The Saccharomyces cerevisiae mitochondrial carrier YGR257Cp (Mtm1p) is an integral membrane protein that plays an essential role in mitochondrial iron homeostasis and respiratory functions, but its carrier substrate has not previously been identified. Large amounts of pure protein are required for biochemical characterization, including substrate screening. Functional complementation of a Saccharomyces knockout by expression of TwinStrep tagged YGR257Cp demonstrates that an affinity tag does not interfere with protein function, but the expression level is very low. Heterologous expression in Pichia pastoris improves the yield but the product is heterogeneous. Expression has been screened in several Escherichia coli hosts, optimizing yield by modifying induction conditions and supplementing with rare tRNAs to overcome codon bias in the eukaryotic gene. Detection of an additional N-terminal truncation product in E. coli reveals the presence of a secondary intracistronic translation initiation site, which can be eliminated by silent mutagenesis of an alternative (Leu) initiation codon, resulting in production of a single, full-length polypeptide (∼30% of the total protein) as insoluble inclusion bodies. Purified inclusion bodies were successfully refolded and affinity purified, yielding approximately 40mg of pure, soluble product per liter of culture. Refolded YGR257Cp binds pyridoxal 5'-phosphate tightly (KD<1μM), supporting a new hypothesis that the mitochondrial carrier YGR237Cp and its homologs function as high affinity PLP transporters in mitochondria, providing the first evidence for this essential transport function in eukaryotes.
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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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Evidence ID | Analyze ID | File | Description |
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