The Hap2/3/4/5 complex is a heme-activated, CCAATT binding, global transcriptional activator of genes involved in respiration and mitochondrial biogenesis in the yeast species Saccharomyces cerevisiae. Hap4 is the regulatory subunit of the complex and its levelsdetermine the activity of the complex. Hap4 is known to play a signaling role in response toenvironmental conditions; however, little is known about the regulation of Hap4 levels or how it responses to a cell’s functional state. The activity of the Hap2-5 complex is known to be reduced in respiratory-deficient cells. In Liu Lab, it has previously been found that a link between Hap4 stability, mediated through 26S proteasome-dependent degradation, and dependence on mitochondrial functional state plays a regulatory role on downstream targets of the Hap complex. However, the mechanism behind this regulation is still largely unknown. In normally functioning yeast cells, Hap4 is a highly unstable protein with a half-life of ~10 min. We have observed that loss of mitochondrial DNA in respiratory deficient rho 0 cells has a role in the further destabilization of Hap4 to a half-life of ~4 min through the ubiquitin-proteasome pathway. Through the screening of a collection of mutants defective in E2 ubiquitin-conjugating enzymes, we show that Hap4 is greatly stabilized in ubc1Δubc4Δ double mutant cells. We also show that Hap4 stabilization in the ubc1Δubc4Δ mutant leads to increased activity of the Hap2-5 complex, indicating that mitochondrial biogenesis in yeast is regulated by the functional state of mitochondria through ubiquitin/proteasome-dependent degradation of Hap4. Furthermore, studies on Hap4 mutants involving two highly conserved cysteine residues led to a proposed mechanism behind the regulation of Ubc4 activity towards Hap4 in response to changes in the cellular redox state.

The CCAAT binding-factor (CBF) is a transcriptional activator conserved in eukaryotes. The CBF in Saccharomyces cerevisiae is a multimeric heteromer termed the Hap2/3/4/5 complex. Hap4, which contains the activation domain of the complex, is also the regulatory subunit and is known to be transcriptionally controlled by carbon sources. However, little is known about Hap4 regulation. In this report, I identify mechanisms by which Hap4 is regulated, including: (1) transcriptional regulation via two short upstream open reading frames (uORFs) in the 5' leader sequence of HAP4 mRNA; (2) proteasome-dependent degradation of Hap4; and (3) identification of two negative regulators of HAP4 expression, CYC8 and SIN4. I also report differential patterns of Hap4 cellular localization which depends on (1) carbon sources, (2) abundance of Hap4 protein, and (3) presence or absence of mitochondrial DNA (mtDNA).

Nutrient availability regulates eukaryotic cell growth. This study focuses on two signaling pathways, involved in sensing amino acids and carbon sources, which allow cells to respond appropriately to their presence. The first part of this study shows that Ssy1, a plasma membrane localized sensor in the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway, can detect 19 common L-amino acids with different potencies and affinities based on the physiochemical structure of amino acids. Substituents around alpha carbon are critical for amino acid sensing by Ssy1. Furthermore, a high concentration of cysteine is toxic to cells. Inactivation of SPS signaling confers resistance to cysteine. The second part focuses on the regulation of Hap4, the regulatory subunit of the Hap2/3/4/5 transcriptional factor complex. Many components of the 25-subunit Mediator complex negatively regulate HAP4 expression. Srb8 undergoes post-translational modification in response to changes of the carbon source. Gal11 and Med3 positively regulate HAP4 expression.