A mechanism of oxygen sensing in yeast. Multiple oxygen-responsive steps in the heme biosynthetic pathway affect Hap1 activity

Heme plays central roles in oxygen sensing and utilization in many living organisms. In yeast, heme mediates the effect of oxygen on the expression of many genes involved in using or detoxifying oxygen. However, a direct link between intracellular heme level and oxygen concentration has not been vigorously established. In this report, we have examined the relationships among oxygen levels, heme levels, Hap1 activity, and HAP1 expression. We found that Hap1 activity is controlled in vivo by heme and not by its precursors and that heme activates Hap1 even in anoxic cells. We also found that Hap1 activity exhibits the same oxygen dose-response curves as Hap1-dependent aerobic genes and that these dose-response curves have a sharp break at approximately 1 microM O2. The results show that the intracellular signaling heme level, reflected as Hap1 activity, is closely correlated with oxygen concentration. Furthermore, we found that bypass of all heme synthetic steps but ferrochelatase by deuteroporphyrin IX does not circumvent the need for oxygen in Hap1 full activation by heme, suggesting that the last step of heme synthesis, catalyzed by ferrochelatase, is also subjected to oxygen control. Our results show that multiple heme synthetic steps can sense oxygen concentration and provide significant insights into the mechanism of oxygen sensing in yeast.     

The yeast UME6 gene product is required for transcriptional repression mediated by the CAR1 URS1 repressor binding site.

URS1 is known to be a repressor binding site in Saccharomyces cerevisiae that negatively regulates expression of many genes including CAR1 (arginase), several required for sporulation, mating type switching, inositol metabolism, and oxidative carbon metabolism. In addition to the proteins previously shown to directly bind to the URS1 site, we show here that the UME6 gene product is required for URS1 to mediate repression of gene expression in the absence of inducer. We also show that mutations in the CAR80 (CARGRI) gene are allelic to those in UME6.     

Multiple domains mediate heme control of the yeast activator HAP1

The activity of the yeast activator HAP1in vivo requires heme. A heme responsive domain of HAP1 was identified previously. It is adjacent to the DNA-binding domain, and appears to block DNA binding in the absence of heme. Here we describe a novel genetic selection which yielded mutants of HAP1 that are independent of heme when assayed for activation. These mutants define a second region of HAP1, close to the activation domain, which also controls its response to heme.