Ras proteins, fungal mating pheromones, and other proteins terminating in the sequence CaaX (where C is Cys, a is any aliphatic amino acid, and X is the C-terminal residue) are posttranslationally prenylated. Farnesyl-protein transferase (FPTase) transfers the farnesyl moiety of farnesyl pyrophosphate (FPP) to the thiol of the CaaX box cysteine in a reaction that requires Zn2+ and Mg2+. We have created mutations in conserved amino acids of the yeast Ram1 protein to identify residues important for Zn2+-dependent FPTase activity. Wild-type and mutant Ram1 proteins were expressed as operon fusions in bacteria, and FPTase activity was measured. Mutations in conserved residues Glu256, His258, Asp307, Cys309, Asp360, and His363 reduce FPTase activity. Asp307, Cys309, and His363 correspond to the residues that have been shown to coordinate Zn2+ in mammalian FPTase. The H258N mutant enzyme exhibited an increased sensitivity to the Zn2+ chelator 1,10-phenanthroline, required higher concentrations of Zn2+ to restore activity to the apoenzyme, and had a 10-fold reduction in catalytic efficiency. The decreases in FPTase activity observed do not appear to be caused by major structural perturbations because the mutants were stably expressed and retained the ability to interact with Ram2p during purification. The FPTase activity of the mutants measured in vitro correlated well with their ability to complement the mating and growth defects of a ram1Delta strain in vivo.
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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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