Mannan outer chain N-glycan structures are yeast/fungal-specific typically found on secreted and cell wall glycoproteins. Mannan outer chains consist of an alpha-1,6 polymannose backbone attached to a Man(8-10)(GlcNAc)(2) core. The backbone contains branches of alpha-1,2 mannose residues, terminated with alpha-1,3 mannose and decorated with alpha-1,2 mannose phosphate. Mannan biosynthesis starts in the Golgi with the initial polymerization of the alpha-1,6 linked mannose backbone by the M-Pol I complex. Constructs encoding soluble portions of the M-Pol I subunits, Mnn9p and Van1p from Saccharomyces cerevisiae, were expressed in Pichia pastoris. Both subunits had to be expressed in the same strain to obtain the recombinant proteins. Recombinant M-Pol I was made only by the KM71 strain transformed with two vectors: one encoding Mnn9p and the other encoding Van1p. Soluble secreted M-Pol I was purified by sequential chromatography on DEAE-Trisacryl, GDP-Hexanolamine-Sepharose and Superdex 200. Characterization of the purified complex indicates that recombinant M-Pol 1 is a Mnn9p-Van1p heterodimer. Purified M-Pol I was active with alpha-1,6 mannobiose as acceptor and GDP-mannose as donor. HPLC identified five products confirmed to be 3-7 mannose residues long. Digestion with linkage-specific alpha-mannosidases revealed that the linkage formed is exclusively alpha-1,6. No alpha-1,2 mannosyltransferase activity, reported previously for M-Pol I immunoprecipitates from cell extracts was detected. These results provide further information on the role of M-Pol I in mannan biosynthesis.
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| Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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| Site | Modification | Modifier | Source | Reference |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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| Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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| Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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