Screen for temperature-sensitive mutants of non-essential yeast genes

Abstract

Yeast deletion mutants of crucial genes are often associated with a number of secondary defects, which hamper the analysis of primary protein function. Therefore, temperature-sensitive mutants are valuable tools to evaluate protein function in a focused and often reversible manner. However, temperature-sensitive mutants are uncommon for non-essential genes that nevertheless may have strong defects. Here we describe a screening method for generating temperature-sensitive mutants of non-essential genes in synthetic lethal backgrounds of Saccharomyces cerevisiae. As proof of principle, we describe a successful screen for the yeast mitochondrial inner membrane protease iAAA subunit Yme1 utilizing two screening approaches: a random mutagenesis and rational design approach. We then describe how candidate temperature-sensitive mutants are validated.

Keywords: Mitochondria; Protease; Proteolysis; Saccharomyces cerevisiae; Temperature-sensitive screen; Yeast; Yme1; i-AAA.