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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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The HOG1 gene  does not complement the  Ldr− phenotype in TRY220.  (a) WT (SEY6210 pMNN1-s),  TRY220 pMNN1-s, and  TRY220 pMNN1-s pTR-26  were streaked out in duplicate and tested by colony  blot. (b) Summary table of  results from testing TRY120  (hog1-11) and TRY220 (with  or without) a plasmid borne  copy of the HOG1 gene for  growth on 1 M sorbitol plates  and for Mnn1-s secretion by  colony blot. Other ldr mutants were tested for growth  on 1 M sorbitol. s indicates  Mnn1-s was secreted and ns  indicates Mnn1-s was not secreted.
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Figure 4: The HOG1 gene does not complement the Ldr− phenotype in TRY220. (a) WT (SEY6210 pMNN1-s), TRY220 pMNN1-s, and TRY220 pMNN1-s pTR-26 were streaked out in duplicate and tested by colony blot. (b) Summary table of results from testing TRY120 (hog1-11) and TRY220 (with or without) a plasmid borne copy of the HOG1 gene for growth on 1 M sorbitol plates and for Mnn1-s secretion by colony blot. Other ldr mutants were tested for growth on 1 M sorbitol. s indicates Mnn1-s was secreted and ns indicates Mnn1-s was not secreted.

Mentions: hog1 mutants are unable to grow in conditions of osmotic stress such as agar plates containing 1.0 M sorbitol. We found that TRY120 was unable to grow on 1 M sorbitol plates (Figs. 3 C and 4 B) and the HOG1 gene expressed from a single-copy plasmid was able to complement the growth defect of TRY120 on 1 M sorbitol (Fig. 4 B). In addition, a diploid made by crossing TRY120 with a hog1Δ strain was also unable to grow on 1 M sorbitol (Fig. 3 C), and when this diploid was sporulated and the segregants examined by random spore analysis, 0 out of 209 segregants were able to grow on 1.5 M sorbitol plates confirming that TRY120 carries a mutant allele of HOG1. We now refer to this allele as hog1-11. Surprisingly, when one of the other ldr mutants, TRY220, was transformed with a plasmid containing the HOG1 gene, the Ldr− phenotype was not complemented (Fig. 4, A and B). In addition, TRY220 did not show a growth defect on 1 M sorbitol plates. These data indicated that the Ldr− phenotype exhibited by TRY220 was caused by a mutation in a gene other than HOG1. The other four ldr mutants isolated in this screen (TRY3 through TRY6 or their derivatives) were also found to grow on 1 M sorbitol (Fig. 4 B) suggesting that these strains also did not carry mutant alleles of the HOG1 gene. To clearly determine the number of genes represented by the remaining five ldr mutants, we analyzed tetrads generated from intercrossing these mutant strains. The results from this linkage analysis showed that all five mutants carried mutant alleles of the same gene, LDR1, which we refer to as ldr1-2 (TRY2) through ldr1-6 (TRY6; data not shown). The failure to produce diploids that were complemented for the Ldr− phenotype when derivatives of TRY1 (hog1-11) are crossed to any of the remaining five ldr mutants (Fig. 2 C, TRY121/ TRY320) appears to result from nonallelic noncomplementation, a genetic interaction that suggests a functional interaction between the encoded proteins. This is a specific genetic interaction between ldr1 and hog1, since a pmr1Δ strain complemented all of the mutants.


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)

The HOG1 gene  does not complement the  Ldr− phenotype in TRY220.  (a) WT (SEY6210 pMNN1-s),  TRY220 pMNN1-s, and  TRY220 pMNN1-s pTR-26  were streaked out in duplicate and tested by colony  blot. (b) Summary table of  results from testing TRY120  (hog1-11) and TRY220 (with  or without) a plasmid borne  copy of the HOG1 gene for  growth on 1 M sorbitol plates  and for Mnn1-s secretion by  colony blot. Other ldr mutants were tested for growth  on 1 M sorbitol. s indicates  Mnn1-s was secreted and ns  indicates Mnn1-s was not secreted.
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Figure 4: The HOG1 gene does not complement the Ldr− phenotype in TRY220. (a) WT (SEY6210 pMNN1-s), TRY220 pMNN1-s, and TRY220 pMNN1-s pTR-26 were streaked out in duplicate and tested by colony blot. (b) Summary table of results from testing TRY120 (hog1-11) and TRY220 (with or without) a plasmid borne copy of the HOG1 gene for growth on 1 M sorbitol plates and for Mnn1-s secretion by colony blot. Other ldr mutants were tested for growth on 1 M sorbitol. s indicates Mnn1-s was secreted and ns indicates Mnn1-s was not secreted.
Mentions: hog1 mutants are unable to grow in conditions of osmotic stress such as agar plates containing 1.0 M sorbitol. We found that TRY120 was unable to grow on 1 M sorbitol plates (Figs. 3 C and 4 B) and the HOG1 gene expressed from a single-copy plasmid was able to complement the growth defect of TRY120 on 1 M sorbitol (Fig. 4 B). In addition, a diploid made by crossing TRY120 with a hog1Δ strain was also unable to grow on 1 M sorbitol (Fig. 3 C), and when this diploid was sporulated and the segregants examined by random spore analysis, 0 out of 209 segregants were able to grow on 1.5 M sorbitol plates confirming that TRY120 carries a mutant allele of HOG1. We now refer to this allele as hog1-11. Surprisingly, when one of the other ldr mutants, TRY220, was transformed with a plasmid containing the HOG1 gene, the Ldr− phenotype was not complemented (Fig. 4, A and B). In addition, TRY220 did not show a growth defect on 1 M sorbitol plates. These data indicated that the Ldr− phenotype exhibited by TRY220 was caused by a mutation in a gene other than HOG1. The other four ldr mutants isolated in this screen (TRY3 through TRY6 or their derivatives) were also found to grow on 1 M sorbitol (Fig. 4 B) suggesting that these strains also did not carry mutant alleles of the HOG1 gene. To clearly determine the number of genes represented by the remaining five ldr mutants, we analyzed tetrads generated from intercrossing these mutant strains. The results from this linkage analysis showed that all five mutants carried mutant alleles of the same gene, LDR1, which we refer to as ldr1-2 (TRY2) through ldr1-6 (TRY6; data not shown). The failure to produce diploids that were complemented for the Ldr− phenotype when derivatives of TRY1 (hog1-11) are crossed to any of the remaining five ldr mutants (Fig. 2 C, TRY121/ TRY320) appears to result from nonallelic noncomplementation, a genetic interaction that suggests a functional interaction between the encoded proteins. This is a specific genetic interaction between ldr1 and hog1, since a pmr1Δ strain complemented all of the mutants.

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