Characterization of Saccharomyces cerevisiae Kar4p in Meiotic Transcription and Translation

Abstract

The budding yeast Saccharomyces cerevisiae undergoes cellular differentiation during both mating and meiosis. The mating pathway is regulated by exposure to pheromones, whereas meiosis is regulated by integration of multiple signaling pathways that assess the cellular state and nutritional environment. For mating, Kar4p acts with Ste12p to activate expression of a subset of genes required for nuclear fusion. However, Kar4p is also required for meiosis. Previous work supports Kar4p as having regulatory roles in meiosis and sporulation. Overexpression of the master meiotic transcriptional activator Ime1p suppressed the kar4Δ meiosis defect. However, to suppress the kar4Δ sporulation defect co-overexpression of RIM4 and IME1 was required.

Based on Kar4p’s role in mating and the suppression by Ime1p, Kar4p might be a co-factor required to properly regulate transcription of meiotic genes. Using an inducible gene expression system and DNA gene expression microarrays, the kar4Δ meiotic transcriptional profile was characterized and a set of genes whose expression is KAR4-dependent was identified.

Rim4p is a known translational regulator. Kar4p might be required to properly regulate translation of meiotic genes. Evidence from immunoblotting showed Ime2p levels are low in kar4Δ cells. Rim4p overexpression restored Ime2p expression to normal levels and dynamics. To determine if other meiotic proteins are KAR4-dependent, whole proteome mass spectrometry was performed. By comparing the meiotic proteome data with the gene expression data, a set of genes was identified whose protein levels are KAR4-dependent. We hypothesize that Kar4p is required for their efficient translation.

To identify physical interactions and elucidate molecular mechanisms of Kar4p’s meiotic regulatory roles, co-immunoprecipitation and mass spectrometry was performed. The data suggest that Kar4p interacts with Ime4p and Mum2p, two members of the yeast meiotic methylation complex, as well as with Ygl036p, a protein of previously unknown function. We found that Ygl036p is required for sporulation.

Taken together, this thesis proposes that Kar4p regulates meiosis and sporulation in two ways: first, by enhancing the activity of Ime1p and turning on a subset of its transcriptional targets; secondly, by enhancing the translational efficiency, possibly acting in a new complex with Mum2p and Ygl036p.