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Sequence dependence of protein isoprenylation
S L Moores1, M D Schaber, S D Mosser
1Department of Cancer Research, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486.
The Journal of Biological Chemistry
|August 5, 1991
Summary
Researchers identified three distinct enzymes that attach isoprenoid molecules to proteins, a process called protein isoprenylation. These enzymes, farnesyl-protein transferase (FTase) and two geranyl-geranyl-protein transferases (GGTase-I and GGTase-II), show selectivity for different protein substrates and isoprenoid compounds.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Protein post-translational modification is crucial for cellular function.
- Isoprenoid modification, specifically farnesylation and geranylgeranylation, targets C-terminal cysteine residues.
- These modifications are mediated by specific enzymes involved in isoprenoid biosynthesis.
Purpose of the Study:
- To identify and characterize the enzymes responsible for protein isoprenylation in bovine brain cytosol.
- To investigate the substrate specificity and determinants of farnesyl-protein transferase (FTase) and geranyl-geranyl-protein transferases (GGTases).
- To explore the relationship between FTase and GGTase activities and their genetic underpinnings.
Main Methods:
- Chromatographic resolution of cytosolic proteins from bovine brain.
- Assays to determine farnesyl-protein transferase (FTase) and geranyl-geranyl-protein transferase (GGTase) activities.
- Studies using Saccharomyces cerevisiae strains with mutations in FTase activity (ram1, ram2).
- Competition assays using synthetic peptides mimicking C-terminal protein sequences.
Main Results:
- Three distinct isoprenyl-protein transferases were identified: FTase, GGTase-I, and GGTase-II.
- FTase modified Ras protein, GGTase-I modified a chimeric Ras, and GGTase-II modified YPT1.
- The CAAX box motif at the C-terminus of protein substrates is critical for FTase and GGTase-I recognition.
- FTase and GGTase-I exhibit complementary substrate affinities, with C-terminal leucine influencing substrate preference.
- GGTase-I activity was reduced in a yeast strain lacking FTase activity, suggesting shared genetic features.
- GGTase-II interaction with YPT1 appears to involve more than just the C-terminal sequence.
Conclusions:
- Distinct enzymes, FTase, GGTase-I, and GGTase-II, mediate protein isoprenylation with specific substrate selectivities.
- The CAAX box is a key determinant for FTase and GGTase-I substrate recognition, with sequence variations dictating modification type.
- These findings elucidate the enzymatic machinery and specificity governing protein isoprenylation.