Current models evoke the plasma membrane (PM) as the exclusive platform from which Ras regulates signalling. We developed a fluorescent probe that reports where and when Ras is activated in living cells. We show that oncogenic H-Ras and N-Ras engage Raf-1 on the Golgi and that endogenous Ras and unpalmitoylated H-Ras are activated in response to mitogens on the Golgi and endoplasmic reticulum (ER), respectively. We also demonstrate that H-Ras that is restricted to the ER can activate the Erk pathway and transform fibroblasts, and that Ras localized on different membrane compartments differentially engages various signalling pathways. Thus, Ras signalling is not limited to the PM, but also proceeds on the endomembrane.

Heme is a key molecule in mediating the effects of oxygen on various molecular and cellular processes in many living organisms. In the yeast Saccharomyces cerevisiae, heme serves as a secondary signal for oxygen; intracellular heme synthesis directly correlates with oxygen tension in the environment. In yeast, oxygen sensing and heme signaling are primarily mediated by the heme activator protein Hap1, which, in response to heme, activates the transcription of genes required for respiration and for controlling oxidative damage. Heme regulation of many genes required for anaerobic growth is mediated by the aerobic repressor Rox1, whose expression is controlled by heme. In this review, we summarize recent knowledge about (i) how heme synthesis may be controlled by oxygen tension, (ii) how heme precisely and stringently controls Hap1 activity and (iii) whether other transcriptional activators can also mediate heme action.

Apart from serving as a prosthetic group in globins and enzymes, heme is a key regulator controlling a wide range of molecular and cellular processes involved in oxygen sensing and utilization. To gain insights into molecular mechanisms of heme signaling and oxygen sensing in eukaryotes, we investigated the yeast hemeresponsive transcriptional activator HAP1. HAP1 activity is regulated precisely and tightly by heme. Here we show that in the absence of heme, HAP1 forms a biochemically distinctive higher-order complex. Our data suggest that this complex contains HAP1 and four other cellular proteins including Hsp82 and Ydj1. The formation of this complex is directly correlated with HAP1 repression in the absence of heme, and mutational or heme disruption of the complex correlates with HAP1 activation, suggesting that this complex is responsible for heme regulation of HAP1 activity. Further, we determined HAP1 domains required for heme regulation: three domains-the dimerization domain, the heme domain, and the HRM7 (heme-responsive motif 7) domain-cooperate to form the higher-order complex and mediate heme regulation. Strikingly, we uncovered a novel function for the HAP1 dimerization domain: it not only allows dimerization but also provides critical functions in heme regulation and transcriptional activation. Our studies provide significant insights into the molecular events leading to heme activation of HAP1 and may shed light on molecular mechanisms of various heme-controlled biological processes in diverse organisms.

In Saccharomyces cerevisiae, heme directly mediates the effects of oxygen on transcription through the heme activator protein Hap1. In the absence of heme, Hap1 is bound by at least four cellular proteins, including Hsp90 and Ydj1, forming a higher-order complex, termed HMC, and its activity is repressed. Here we purified the HMC and showed by mass spectrometry that two previously unidentified major components of the HMC are the Ssa-type Hsp70 molecular chaperone and Sro9 proteins. In vivo functional analysis, combined with biochemical analysis, strongly suggests that Ssa proteins are critical for Hap1 repression in the absence of heme. Ssa may repress the activities of both Hap1 DNA-binding and activation domains. The Ssa cochaperones Ydj1 and Sro9 appear to assist Ssa in Hap1 repression, and only Ydj1 residues 1 to 172 containing the J domain are required for Hap1 repression. Our results suggest that Ssa-Ydj1 and Sro9 act together to mediate Hap1 repression in the absence of heme and that molecular chaperones promote heme regulation of Hap1 by a mechanism distinct from the mechanism of steroid signaling.

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