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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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Examination of other mutants in the HOG pathway for  the Ldr− phenotype. Strains harboring  mutations in PBS2  (a) SSK2 SSK22 SHO1 (b) SSK1 (c) and SHO1 (d) were tested  for Mnn1-s secretion by the colony blot assay. A hog1 mutant and  isogenic wild-type strains were included in each plate. Strains  marked with an asterisk (*) are in the W303 background and all  other strains are in the SEY6210 background. All strains assayed  are carrying pMNN1-s.
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Figure 7: Examination of other mutants in the HOG pathway for the Ldr− phenotype. Strains harboring mutations in PBS2 (a) SSK2 SSK22 SHO1 (b) SSK1 (c) and SHO1 (d) were tested for Mnn1-s secretion by the colony blot assay. A hog1 mutant and isogenic wild-type strains were included in each plate. Strains marked with an asterisk (*) are in the W303 background and all other strains are in the SEY6210 background. All strains assayed are carrying pMNN1-s.

Mentions: The fact that the Hog1p MAPK is required for proper localization of Mnn1p in the Golgi complex in the absence of hyperosmotic stress raised the question as to whether signal transduction through the HOG pathway may have a function in retaining Golgi proteins in nonstressed cells. To test this, a pbs2Δ (MAPKKΔ) mutant was analyzed for the Ldr− phenotype by the colony blotting assay and, like hog1-11 mutants, was found to secrete Mnn1-s (Fig. 7 A), but pbs2Δ did not perturb localization of M39I (Fig. 5). These results implied that the rest of the HOG pathway may be involved in localization of the Mnn1p lumenal domain. To block the HOG pathway response to hyperosmotic stress upstream of Pbs2p, both of the redundant receptor pathways, Sho1p and Sln1p-Ypd1p, must be blocked (Maeda et al., 1995; Wurgler-Murphy and Saito, 1997). A triple mutant was tested in which the genes encoding the redundant MAPKKKs Ssk2p and Ssk22p that function in the Sln1p-Ypd1p receptor pathway and the gene encoding the Sho1p receptor were all three knocked out and therefore has a block in both input branches of the HOG pathway (see Fig. 8). This triple mutant, ssk2Δ ssk22Δ sho1Δ, also mislocalized Mnn1-s as seen in Fig. 7 B. These results strongly suggest that a basal level of signaling through the entire HOG pathway in the absence of osmotic stress is required for Mnn1p localization. However, these results do not rule out the possibility that only one input branch and not both are involved. To test this, each branch was individually knocked out. An ssk1Δ mutation knocks out the Sln1p-Ypd1p osmosensor pathway (Posas et al., 1996) and the sho1Δ mutation blocks the alternative pathway (Maeda et al., 1995). Neither the ssk1Δ mutation alone nor the sho1Δ mutation alone resulted in Mnn1-s secretion (Fig. 7, C and D). Therefore, a basal level of Hog1p activation through either branch of the pathway is sufficient to maintain proper localization of Mnn1-s.


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)

Examination of other mutants in the HOG pathway for  the Ldr− phenotype. Strains harboring  mutations in PBS2  (a) SSK2 SSK22 SHO1 (b) SSK1 (c) and SHO1 (d) were tested  for Mnn1-s secretion by the colony blot assay. A hog1 mutant and  isogenic wild-type strains were included in each plate. Strains  marked with an asterisk (*) are in the W303 background and all  other strains are in the SEY6210 background. All strains assayed  are carrying pMNN1-s.
© Copyright Policy
Related In: Results  -  Collection

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Figure 7: Examination of other mutants in the HOG pathway for the Ldr− phenotype. Strains harboring mutations in PBS2 (a) SSK2 SSK22 SHO1 (b) SSK1 (c) and SHO1 (d) were tested for Mnn1-s secretion by the colony blot assay. A hog1 mutant and isogenic wild-type strains were included in each plate. Strains marked with an asterisk (*) are in the W303 background and all other strains are in the SEY6210 background. All strains assayed are carrying pMNN1-s.
Mentions: The fact that the Hog1p MAPK is required for proper localization of Mnn1p in the Golgi complex in the absence of hyperosmotic stress raised the question as to whether signal transduction through the HOG pathway may have a function in retaining Golgi proteins in nonstressed cells. To test this, a pbs2Δ (MAPKKΔ) mutant was analyzed for the Ldr− phenotype by the colony blotting assay and, like hog1-11 mutants, was found to secrete Mnn1-s (Fig. 7 A), but pbs2Δ did not perturb localization of M39I (Fig. 5). These results implied that the rest of the HOG pathway may be involved in localization of the Mnn1p lumenal domain. To block the HOG pathway response to hyperosmotic stress upstream of Pbs2p, both of the redundant receptor pathways, Sho1p and Sln1p-Ypd1p, must be blocked (Maeda et al., 1995; Wurgler-Murphy and Saito, 1997). A triple mutant was tested in which the genes encoding the redundant MAPKKKs Ssk2p and Ssk22p that function in the Sln1p-Ypd1p receptor pathway and the gene encoding the Sho1p receptor were all three knocked out and therefore has a block in both input branches of the HOG pathway (see Fig. 8). This triple mutant, ssk2Δ ssk22Δ sho1Δ, also mislocalized Mnn1-s as seen in Fig. 7 B. These results strongly suggest that a basal level of signaling through the entire HOG pathway in the absence of osmotic stress is required for Mnn1p localization. However, these results do not rule out the possibility that only one input branch and not both are involved. To test this, each branch was individually knocked out. An ssk1Δ mutation knocks out the Sln1p-Ypd1p osmosensor pathway (Posas et al., 1996) and the sho1Δ mutation blocks the alternative pathway (Maeda et al., 1995). Neither the ssk1Δ mutation alone nor the sho1Δ mutation alone resulted in Mnn1-s secretion (Fig. 7, C and D). Therefore, a basal level of Hog1p activation through either branch of the pathway is sufficient to maintain proper localization of Mnn1-s.

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