library isolates containing the KAR5 gene and flanking DNA, the plasmids were analyzed by restriction mapping (A, AvrII; B, BamHI; R, EcoRI; H, HindIII; K, KpnI; S, SalI; Sc, SacI; X, XhoI; Xb, XbaI; designated to scale). To determine the minimal KAR5 complementing DNA, the indicated subclones were transformed into a kar5-1162 strain (MS2686). Subclones that suppressed the kar5-1162 karyogamy defect are indicated by black bars; open bars indicate no suppression. The minimal complementing DNA subcloned in pMR2710 was used to create the two disruption constructs pMR2741 and pMR2869. In pMR2741, the URA3 gene replaces the deleted KAR5 sequence indicated (kar5-Δ1); in pMR2869, LEU2 replaces deleted KAR5 sequence (kar5-Δ2).
Mentions: To clone the KAR5 gene, the kar5-1162 mutant strain (MS2686) was transformed with a yeast centromere vector– based genomic library (Rose et al., 1987). A total of 86,000 transformants (57 genomic equivalents) were screened by replica plating onto lawns of kar5-1162 cells of the opposite mating type (MS2685) and allowing them to mate for only 3 h before replica plating to media to select for diploids (interrupted plate matings). Several mating-proficient transformants were isolated; two of these suppressed the kar5-1162 mating defect after isolation of the plasmids in E. coli and retransformation back into kar5-1162 cells. Suppression was therefore plasmid-linked, and the genomic DNA inserts on the plasmids had related restriction maps (Fig. 1).

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