Probing the mechanism of FET3 repression by Izh2p overexpression.

We previously reported a role for the IZH2 gene product in metal ion metabolism. Subsequently, Izh2p was also identified as a member of the PAQR family of receptors and, more specifically, as the receptor for the plant protein osmotin. In this report, we investigate the effect of Izh2p on iron homeostasis. We show that overproduction of Izh2p prevents the iron-dependent induction of the Fet3p component of the high-affinity iron-uptake system and is deleterious for growth in iron-limited medium. We demonstrate that the effect of Izh2p requires cAMP-dependent kinase and AMP-dependent kinase and is not mediated by general inhibition of the Aft1p iron-responsive transcriptional activator. We also show that Izh2p-overproduction negatively regulates Nrg1p/Nrg2p- and Msn2p/Msn4p-dependent reporters. Furthermore, we show that the Nrg1p/Nrg2p and Msn2p/Msn4p pairs are epistatic to each other with respect to their effects on FET3 expression. Finally, we show that the mechanism by which PAQR receptors activate signal transduction pathways is likely to be conserved from yeast to humans.

Heterologous expression of human mPRalpha, mPRbeta and mPRgamma in yeast confirms their ability to function as membrane progesterone receptors.

The nuclear progesterone receptor (nPR) mediates many of the physiological effects of progesterone by regulating the expression of genes, however, progesterone also exerts non-transcriptional (non-genomic) effects that have been proposed to rely on a receptor that is distinct from nPR. Several members of the progestin and AdipoQ-Receptor (PAQR) family were recently identified as potential mediators of these non-genomic effects. Membranes from cells expressing these proteins, called mPRalpha, mPRbeta and mPRgamma, were shown to specifically bind progesterone and have G-protein coupled receptor (GPCR) characteristics, although other studies dispute these findings. To clarify the role of these mPRs in non-genomic progesterone signaling, we established an assay for PAQR functional evaluation using heterologous expression in Saccharomyces cerevisiae. Using this assay, we demonstrate unequivocally that mPRalpha, mPRbeta and mPRgamma can sense and respond to progesterone with EC(50) values that are physiologically relevant. Agonist profiles also show that mPRalpha, mPRbeta and mPRgamma are activated by ligands, such as 17alpha-hydroxyprogesterone, that are known to activate non-genomic pathways but not nPR. These results strongly suggest that these receptors may indeed function as the long-sought-after membrane progesterone receptors. Additionally, we show that two uncharacterized PAQRs, PAQR6 and PAQR9, are also capable of responding to progesterone. These mPR-like PAQRs have been renamed as mPRdelta (PAQR6) and mPRvarepsilon (PAQR9). Additional characterization of mPRgamma and mPRalpha indicates that their progesterone-dependent signaling in yeast does not require heterotrimeric G-proteins, thus calling into question the characterization of the mPRs as a novel class of G-protein coupled receptor.

Metalloregulation of yeast membrane steroid receptor homologs.

Zinc is an essential micronutrient that can also be toxic. An intricate mechanism exists in yeast that maintains cellular zinc within an optimal range. The centerpiece of this mechanism is the Zap1p protein, a transcription factor that senses zinc deficiency and responds by up-regulating genes involved in zinc metabolism. A microarray screen for novel Zap1p target genes suggested a role in zinc homeostasis for four homologous yeast genes. The expression of two of these genes, YDR492w and YOL002c, suggested direct regulation by Zap1p, whereas the expression of YOL002c and a third homologous gene, YOL101c, was induced by high zinc. YDR492w and YOL002c are confirmed to be direct Zap1p target genes. The induction of YOL002c and YOL101c by toxic metal ion exposure is shown to be mediated by the Mga2p hypoxia sensor. Furthermore, YOL101c is induced by deletion of the Aft1p iron-responsive transcription factor. These three genes, along with a fourth yeast homolog, YLR023c, have phenotypic effects on zinc tolerance and Zap1p activity. Because of their metalloregulation, zinc-related phenotypes, and highly conserved motifs containing potential metal-binding residues, this family has been renamed the IZH gene family (Implicated in Zinc Homeostasis). Furthermore, these genes are regulated by exogenous fatty acids, suggesting a dual role in lipid metabolism. The IZH genes encode membrane proteins that belong to a ubiquitous protein family that includes hemolysin III and vertebrate membrane steroid receptors. We propose that the IZH genes affect zinc homeostasis either directly or indirectly by altering sterol metabolism.