Extramitochondrial citrate synthase activity in bakers' yeast.

Area of Science:

• Biochemistry
• Molecular Biology
• Yeast Genetics

Background:

• Citrate synthase (citrate oxaloacetate lyase; EC 4.1.3.7) is a key enzyme in the citric acid cycle.
• The presence and function of multiple citrate synthase isozymes in yeast are not fully understood.

Purpose of the Study:

• To investigate the existence and characteristics of non-mitochondrial citrate synthase in Saccharomyces cerevisiae.
• To determine the functional significance of mitochondrial citrate synthase in yeast growth.

Main Methods:

• Gene ablation using the yeast LEU2 gene insertion.
• Enzyme activity assays.
• Immunological characterization using antibodies against mitochondrial citrate synthase.
• Growth analysis of gene-deleted yeast strains.

Main Results:

• Ablation of the mitochondrial citrate synthase gene revealed a second, non-mitochondrial citrate synthase.
• The non-mitochondrial enzyme exhibited sensitivity to glucose repression.
• This novel enzyme did not cross-react with antibodies against the mitochondrial form.
• Yeast cells lacking mitochondrial citrate synthase showed slightly reduced growth rates but no auxotrophic requirements.

Conclusions:

• Saccharomyces cerevisiae possesses at least two distinct citrate synthase enzymes: one mitochondrial and one non-mitochondrial.
• The non-mitochondrial citrate synthase is regulated by glucose repression.
• Mitochondrial citrate synthase is important for optimal yeast growth but not essential for viability.