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Defective Escherichia coli signal peptides function in yeast
1Department of Biochemistry, State University of New York, Stony Brook 11794.
Molecular Microbiology
|March 1, 1988
Summary
Yeast successfully processed and secreted hybrid proteins containing bacterial signal peptides, even with mutations affecting E. coli secretion. This indicates yeast secretion machinery recognizes bacterial signal sequences but has different specificity for altered sequences.
Area of Science:
- Molecular Biology
- Protein Secretion
- Eukaryotic and Prokaryotic Systems
Background:
- Signal peptides direct proteins to secretion pathways.
- Understanding signal peptide structure-function relationships is crucial for protein engineering and biotechnology.
Purpose of the Study:
- To investigate the in vivo function of bacterial signal peptides in a eukaryotic system (yeast).
- To determine if yeast secretion machinery recognizes and processes bacterial signal peptides, and how mutations affect this process.
Main Methods:
- Cloning of a hybrid gene encoding E. coli lipoprotein signal peptide fused to E. coli beta-lactamase into yeast.
- Construction and testing of E. coli lipoprotein signal peptide mutants with altered structural features (charge, hydrophobicity, helicity, cleavage site).
- Induction of hybrid protein expression in yeast using the GAL10 promoter and analysis of protein processing and translocation.
Main Results:
- Yeast processed and translocated hybrid proteins with both wild-type and mutant bacterial signal peptides, with approximately 36% of total hybrid protein being processed.
- Mutant signal peptides that altered processing in E. coli did not significantly affect processing or translocation in yeast.
- Signal peptides were cleaved at a conserved site in yeast, one residue from the E. coli signal peptidase II site.
- Mature lipo-beta-lactamase was translocated into the yeast periplasm.
Conclusions:
- Yeast protein secretion machinery recognizes bacterial lipoprotein signal sequences in vivo.
- Yeast exhibits different specificity towards altered signal sequences compared to E. coli.
- This study provides insights into the conserved and divergent aspects of protein translocation across the cytoplasmic membrane in prokaryotes and eukaryotes.