Pregnenolone esterification in Saccharomyces cerevisiae. A potential detoxification mechanism

Area of Science:

• Biochemistry
• Molecular Biology
• Yeast Genetics

Background:

• Steroid metabolism in yeast is not fully understood.
• The yeast Saccharomyces cerevisiae is a model organism for studying cellular processes.
• Steroid compounds can exhibit toxicity to cells.

Purpose of the Study:

• To identify the enzyme responsible for pregnenolone esterification in Saccharomyces cerevisiae.
• To elucidate the function of the identified enzyme in steroid metabolism and detoxification.
• To investigate the role of this enzyme in cellular response to steroid stress.

Main Methods:

• Enzyme activity assays for acetyl-CoA:pregnenolone acetyltransferase (APAT).
• Protein purification using SDS/polyacrylamide gel electrophoresis.
• Peptide sequencing and mass spectrometry for protein identification.
• Gene disruption (mutant creation) to assess gene function.
• Steroid toxicity assays in yeast strains with and without specific transporters.

Main Results:

• Pregnenolone is converted to its acetate ester by APAT in yeast.
• The gene ATF2 encodes the APAT enzyme (Atf2p), a 62-kDa protein.
• Disruption of ATF2 abolished APAT activity and pregnenolone esterification.
• Loss of ATF2 function led to pregnenolone toxicity, exacerbated by the absence of PDR5 and SNQ2 transporters.

Conclusions:

• Atf2p (APAT) is identified as the key enzyme for pregnenolone esterification in Saccharomyces cerevisiae.
• Atf2p plays a significant role in the detoxification of 3beta-hydroxysteroids.
• Atf2p functions in conjunction with efflux pumps Pdr5p and Snq2p for effective steroid detoxification.