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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) Functional model for the compartmental organization of the yeast Golgi complex (Graham and Emr, 1991; Gaynor  et al., 1994). (b) Schematic of the yeast α-1,3-mannosyltransferase (Mnn1p) and the fusion proteins used in this report. Wild-type Mnn1p, a type II integral membrane protein, is shown at the  top. The lumenal domain is expressed in the secretory pathway as  a soluble protein (Mnn1-s) by exchanging the transmembrane domain (TMD) and cytoplasmic tail (CT) for the cleavable signal  peptide of CPY. The Mnn1p TMD and CT are fused to the secreted enzyme invertase and the resulting fusion protein (M39I)  is used to examine TMD mediated localization; previous studies  have indicated that the CT does not contribute to the localization  of Mnn1p (Graham and Krasnov, 1995).
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Figure 1: (a) Functional model for the compartmental organization of the yeast Golgi complex (Graham and Emr, 1991; Gaynor et al., 1994). (b) Schematic of the yeast α-1,3-mannosyltransferase (Mnn1p) and the fusion proteins used in this report. Wild-type Mnn1p, a type II integral membrane protein, is shown at the top. The lumenal domain is expressed in the secretory pathway as a soluble protein (Mnn1-s) by exchanging the transmembrane domain (TMD) and cytoplasmic tail (CT) for the cleavable signal peptide of CPY. The Mnn1p TMD and CT are fused to the secreted enzyme invertase and the resulting fusion protein (M39I) is used to examine TMD mediated localization; previous studies have indicated that the CT does not contribute to the localization of Mnn1p (Graham and Krasnov, 1995).

Mentions: The Golgi complex is at the center of the eukaryotic secretory pathway. It is responsible for sorting proteins that are to be secreted from the cell, transported forward (anterograde) to the plasma membrane, endosome, or lysosome/vacuole and backward (retrograde) to the ER. In addition to its central role in protein sorting, the Golgi complex contains an array of glycosyltransferases that elongate the core O- and N-linked carbohydrate structures added to glycoproteins in the ER. It also possesses proteases that cleave specific proteins as they pass through this organelle. These different Golgi enzymes are enriched in distinct subcompartments, referred to as the cis, medial, trans, and TGN that together compose the Golgi complex (Farquhar and Palade, 1998). Although the cisternae that compose the Golgi complex of S. cerevisiae are not arranged in an ordered stack as seen in electron micrographs of the Golgi complex in higher eukaryotes, the yeast Golgi is functionally ordered in a similar manner. Like the mammalian Golgi, transport of proteins through the yeast Golgi can be followed by observing the posttranslational modification of cargo proteins, which include N- and O-linked carbohydrate additions as well as proteolytic cleavage events. A model for the compartmental organization of the yeast Golgi complex based on a functional analysis of posttranslational modification events is shown in Fig. 1 A (Gaynor et al., 1994; Graham and Emr, 1991).


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) Functional model for the compartmental organization of the yeast Golgi complex (Graham and Emr, 1991; Gaynor  et al., 1994). (b) Schematic of the yeast α-1,3-mannosyltransferase (Mnn1p) and the fusion proteins used in this report. Wild-type Mnn1p, a type II integral membrane protein, is shown at the  top. The lumenal domain is expressed in the secretory pathway as  a soluble protein (Mnn1-s) by exchanging the transmembrane domain (TMD) and cytoplasmic tail (CT) for the cleavable signal  peptide of CPY. The Mnn1p TMD and CT are fused to the secreted enzyme invertase and the resulting fusion protein (M39I)  is used to examine TMD mediated localization; previous studies  have indicated that the CT does not contribute to the localization  of Mnn1p (Graham and Krasnov, 1995).
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Related In: Results  -  Collection

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Figure 1: (a) Functional model for the compartmental organization of the yeast Golgi complex (Graham and Emr, 1991; Gaynor et al., 1994). (b) Schematic of the yeast α-1,3-mannosyltransferase (Mnn1p) and the fusion proteins used in this report. Wild-type Mnn1p, a type II integral membrane protein, is shown at the top. The lumenal domain is expressed in the secretory pathway as a soluble protein (Mnn1-s) by exchanging the transmembrane domain (TMD) and cytoplasmic tail (CT) for the cleavable signal peptide of CPY. The Mnn1p TMD and CT are fused to the secreted enzyme invertase and the resulting fusion protein (M39I) is used to examine TMD mediated localization; previous studies have indicated that the CT does not contribute to the localization of Mnn1p (Graham and Krasnov, 1995).
Mentions: The Golgi complex is at the center of the eukaryotic secretory pathway. It is responsible for sorting proteins that are to be secreted from the cell, transported forward (anterograde) to the plasma membrane, endosome, or lysosome/vacuole and backward (retrograde) to the ER. In addition to its central role in protein sorting, the Golgi complex contains an array of glycosyltransferases that elongate the core O- and N-linked carbohydrate structures added to glycoproteins in the ER. It also possesses proteases that cleave specific proteins as they pass through this organelle. These different Golgi enzymes are enriched in distinct subcompartments, referred to as the cis, medial, trans, and TGN that together compose the Golgi complex (Farquhar and Palade, 1998). Although the cisternae that compose the Golgi complex of S. cerevisiae are not arranged in an ordered stack as seen in electron micrographs of the Golgi complex in higher eukaryotes, the yeast Golgi is functionally ordered in a similar manner. Like the mammalian Golgi, transport of proteins through the yeast Golgi can be followed by observing the posttranslational modification of cargo proteins, which include N- and O-linked carbohydrate additions as well as proteolytic cleavage events. A model for the compartmental organization of the yeast Golgi complex based on a functional analysis of posttranslational modification events is shown in Fig. 1 A (Gaynor et al., 1994; Graham and Emr, 1991).

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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