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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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The KAR5 sequence. (A) The DNA sequence of the  2.8-kb minimal complementing region was determined. All yeast  DNA sequences on this fragment upstream of the structural gene  are shown (−259 to +3). This region contained all sequence elements required for the regulation of the KAR5 gene. Within this  region are two PREs at position −140 (TGTGTCA) and −122  (TGTTTTA) that match the consensus PRE (TGTTTCA) in 6/7  residues. The nucleotide numbers are indicated to the left of sequence. A potential transcriptional TATA element is underlined;  the PRE homologous sequences are indicated by the reverse font.  (B) The translated protein corresponding to KAR5 (YMR065W)  is shown. The KAR5 structural gene began at nucleotide 260 and  ended at nucleotide 1762 and potentially encodes a 504–amino  acid protein. The hydrophobic regions are indicated by the reverse font, and the potential coiled-coils are indicated by double  underscoring. The one difference between our DNA sequence  and that produced by the Yeast Genome Sequence Consortium is  indicated at residue 5, which is a leucine in our sequence. The  amino acid residue number is indicated on the left.
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Figure 2: The KAR5 sequence. (A) The DNA sequence of the 2.8-kb minimal complementing region was determined. All yeast DNA sequences on this fragment upstream of the structural gene are shown (−259 to +3). This region contained all sequence elements required for the regulation of the KAR5 gene. Within this region are two PREs at position −140 (TGTGTCA) and −122 (TGTTTTA) that match the consensus PRE (TGTTTCA) in 6/7 residues. The nucleotide numbers are indicated to the left of sequence. A potential transcriptional TATA element is underlined; the PRE homologous sequences are indicated by the reverse font. (B) The translated protein corresponding to KAR5 (YMR065W) is shown. The KAR5 structural gene began at nucleotide 260 and ended at nucleotide 1762 and potentially encodes a 504–amino acid protein. The hydrophobic regions are indicated by the reverse font, and the potential coiled-coils are indicated by double underscoring. The one difference between our DNA sequence and that produced by the Yeast Genome Sequence Consortium is indicated at residue 5, which is a leucine in our sequence. The amino acid residue number is indicated on the left.

Mentions: To analyze the KAR5 gene, the DNA of the smallest (2.8-kb HindIII) complementing subclone (pMR2710) was sequenced. The complementing subclone contained only one complete open reading frame of 1,512 bp, encoding a protein predicted to be 504 amino acids in length (Fig. 2 B) with a molecular mass of 58.4 kD. The two disruption alleles (pMR2741 and pMR2869) delete large segments of this open reading frame (see Fig. 1), and neither complemented the kar5-1162 karyogamy defect. Using the 2.8-kb subclone as the probe, the KAR5 gene was physically mapped by hybridization with whole chromosome blots and lambda prime clone filters (Riles et al., 1993) to chromosome XIII near the ADH3 gene. The gene has also been sequenced by the Saccharomyces genome sequencing project and corresponds to hypothetical protein YMR065W. The two sequences of KAR5 concur, with the exception of 1 bp difference at residue 5. Our sequence predicts a leucine at this site, whereas the sequencing project predicts an arginine.


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

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

The KAR5 sequence. (A) The DNA sequence of the  2.8-kb minimal complementing region was determined. All yeast  DNA sequences on this fragment upstream of the structural gene  are shown (−259 to +3). This region contained all sequence elements required for the regulation of the KAR5 gene. Within this  region are two PREs at position −140 (TGTGTCA) and −122  (TGTTTTA) that match the consensus PRE (TGTTTCA) in 6/7  residues. The nucleotide numbers are indicated to the left of sequence. A potential transcriptional TATA element is underlined;  the PRE homologous sequences are indicated by the reverse font.  (B) The translated protein corresponding to KAR5 (YMR065W)  is shown. The KAR5 structural gene began at nucleotide 260 and  ended at nucleotide 1762 and potentially encodes a 504–amino  acid protein. The hydrophobic regions are indicated by the reverse font, and the potential coiled-coils are indicated by double  underscoring. The one difference between our DNA sequence  and that produced by the Yeast Genome Sequence Consortium is  indicated at residue 5, which is a leucine in our sequence. The  amino acid residue number is indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: The KAR5 sequence. (A) The DNA sequence of the 2.8-kb minimal complementing region was determined. All yeast DNA sequences on this fragment upstream of the structural gene are shown (−259 to +3). This region contained all sequence elements required for the regulation of the KAR5 gene. Within this region are two PREs at position −140 (TGTGTCA) and −122 (TGTTTTA) that match the consensus PRE (TGTTTCA) in 6/7 residues. The nucleotide numbers are indicated to the left of sequence. A potential transcriptional TATA element is underlined; the PRE homologous sequences are indicated by the reverse font. (B) The translated protein corresponding to KAR5 (YMR065W) is shown. The KAR5 structural gene began at nucleotide 260 and ended at nucleotide 1762 and potentially encodes a 504–amino acid protein. The hydrophobic regions are indicated by the reverse font, and the potential coiled-coils are indicated by double underscoring. The one difference between our DNA sequence and that produced by the Yeast Genome Sequence Consortium is indicated at residue 5, which is a leucine in our sequence. The amino acid residue number is indicated on the left.
Mentions: To analyze the KAR5 gene, the DNA of the smallest (2.8-kb HindIII) complementing subclone (pMR2710) was sequenced. The complementing subclone contained only one complete open reading frame of 1,512 bp, encoding a protein predicted to be 504 amino acids in length (Fig. 2 B) with a molecular mass of 58.4 kD. The two disruption alleles (pMR2741 and pMR2869) delete large segments of this open reading frame (see Fig. 1), and neither complemented the kar5-1162 karyogamy defect. Using the 2.8-kb subclone as the probe, the KAR5 gene was physically mapped by hybridization with whole chromosome blots and lambda prime clone filters (Riles et al., 1993) to chromosome XIII near the ADH3 gene. The gene has also been sequenced by the Saccharomyces genome sequencing project and corresponds to hypothetical protein YMR065W. The two sequences of KAR5 concur, with the exception of 1 bp difference at residue 5. Our sequence predicts a leucine at this site, whereas the sequencing project predicts an arginine.

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