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The high osmolarity glycerol response (HOG) MAP kinase pathway controls localization of a yeast golgi glycosyltransferase.

Reynolds TB, Hopkins BD, Lyons MR, Graham TR - J. Cell Biol. (1998)

Bottom Line: Biol.We have found that basal signaling through the HOG pathway is required to localize Mnn1-s to the Golgi in standard osmotic conditions.Mutations in HOG1 and LDR1 also perturb localization of intact Mnn1p, resulting in its loss from early Golgi compartments and a concomitant increase of Mnn1p in later Golgi compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.

ABSTRACT
The yeast alpha-1,3-mannosyltransferase (Mnn1p) is localized to the Golgi by independent transmembrane and lumenal domain signals. The lumenal domain is localized to the Golgi complex when expressed as a soluble form (Mnn1-s) by exchange of its transmembrane domain for a cleavable signal sequence (Graham, T. R., and V. A. Krasnov. 1995. Mol. Biol. Cell. 6:809-824). Mutants that failed to retain the lumenal domain in the Golgi complex, called lumenal domain retention (ldr) mutants, were isolated by screening mutagenized yeast colonies for those that secreted Mnn1-s. Two genes were identified by this screen, HOG1, a gene encoding a mitogen-activated protein kinase (MAPK) that functions in the high osmolarity glycerol (HOG) pathway, and LDR1. We have found that basal signaling through the HOG pathway is required to localize Mnn1-s to the Golgi in standard osmotic conditions. Mutations in HOG1 and LDR1 also perturb localization of intact Mnn1p, resulting in its loss from early Golgi compartments and a concomitant increase of Mnn1p in later Golgi compartments.

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Strain TRY120 carries a mutant allele of HOG1. (a)  Colony blot showing complementation of the TRY120 Ldr− phenotype by pTR-26 and pHOG1. TRY120 pHOG1, TRY120 pTR-26, TRY120 pRS315, and WT (SEY6210 pRS315) were streaked  out in duplicate and examined by colony blot. All strains carry  pMNN1-s. (b) Primers that annealed to vector sequences on  pTR-26 that flanked the yeast genomic insert were used to sequence the ends of the insert. These sequences were used in a  BLAST search of the complete yeast genome database to reveal  the genomic insert represented diagrammatically. The HOG1  gene is depicted as an open box and sites used to subclone it into  the pRS313 vector to make pHOG1 are shown as well. (c) A diploid made from TRY120 crossed to AMY36 (hog1Δ) fails to grow  on 1.0 M sorbitol plates. Wild-type strains SEY6210 and FY250  (WT*) were streaked onto a YPD plate containing 1.0 M sorbitol  along with haploid strains JSY2092 (SEY6210 pbsΔ), AMY36  (hog1Δ*), and TRY120. Diploids were made by crossing TRY120  with JSY2092 (TRY120/JSY2092) and AMY36 (TRY120/ AMY36) and these diploids were also streaked on the 1.0 M sorbitol to test for complementation of the pbs2Δ- and hog1Δ-associated growth defects, respectively.
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Figure 3: Strain TRY120 carries a mutant allele of HOG1. (a) Colony blot showing complementation of the TRY120 Ldr− phenotype by pTR-26 and pHOG1. TRY120 pHOG1, TRY120 pTR-26, TRY120 pRS315, and WT (SEY6210 pRS315) were streaked out in duplicate and examined by colony blot. All strains carry pMNN1-s. (b) Primers that annealed to vector sequences on pTR-26 that flanked the yeast genomic insert were used to sequence the ends of the insert. These sequences were used in a BLAST search of the complete yeast genome database to reveal the genomic insert represented diagrammatically. The HOG1 gene is depicted as an open box and sites used to subclone it into the pRS313 vector to make pHOG1 are shown as well. (c) A diploid made from TRY120 crossed to AMY36 (hog1Δ) fails to grow on 1.0 M sorbitol plates. Wild-type strains SEY6210 and FY250 (WT*) were streaked onto a YPD plate containing 1.0 M sorbitol along with haploid strains JSY2092 (SEY6210 pbsΔ), AMY36 (hog1Δ*), and TRY120. Diploids were made by crossing TRY120 with JSY2092 (TRY120/JSY2092) and AMY36 (TRY120/ AMY36) and these diploids were also streaked on the 1.0 M sorbitol to test for complementation of the pbs2Δ- and hog1Δ-associated growth defects, respectively.

Mentions: The wild-type gene represented by the ldr mutation in TRY120 was cloned by screening a single-copy yeast genomic library for plasmids which complemented the Ldr− mutant phenotype of TRY120. This was done by analyzing transformed colonies for those that produced a lighter signal on colony blots relative to neighboring noncomplemented colonies. TRY120 was used because the mutation it carried gave the strongest Ldr− phenotype in both mating types after two backcrosses. A single clone was isolated from 15,000 transformants that could complement the Ldr− phenotype in a plasmid-linked manner (Fig. 3 A). The ends of the insert were sequenced and compared with the yeast genome sequence to identify the chromosome XII fragment shown in Fig. 3 B. Deletion analysis and subcloning revealed that the HOG1 gene was responsible for complementing the Ldr− phenotype (Fig. 3 A and Materials and Methods).


The high osmolarity glycerol response (HOG) MAP kinase pathway controls localization of a yeast golgi glycosyltransferase.

Reynolds TB, Hopkins BD, Lyons MR, Graham TR - J. Cell Biol. (1998)

Strain TRY120 carries a mutant allele of HOG1. (a)  Colony blot showing complementation of the TRY120 Ldr− phenotype by pTR-26 and pHOG1. TRY120 pHOG1, TRY120 pTR-26, TRY120 pRS315, and WT (SEY6210 pRS315) were streaked  out in duplicate and examined by colony blot. All strains carry  pMNN1-s. (b) Primers that annealed to vector sequences on  pTR-26 that flanked the yeast genomic insert were used to sequence the ends of the insert. These sequences were used in a  BLAST search of the complete yeast genome database to reveal  the genomic insert represented diagrammatically. The HOG1  gene is depicted as an open box and sites used to subclone it into  the pRS313 vector to make pHOG1 are shown as well. (c) A diploid made from TRY120 crossed to AMY36 (hog1Δ) fails to grow  on 1.0 M sorbitol plates. Wild-type strains SEY6210 and FY250  (WT*) were streaked onto a YPD plate containing 1.0 M sorbitol  along with haploid strains JSY2092 (SEY6210 pbsΔ), AMY36  (hog1Δ*), and TRY120. Diploids were made by crossing TRY120  with JSY2092 (TRY120/JSY2092) and AMY36 (TRY120/ AMY36) and these diploids were also streaked on the 1.0 M sorbitol to test for complementation of the pbs2Δ- and hog1Δ-associated growth defects, respectively.
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Related In: Results  -  Collection

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Figure 3: Strain TRY120 carries a mutant allele of HOG1. (a) Colony blot showing complementation of the TRY120 Ldr− phenotype by pTR-26 and pHOG1. TRY120 pHOG1, TRY120 pTR-26, TRY120 pRS315, and WT (SEY6210 pRS315) were streaked out in duplicate and examined by colony blot. All strains carry pMNN1-s. (b) Primers that annealed to vector sequences on pTR-26 that flanked the yeast genomic insert were used to sequence the ends of the insert. These sequences were used in a BLAST search of the complete yeast genome database to reveal the genomic insert represented diagrammatically. The HOG1 gene is depicted as an open box and sites used to subclone it into the pRS313 vector to make pHOG1 are shown as well. (c) A diploid made from TRY120 crossed to AMY36 (hog1Δ) fails to grow on 1.0 M sorbitol plates. Wild-type strains SEY6210 and FY250 (WT*) were streaked onto a YPD plate containing 1.0 M sorbitol along with haploid strains JSY2092 (SEY6210 pbsΔ), AMY36 (hog1Δ*), and TRY120. Diploids were made by crossing TRY120 with JSY2092 (TRY120/JSY2092) and AMY36 (TRY120/ AMY36) and these diploids were also streaked on the 1.0 M sorbitol to test for complementation of the pbs2Δ- and hog1Δ-associated growth defects, respectively.
Mentions: The wild-type gene represented by the ldr mutation in TRY120 was cloned by screening a single-copy yeast genomic library for plasmids which complemented the Ldr− mutant phenotype of TRY120. This was done by analyzing transformed colonies for those that produced a lighter signal on colony blots relative to neighboring noncomplemented colonies. TRY120 was used because the mutation it carried gave the strongest Ldr− phenotype in both mating types after two backcrosses. A single clone was isolated from 15,000 transformants that could complement the Ldr− phenotype in a plasmid-linked manner (Fig. 3 A). The ends of the insert were sequenced and compared with the yeast genome sequence to identify the chromosome XII fragment shown in Fig. 3 B. Deletion analysis and subcloning revealed that the HOG1 gene was responsible for complementing the Ldr− phenotype (Fig. 3 A and Materials and Methods).

Bottom Line: Biol.We have found that basal signaling through the HOG pathway is required to localize Mnn1-s to the Golgi in standard osmotic conditions.Mutations in HOG1 and LDR1 also perturb localization of intact Mnn1p, resulting in its loss from early Golgi compartments and a concomitant increase of Mnn1p in later Golgi compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.

ABSTRACT
The yeast alpha-1,3-mannosyltransferase (Mnn1p) is localized to the Golgi by independent transmembrane and lumenal domain signals. The lumenal domain is localized to the Golgi complex when expressed as a soluble form (Mnn1-s) by exchange of its transmembrane domain for a cleavable signal sequence (Graham, T. R., and V. A. Krasnov. 1995. Mol. Biol. Cell. 6:809-824). Mutants that failed to retain the lumenal domain in the Golgi complex, called lumenal domain retention (ldr) mutants, were isolated by screening mutagenized yeast colonies for those that secreted Mnn1-s. Two genes were identified by this screen, HOG1, a gene encoding a mitogen-activated protein kinase (MAPK) that functions in the high osmolarity glycerol (HOG) pathway, and LDR1. We have found that basal signaling through the HOG pathway is required to localize Mnn1-s to the Golgi in standard osmotic conditions. Mutations in HOG1 and LDR1 also perturb localization of intact Mnn1p, resulting in its loss from early Golgi compartments and a concomitant increase of Mnn1p in later Golgi compartments.

Show MeSH
Related in: MedlinePlus