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Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae
Published on: February 16, 2015
Efficient secretion in yeast based on fragments from K1 killer preprotoxin
C P Cartwright1, Y S Zhu, D J Tipper
1Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester 01655.
Yeast (Chichester, England)
|April 1, 1992
Summary
Secretion of beta-lactamase in yeast is enhanced by fusing it to specific K1 killer toxin precursor (ppTox) segments. Shorter fusions and those targeting the Golgi pathway improve secretion efficiency, suggesting potential for foreign protein production.
Area of Science:
- Molecular and Cellular Biology
- Biochemistry
- Yeast Genetics
Background:
- The K1 killer toxin from Saccharomyces cerevisiae is a heterodimeric protein composed of alpha and beta subunits derived from a single preprotoxin (ppTox).
- Kex2 protease is crucial for processing ppTox, mediating cleavage at specific sites to release mature subunits.
- Secretion of heterologous proteins in yeast is often limited by inefficient processing and transport.
Purpose of the Study:
- To investigate the role of ppTox structure and Kex2 processing in the secretion of beta-lactamase (bla).
- To identify ppTox domains that facilitate efficient translocation and secretion of fused proteins.
- To assess the potential of using modified ppTox as a secretion system for foreign proteins in yeast.
Main Methods:
- Construction of various beta-lactamase fusion proteins with different segments of the K1 killer toxin preprotoxin (ppTox).
- Expression of these fusion proteins in Saccharomyces cerevisiae strains, including Kex2-proficient and Kex2-deficient mutants.
- Quantification of secreted beta-lactamase activity and protein levels.
- Analysis of secretion efficiency based on fusion construct length and Kex2 processing.
Main Results:
- Fusion of beta-lactamase to the N-terminus of ppTox (SP-beta la) resulted in a 10-fold increase in activity and efficient secretion, even in kex2-null strains.
- Fusions at earlier ppTox sites (Ala34 or Ala46) also led to efficient secretion in both KEX2 and kex2-null strains.
- Longer fusion constructs showed reduced secretion efficiency, with uncleaved protein potentially being diverted to the vacuole.
Conclusions:
- The efficiency of beta-lactamase secretion is primarily determined by the efficient translocation of the fusion protein to the Golgi apparatus, where Kex2 is active.
- Shorter ppTox-beta-lactamase fusions or those targeted to specific processing pathways enhance secretion.
- Modified K1 killer toxin precursors offer a promising strategy for the high-level secretion of foreign proteins in yeast.
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