Oligomerization of Hamster UDP-GlcNAc:Dolichol-P GlcNAc-1-P Transferase, an Enzyme with Multiple Transmembrane Spans

Dan, Ning; Lehrman, Mark A.

doi:10.1074/jbc.272.22.14214

Cited by 30 publications

(25 citation statements)

References 28 publications

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“…3A), indicating that hAlg7p interacts with itself to form a dimer (or oligomer). This observation coincided with a previous report on hamster GPT (15). Co-transformants with hAlg14 prey also grew on both selective media (Fig.…”

Section: C-1 Halg7p Halg14p and Halg13p Form A Gpt/nagt Complexsupporting

confidence: 93%

Physical Interactions among Human Glycosyltransferases Involved in Dolichol-Linked Oligosaccharide Biosynthesis

Takahashi¹,

Gao²

2012

TIGG

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In eukaryotic glycoproteins, the N-glycan plays an important role with respect to both their structure and function. On the rough endoplasmic reticulum (rER) membrane, the N-glycan precursor is biosynthesized as a dolichol-linked oligosaccharide (DLO), which consists of fourteen sugars linked to pyrophosphoryl-dolichol. For completion of fulllength DLO (Glc 3 Man 9 GlcNAc 2 -PP-dolichol), activities of at least eleven glycosyltransferases localized on the rER membrane are essential. Although twelve human genes for these enzymes have been identified, any physical interactions among them have not yet been systematically analyzed. In this review, we describe several physical interactions among them that were uncovered using the yeast split-ubiquitin system. Moreover, on the basis of observations obtained by this technique, novel models for networking among human glycosyltransferases can be proposed.

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Section: C-1 Halg7p Halg14p and Halg13p Form A Gpt/nagt Complexsupporting

confidence: 93%

Physical Interactions among Human Glycosyltransferases Involved in Dolichol-Linked Oligosaccharide Biosynthesis

Takahashi¹,

Gao²

2012

TIGG

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“…This enzyme initiates N-linked glycosylation by catalyzing the synthesis of GlcNAc-P-P-dolichol. Lehrman and coworkers (42,46) demonstrated that GPT is a multimeric enzyme with multiple, most likely 10 transmembrane spans. The largest hydrophilic segment, located between TM 9 and TM 10, is facing the cytoplasm.…”

Section: Discussionmentioning

confidence: 99%

Transmembrane Topology of Pmt1p, a Member of an Evolutionarily Conserved Family of Protein O-Mannosyltransferases

Strahl‐Bolsinger

Scheinost

1999

Journal of Biological Chemistry

100

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The identification of the evolutionarily conserved family of dolichyl-phosphate-D-mannose:protein O-mannosyltransferases (Pmts) revealed that protein O-mannosylation plays an essential role in a number of physiologically important processes. Strikingly, all members of the Pmt protein family share almost identical hydropathy profiles; a central hydrophilic domain is flanked by amino-and carboxyl-terminal sequences containing several putative transmembrane helices. This pattern is of particular interest because it diverges from structural models of all glycosyltransferases characterized so far. Here, we examine the transmembrane topology of Pmt1p, an integral membrane protein of the endoplasmic reticulum, from Saccharomyces cerevisiae. Structural predictions were directly tested by site-directed mutagenesis of endogenous N-glycosylation sites, by fusing a topology-sensitive monitor protein domain to carboxyl-terminal truncated versions of the Pmt1 protein and, in addition, by N-glycosylation scanning. Based on our results we propose a seven-transmembrane helical model for the yeast Pmt1p mannosyltransferase. The Pmt1p amino terminus faces the cytoplasm, whereas the carboxyl terminus faces the lumen of the endoplasmic reticulum. A large hydrophilic segment that is oriented toward the lumen of the endoplasmic reticulum is flanked by five amino-terminal and two carboxyl-terminal membrane spanning domains. We could demonstrate that this central loop is essential for the function of Pmt1p.Glycosylation is one of the most elaborate covalent protein modifications known. The carbohydrate chains can be coupled to the protein through either an N-or O-glycosidic bond. Protein O-mannosylation, originally observed in fungi (1), is initiated at the endoplasmic reticulum by protein mannosyltransferases (Pmts) 1 that catalyze the transfer of a mannosyl residue from dolichyl phosphate-activated mannose (Dol-PMan) to serine or threonine residues of nascent proteins entering the secretory pathway; in the Golgi apparatus additional sugars are added to the O-linked mannose with GDP-mannose serving as carbohydrate donor (2, 3). Dol-P-Man-dependent O-glycosylation of secreted proteins is a general feature of yeasts and filamentous fungi (4).The key enzyme of protein O-mannosylation, the Dol-P-Man: protein O-mannosyltransferase Pmt1p, was purified from Saccharomyces cerevisiae following the enzyme activity, and the corresponding gene was cloned (5, 6). Pmt1p is an integral membrane glycoprotein located at the ER (5, 7-9). Based on homology to Pmt1p, a family of seven protein O-mannosyltransferases (Pmt1p-Pmt7p) has been identified (10 -13). Thus far, protein O-mannosyltransferase activity has been demonstrated for Pmt1p, Pmt2p, Pmt3p, Pmt4p, and Pmt6p (13, 14). The individual mannosyltransferases recognize specific protein substrates that might explain the presence of more than one transferase in S. cerevisiae (14). Moreover, Pmtp orthologues have been identified from other yeasts (4), from the opportunistic fungal pathogen Candida alb...

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“…3 Thus, the clustering of O-linked sugars requires high efficiency sugar transfer, which might be provided by mannosyltransferase complexes. That oligomerization enhances enzyme function has been proven for other glycosyltransferases such as UDPGlcNAc:dolichol-P GlcNAc-1-P transferase (50) or the mannosyltransferase complexes M-Pol I and M-Pol II (51)(52)(53). Furthermore, even though members of the PMT1 and PMT2 subfamily act on the same protein substrate (14,25), they might actually O-mannosylate different serine and threonine residues within one and the same protein.…”

Section: Heteromeric Protein Complexes Between Pmt1 and Pmt2 Subfamilmentioning

confidence: 98%

Members of the Evolutionarily Conserved PMT Family of ProteinO-Mannosyltransferases Form Distinct Protein Complexes among Themselves

Girrbach

Strahl

2003

Journal of Biological Chemistry

134

152

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Protein O-mannosyltransferases (PMTs) initiate the assembly of O-mannosyl glycans, an essential protein modification. Since PMTs are evolutionarily conserved in fungi but are absent in green plants, the PMT family is a putative target for new antifungal drugs, particularly in fighting the threat of phytopathogenic fungi. The PMT family is phylogenetically classified into PMT1, PMT2, and PMT4 subfamilies, which differ in protein substrate specificity. In the model organism Saccharomyces cerevisiae as well as in many other fungi the PMT family is highly redundant, and only the simultaneous deletion of PMT1/PMT2 and PMT4 subfamily members is lethal. In this study we analyzed the molecular organization of PMT family members in S. cerevisiae. We show that members of the PMT1 subfamily (Pmt1p and Pmt5p) interact in pairs with members of the PMT2 subfamily (Pmt2p and Pmt3p) and that Pmt1p-Pmt2p and Pmt5p-Pmt3p complexes represent the predominant forms. Under certain physiological conditions, however, Pmt1p interacts also with Pmt3p, and Pmt5p with Pmt2p, suggesting a compensatory cooperation that guarantees the maintenance of O-mannosylation. Unlike the PMT1/PMT2 subfamily members, the single member of the PMT4 subfamily (Pmt4p) acts as a homomeric complex. Using mutational analyses we demonstrate that the same conserved protein domains underlie both heteromeric and homomeric interactions, and we identify an invariant arginine residue of transmembrane domain two as essential for the formation and/or stability of PMT complexes in general. Our data suggest that protein-protein interactions between the PMT family members offer a point of attack to shut down overall protein O-mannosylation in fungi.Protein O-mannosylation is an evolutionarily conserved protein modification of fundamental importance in many eukaryotes. In yeasts and fungi, the attachment of O-linked mannosyl residues to proteins of the secretory pathway is essential for cell viability (1). In particular it is indispensable for cell wall integrity and normal cellular morphogenesis (2-4). Impairment of O-mannosylation also affects the stability, localization, and/or proper function of individual proteins (5-10). Furthermore, aberrant O-mannosylation can interfere with the retrograde transport of misfolded proteins across the membrane of the endoplasmic reticulum (ER) 1 (11). O-mannosylation is not only important in yeast, but also in mammals. It was recently shown that in humans, O-mannosyl glycosylation represents a new pathomechanism for muscular dystrophy and neuronal migration disorders (12, 13).In yeast and fungi, O-mannosylation is initiated in the lumen of the ER by an essential family of protein O-mannosyltransferases (PMTs). These enzymes catalyze the transfer of mannose from dolichyl phosphate-activated mannose (Dol-PMan) to serine or threonine residues of secretory proteins (2). In Saccharomyces cerevisiae, a total of seven PMT family members (Pmt1-7p) have been identified, which share almost identical hydropathy profiles that predict the PMTs to b...

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Oligomerization of Hamster UDP-GlcNAc:Dolichol-P GlcNAc-1-P Transferase, an Enzyme with Multiple Transmembrane Spans

Cited by 30 publications

References 28 publications

Physical Interactions among Human Glycosyltransferases Involved in Dolichol-Linked Oligosaccharide Biosynthesis

Physical Interactions among Human Glycosyltransferases Involved in Dolichol-Linked Oligosaccharide Biosynthesis

Transmembrane Topology of Pmt1p, a Member of an Evolutionarily Conserved Family of Protein O-Mannosyltransferases

Members of the Evolutionarily Conserved PMT Family of ProteinO-Mannosyltransferases Form Distinct Protein Complexes among Themselves

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