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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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Sucrose gradient fractionation profiles of Golgi membranes from wild-type, hog1-11, and ldr1-2 cells. Differential centrifugation, sucrose gradient fractionation, and enzyme assays  were performed as described in Materials and Methods. Fractionation profiles of Kex2p, GDPase, and Mnn1p from (a) WT  (SEY6210), (b) TRY120 (hog1-11), and (c) TRY220 (ldr1-2) cells  were generated from the average of three experiments performed  for each strain. The amount of each marker in a particular fraction was plotted as percentage of the total found in all the fractions. Quantitation of Western blots was done as described in  Materials and Methods.
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Figure 6: Sucrose gradient fractionation profiles of Golgi membranes from wild-type, hog1-11, and ldr1-2 cells. Differential centrifugation, sucrose gradient fractionation, and enzyme assays were performed as described in Materials and Methods. Fractionation profiles of Kex2p, GDPase, and Mnn1p from (a) WT (SEY6210), (b) TRY120 (hog1-11), and (c) TRY220 (ldr1-2) cells were generated from the average of three experiments performed for each strain. The amount of each marker in a particular fraction was plotted as percentage of the total found in all the fractions. Quantitation of Western blots was done as described in Materials and Methods.

Mentions: A significant alteration in the percentage of Mnn1p in the P13 fraction from the mutants was not observed, suggesting that Mnn1p was not accumulating in the ER, vacuolar membranes, or plasma membrane of the mutants. Mnn1p was enriched in the P100 from the wild-type cells as reported previously (Graham et al., 1994) and was also enriched to a similar extent in the P100 derived from the mutant strains (data not shown). However, the distribution of Mnn1p in the sucrose gradient fractions from the mutants differed significantly from the wild-type profile (Fig. 6, A–C). A peak containing 30–70% of Mnn1p is typically found in the less dense sucrose fractions (1–4) and cofractionates with the major peak of GDPase in gradients from wild-type cells (Fig. 6 A). This first peak of Mnn1p is nearly absent in gradients containing Golgi membranes from the mutants (Fig. 6, B and C). The Mnn1p lost from the less dense Golgi fractions in the mutants appears to have shifted to the more dense fractions that contain the TGN marker Kex2p. A similar profile to that shown in Fig. 6 B was observed for Golgi membranes isolated from hog1Δ cells.


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)

Sucrose gradient fractionation profiles of Golgi membranes from wild-type, hog1-11, and ldr1-2 cells. Differential centrifugation, sucrose gradient fractionation, and enzyme assays  were performed as described in Materials and Methods. Fractionation profiles of Kex2p, GDPase, and Mnn1p from (a) WT  (SEY6210), (b) TRY120 (hog1-11), and (c) TRY220 (ldr1-2) cells  were generated from the average of three experiments performed  for each strain. The amount of each marker in a particular fraction was plotted as percentage of the total found in all the fractions. Quantitation of Western blots was done as described in  Materials and Methods.
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Related In: Results  -  Collection

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Figure 6: Sucrose gradient fractionation profiles of Golgi membranes from wild-type, hog1-11, and ldr1-2 cells. Differential centrifugation, sucrose gradient fractionation, and enzyme assays were performed as described in Materials and Methods. Fractionation profiles of Kex2p, GDPase, and Mnn1p from (a) WT (SEY6210), (b) TRY120 (hog1-11), and (c) TRY220 (ldr1-2) cells were generated from the average of three experiments performed for each strain. The amount of each marker in a particular fraction was plotted as percentage of the total found in all the fractions. Quantitation of Western blots was done as described in Materials and Methods.
Mentions: A significant alteration in the percentage of Mnn1p in the P13 fraction from the mutants was not observed, suggesting that Mnn1p was not accumulating in the ER, vacuolar membranes, or plasma membrane of the mutants. Mnn1p was enriched in the P100 from the wild-type cells as reported previously (Graham et al., 1994) and was also enriched to a similar extent in the P100 derived from the mutant strains (data not shown). However, the distribution of Mnn1p in the sucrose gradient fractions from the mutants differed significantly from the wild-type profile (Fig. 6, A–C). A peak containing 30–70% of Mnn1p is typically found in the less dense sucrose fractions (1–4) and cofractionates with the major peak of GDPase in gradients from wild-type cells (Fig. 6 A). This first peak of Mnn1p is nearly absent in gradients containing Golgi membranes from the mutants (Fig. 6, B and C). The Mnn1p lost from the less dense Golgi fractions in the mutants appears to have shifted to the more dense fractions that contain the TGN marker Kex2p. A similar profile to that shown in Fig. 6 B was observed for Golgi membranes isolated from hog1Δ cells.

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