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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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(a) Strains carrying  mutations in the PMR1 gene  mislocalize and secrete Mnn1-s. Strains were streaked out and  then analyzed by the colony blot assay as described in Materials  and Methods. All of the strains except WT 2μ MNN1-s are carrying pMNN1-s. Strains marked with an * indicate that it is a strain  with a genetic background other than SEY6210. The strain  abbreviations in the figure are as follows: WT 2μ MNN1-s  (SEY6210 pRS426 MNN1-s); WT (SEY6210); arf1Δ (6210  arf1Δ); chc1Δ* (GPY1103); pmr1Δ* (L3865); pmr1Δ (SEY6210  pmr1Δ); ret1-1 (TGY381); WT* (TGY383). (b) Summary of results from colony blot experiments with different secretory pathway mutants tested. All strains carry pMNN1-s. All vps strains  are in the SEY6210 background. mnn2* and mnn5* are the LB1-16A and LB65-5D strains respectively. (c) The results of a complementation test between two ldr mutants (TRY121 and  TRY320) is shown. All strains carry the pMNN1-s plasmid. (d)  The percent Mnn1-s secreted from each strain was determined by  Western blot as described in Materials and Methods. Mnn1-s is  expressed at a slightly higher level than Mnn1p and has a different mobility by SDS-PAGE due to differences in N-glycosylation. This allowed us to distinguish Mnn1-s from endogenous  Mnn1p in these experiments.
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Figure 2: (a) Strains carrying mutations in the PMR1 gene mislocalize and secrete Mnn1-s. Strains were streaked out and then analyzed by the colony blot assay as described in Materials and Methods. All of the strains except WT 2μ MNN1-s are carrying pMNN1-s. Strains marked with an * indicate that it is a strain with a genetic background other than SEY6210. The strain abbreviations in the figure are as follows: WT 2μ MNN1-s (SEY6210 pRS426 MNN1-s); WT (SEY6210); arf1Δ (6210 arf1Δ); chc1Δ* (GPY1103); pmr1Δ* (L3865); pmr1Δ (SEY6210 pmr1Δ); ret1-1 (TGY381); WT* (TGY383). (b) Summary of results from colony blot experiments with different secretory pathway mutants tested. All strains carry pMNN1-s. All vps strains are in the SEY6210 background. mnn2* and mnn5* are the LB1-16A and LB65-5D strains respectively. (c) The results of a complementation test between two ldr mutants (TRY121 and TRY320) is shown. All strains carry the pMNN1-s plasmid. (d) The percent Mnn1-s secreted from each strain was determined by Western blot as described in Materials and Methods. Mnn1-s is expressed at a slightly higher level than Mnn1p and has a different mobility by SDS-PAGE due to differences in N-glycosylation. This allowed us to distinguish Mnn1-s from endogenous Mnn1p in these experiments.

Mentions: The lumenal domain of Mnn1p (Fig. 1 B, Mnn1-s) is efficiently localized to the Golgi complex independently of the Mnn1p transmembrane domain. However, Mnn1-s is secreted from the cell when overexpressed from a multi-copy plasmid (Graham and Krasnov, 1995). This suggested that Mnn1-s may be retained by a saturable, receptor-mediated mechanism and that mutants that failed to properly sort Mnn1-s would secrete it. To detect such mutants, we used a colony blotting technique (Wilsbach and Payne, 1993) where yeast colonies growing on agar plates were overlaid with nitrocellulose discs for 16–20 h that were then washed free of yeast and probed with antibodies to Mnn1p. The colony blotting technique was tested using wild-type yeast expressing Mnn1-s on a single-copy plasmid as a negative control (Fig. 2 A, WT), and wild-type yeast overexpressing Mnn1-s from a multi-copy plasmid as a positive control (Fig. 2 A, WT 2μ MNN1-s). Colonies harboring the single-copy Mnn1-s plasmid produced a weak background signal and yeast overexpressing Mnn1-s (therefore secreting this protein) were easily detected by the colony blot assay, suggesting that it would be possible to screen for mutants by this method.


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)

(a) Strains carrying  mutations in the PMR1 gene  mislocalize and secrete Mnn1-s. Strains were streaked out and  then analyzed by the colony blot assay as described in Materials  and Methods. All of the strains except WT 2μ MNN1-s are carrying pMNN1-s. Strains marked with an * indicate that it is a strain  with a genetic background other than SEY6210. The strain  abbreviations in the figure are as follows: WT 2μ MNN1-s  (SEY6210 pRS426 MNN1-s); WT (SEY6210); arf1Δ (6210  arf1Δ); chc1Δ* (GPY1103); pmr1Δ* (L3865); pmr1Δ (SEY6210  pmr1Δ); ret1-1 (TGY381); WT* (TGY383). (b) Summary of results from colony blot experiments with different secretory pathway mutants tested. All strains carry pMNN1-s. All vps strains  are in the SEY6210 background. mnn2* and mnn5* are the LB1-16A and LB65-5D strains respectively. (c) The results of a complementation test between two ldr mutants (TRY121 and  TRY320) is shown. All strains carry the pMNN1-s plasmid. (d)  The percent Mnn1-s secreted from each strain was determined by  Western blot as described in Materials and Methods. Mnn1-s is  expressed at a slightly higher level than Mnn1p and has a different mobility by SDS-PAGE due to differences in N-glycosylation. This allowed us to distinguish Mnn1-s from endogenous  Mnn1p in these experiments.
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Related In: Results  -  Collection

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Figure 2: (a) Strains carrying mutations in the PMR1 gene mislocalize and secrete Mnn1-s. Strains were streaked out and then analyzed by the colony blot assay as described in Materials and Methods. All of the strains except WT 2μ MNN1-s are carrying pMNN1-s. Strains marked with an * indicate that it is a strain with a genetic background other than SEY6210. The strain abbreviations in the figure are as follows: WT 2μ MNN1-s (SEY6210 pRS426 MNN1-s); WT (SEY6210); arf1Δ (6210 arf1Δ); chc1Δ* (GPY1103); pmr1Δ* (L3865); pmr1Δ (SEY6210 pmr1Δ); ret1-1 (TGY381); WT* (TGY383). (b) Summary of results from colony blot experiments with different secretory pathway mutants tested. All strains carry pMNN1-s. All vps strains are in the SEY6210 background. mnn2* and mnn5* are the LB1-16A and LB65-5D strains respectively. (c) The results of a complementation test between two ldr mutants (TRY121 and TRY320) is shown. All strains carry the pMNN1-s plasmid. (d) The percent Mnn1-s secreted from each strain was determined by Western blot as described in Materials and Methods. Mnn1-s is expressed at a slightly higher level than Mnn1p and has a different mobility by SDS-PAGE due to differences in N-glycosylation. This allowed us to distinguish Mnn1-s from endogenous Mnn1p in these experiments.
Mentions: The lumenal domain of Mnn1p (Fig. 1 B, Mnn1-s) is efficiently localized to the Golgi complex independently of the Mnn1p transmembrane domain. However, Mnn1-s is secreted from the cell when overexpressed from a multi-copy plasmid (Graham and Krasnov, 1995). This suggested that Mnn1-s may be retained by a saturable, receptor-mediated mechanism and that mutants that failed to properly sort Mnn1-s would secrete it. To detect such mutants, we used a colony blotting technique (Wilsbach and Payne, 1993) where yeast colonies growing on agar plates were overlaid with nitrocellulose discs for 16–20 h that were then washed free of yeast and probed with antibodies to Mnn1p. The colony blotting technique was tested using wild-type yeast expressing Mnn1-s on a single-copy plasmid as a negative control (Fig. 2 A, WT), and wild-type yeast overexpressing Mnn1-s from a multi-copy plasmid as a positive control (Fig. 2 A, WT 2μ MNN1-s). Colonies harboring the single-copy Mnn1-s plasmid produced a weak background signal and yeast overexpressing Mnn1-s (therefore secreting this protein) were easily detected by the colony blot assay, suggesting that it would be possible to screen for mutants by this method.

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