Rab proteins in mammalian cells, or Ypt1p and Sec4p in yeast, regulate vesicular traffic. Prenylation of these small GTP-binding proteins is required for membrane attachment and subsequent biological activity. Yeast protein geranylgeranyltransferase type-II (PGGTase-II) catalyzes the prenylation of Ypt1p in the presence of an escort protein, Msi4p. The genes encoding the alpha-(BET4) and beta-(BET2) subunits of PGGTase-II were translationally coupled by overlapping the BET4-BET2 stop/start codons and by adding a ribosome-binding site near the 3'-end of BET4 that fused an -EEF C-terminal alpha-tubulin epitope to Bet4p. Active recombinant heterodimer was purified by chromatography on DE52 and anti-alpha-tubulin columns. Recombinant Msi4p with an N-terminal polyhistidine leader was purified on a Ni(2+)-Sepharose column, followed by gel filtration and ion exchange chromatography. An escort protein, Msi4p, was necessary for geranylgeranylation of Ypt1p by yeast PGGTase-II. Michaelis constants for GGPP and Ypt1p were 1.6 and 1.1 microM, respectively; Vmax = 1.7 nmol min-1 mg-1 for yeast PGGTase-II. Typical Michaelis-Menten behavior was also seen for the enzyme for varied concentrations of Msi4p, with a maximal catalytic activity seen for a 10-fold excess of escort protein over enzyme. In contrast to previous reports, PGGTase-II requires both Zn2+ and Mg2+ for maximal activity, although Zn2+ becomes inhibitory at concentrations above approximately 10 microM. Prenylated Ypt1p obtained after incubation of Ypt1p with PGGTase-II, Msi4p, and geranylgeranyl diphosphate was digested with trypsin. The C-terminal peptide fragment from modified Ypt1p was purified by HPLC and analyzed by electrospray mass spectrometry. The mass of the fragment is consistent with the 12-mer C-terminal amino acid fragment predicted from proteolysis by trypsin with both cysteine residues modified by geranylgeranyl moieties.
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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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