Yeast cell-based analysis of human lactate dehydrogenase isoforms

Area of Science:

• Biochemistry
• Molecular Biology
• Yeast Genetics

Background:

• Human lactate dehydrogenase (LDH) is a potential therapeutic target for cancer and contraception.
• Developing robust assay systems is crucial for studying LDH function and inhibitors.
• Saccharomyces cerevisiae offers a powerful platform for genetic analysis and high-throughput screening.

Purpose of the Study:

• To establish a functional yeast cell-based assay system for human lactate dehydrogenase (LDH).
• To investigate the complementation capabilities of different human LDH isoforms (LDHA, LDHB, LDHC) in a Saccharomyces cerevisiae model.
• To characterize the behavior and localization of human LDH isoforms expressed in yeast.

Main Methods:

• Construction of a pdc null mutant Saccharomyces cerevisiae strain unable to perform alcoholic fermentation.
• Introduction and expression of human LDHA, LDHB, and LDHC genes in the engineered yeast.
• Assessment of yeast growth complementation in the presence of an electron transport chain inhibitor.
• Analysis of lactate production and subcellular localization of LDH-RFP fusion proteins.

Main Results:

• Expression of human LDHA and LDHC genes complemented the growth defect of the pdc null yeast mutant, dependent on LDHA catalytic activity.
• Human LDHB did not complement the yeast growth defect but did produce lactate.
• LDHB-RFP fusion protein exhibited blebbing in yeast, while LDHA-RFP and LDHC-RFP showed cytosolic distribution.

Conclusions:

• The engineered yeast system provides a versatile platform for analyzing human LDH function.
• LDHA and LDHC are functionally expressed in yeast and can rescue specific yeast metabolic defects.
• LDHB displays distinct cellular behavior and localization in yeast compared to LDHA and LDHC, suggesting unique properties.