Hypoxia-inducible gene domain 1 proteins in yeast mitochondria protect against proton leak through complex IV.

Hypoxia-inducible gene domain 1 (HIGD1) proteins are small integral membrane proteins, conserved from bacteria to humans, that associate with oxidative phosphorylation supercomplexes. Using yeast as a model organism, we have shown previously that its two HIGD1 proteins, Rcf1 and Rcf2, are required for the generation and maintenance of a normal membrane potential (ΔΨ) across the inner mitochondrial membrane (IMM). We postulated that the lower ΔΨ observed in the absence of the HIGD1 proteins may be due to decreased proton pumping by complex IV (CIV) or enhanced leak of protons across the IMM. Here we measured the ΔΨ generated by complex III (CIII) to discriminate between these possibilities. First, we found that the decreased ΔΨ observed in the absence of the HIGD1 proteins cannot be due to decreased proton pumping by CIV because CIII, operating alone, also exhibited a decreased ΔΨ when HIGD1 proteins were absent. Because CIII can neither lower its pumping stoichiometry nor transfer protons completely across the IMM, this result indicates that HIGD1 protein ablation enhances proton leak across the IMM. Second, we demonstrate that this proton leak occurs through CIV because ΔΨ generation by CIII is restored when CIV is removed from the cell. Third, the proton leak appeared to take place through an inactive population of CIV that accumulates when HIGD1 proteins are absent. We conclude that HIGD1 proteins in yeast prevent CIV inactivation, likely by preventing the loss of lipids bound within the Cox3 protein of CIV.

The yeast mitochondrial proteins Rcf1 and Rcf2 support the enzymology of the cytochrome c oxidase complex and generation of the proton motive force.

The yeast mitochondrial proteins Rcf1 and Rcf2 are associated with a subpopulation of the cytochrome bc1-cytochrome c oxidase supercomplex and have been proposed to play a role in the assembly and/or modulation of the activity of the cytochrome c oxidase (complex IV, CIV). Yeast mutants deficient in either Rcf1 or Rcf2 proteins can use aerobic respiration-based metabolism for growth, but the absence of both proteins results in a strong growth defect. In this study, using assorted biochemical and biophysical analyses of Rcf1/Rcf2 single and double null-mutant yeast cells and mitochondria, we further explored how Rcf1 and Rcf2 support aerobic respiration and growth. We show that the absence of Rcf1 physically reduces the levels of CIV and diminishes the ability of the CIV that is present to maintain a normal mitochondrial proton motive force (PMF). Although the absence of Rcf2 did not noticeably affect the physical content of CIV, the PMF generated by CIV was also lower than normal. Our results indicate that the detrimental effects of the absence of Rcf1 and Rcf2 proteins on the CIV complex are distinct in terms of CIV assembly/accumulation and additive in terms of the ability of CIV to generate PMF. Thus, the combined absence of Rcf1 and Rcf2 alters both CIV physiology and assembly. We conclude that the slow aerobic growth of the Rcf1/Rcf2 double null mutant results from diminished generation of mitochondrial PMF by CIV and limits the level of CIV activity required for maintenance of the PMF and growth under aerobic conditions.

Mitochondrial Voltage-Dependent Anion Channel Protein Por1 Positively Regulates the Nuclear Localization of Saccharomyces cerevisiae AMP-Activated Protein Kinase.

Snf1 protein kinase of the yeast Saccharomyces cerevisiae is a member of the highly conserved eukaryotic AMP-activated protein kinase (AMPK) family, which is involved in regulating responses to energy limitation. Under conditions of carbon/energy stress, such as during glucose depletion, Snf1 is catalytically activated and enriched in the nucleus to regulate transcription. Snf1 catalytic activation requires phosphorylation of its conserved activation loop threonine (Thr210) by upstream kinases. Catalytic activation is also a prerequisite for Snf1's subsequent nuclear enrichment, a process that is mediated by Gal83, one of three alternate ß-subunits of the Snf1 kinase complex. We previously reported that the mitochondrial voltage-dependent anion channel (VDAC) proteins Por1 and Por2 play redundant roles in promoting Snf1 catalytic activation by Thr210 phosphorylation. Here, we show that the por1Δ mutation alone, which by itself does not affect Snf1 Thr210 phosphorylation, causes defects in Snf1 and Gal83 nuclear enrichment and Snf1's ability to stimulate transcription. We present evidence that Por1 promotes Snf1 nuclear enrichment by promoting the nuclear enrichment of Gal83. Overexpression of Por2, which is not believed to have channel activity, can suppress the localization and transcription activation defects of the por1Δ mutant, suggesting that the regulatory role played by Por1 is separable from its channel function. Thus, our findings expand the positive roles of the yeast VDACs in carbon/energy stress signaling upstream of Snf1. Since AMPK/Snf1 and VDAC proteins are conserved in evolution, our findings in yeast may have implications for AMPK regulation in other eukaryotes, including humans. IMPORTANCE AMP-activated protein kinases (AMPKs) sense energy limitation and regulate transcription and metabolism in eukaryotes from yeast to humans. In mammals, AMPK responds to increased AMP-to-ATP or ADP-to-ATP ratios and is implicated in diabetes, heart disease, and cancer. Mitochondria produce ATP and are generally thought to downregulate AMPK. Indeed, some antidiabetic drugs activate AMPK by affecting mitochondrial respiration. ATP release from mitochondria is mediated by evolutionarily conserved proteins known as voltage-dependent anion channels (VDACs). One would therefore expect VDACs to serve as negative regulators of AMPK. However, our experiments in yeast reveal the existence of an opposite relationship. We previously showed that Saccharomyces cerevisiae VDACs Por1 and Por2 positively regulate AMPK/Snf1 catalytic activation. Here, we show that Por1 also plays an important role in promoting AMPK/Snf1 nuclear localization. Our counterintuitive findings could inform research in areas ranging from diabetes to cancer to fungal pathogenesis.

Mitochondrial porin Por1 and its homolog Por2 contribute to the positive control of Snf1 protein kinase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Snf1 is a member of the conserved Snf1/AMP-activated protein kinase (Snf1/AMPK) family involved in regulating responses to energy limitation, which is detected by mechanisms that include sensing adenine nucleotides. Mitochondrial voltage-dependent anion channel (VDAC) proteins, also known as mitochondrial porins, are conserved in eukaryotes from yeast to humans and play key roles in mediating mitochondrial outer membrane permeability to small metabolites, including ATP, ADP, and AMP. We previously recovered the yeast mitochondrial porin Por1 (yVDAC1) from a two-hybrid screen for Snf1-interacting proteins. Here, we present evidence that Snf1 interacts with Por1 and its homolog Por2 (yVDAC2). Cells lacking Por1 and Por2, but not respiratory-deficient rho(0) cells lacking the mitochondrial genome, exhibit reduced Snf1 activation loop phosphorylation in response to glucose limitation. Thus, Por1 and Por2 contribute to the positive control of Snf1 protein kinase. Physical proximity to the VDAC proteins and mitochondrial surface could facilitate Snf1's ability to sense energy limitation.

Rcf1 and Rcf2, members of the hypoxia-induced gene 1 protein family, are critical components of the mitochondrial cytochrome bc1-cytochrome c oxidase supercomplex.

We report that Rcf1 (formerly Aim31), a member of the conserved hypoxia-induced gene 1 (Hig1) protein family, represents a novel component of the yeast cytochrome bc(1)-cytochrome c oxidase (COX) supercomplex. Rcf1 (respiratory supercomplex factor 1) partitions with the COX complex, and evidence that it may act as a bridge to the cytochrome bc(1) complex is presented. Rcf1 interacts with the Cox3 subunit and can do so prior to their assembly into the COX complex. A close proximity of Rcf1 and members of the ADP/ATP carrier (AAC) family was also established. Rcf1 displays overlapping function with another Hig1-related protein, Rcf2 (formerly Aim38), and their joint presence is required for optimal COX enzyme activity and the correct assembly of the cytochrome bc(1)-COX supercomplex. Rcf1 and Rcf2 can independently associate with the cytochrome bc(1)-COX supercomplex, indicating that at least two forms of this supercomplex exist within mitochondria. We provide evidence that the association with the cytochrome bc(1)-COX supercomplex and regulation of the COX complex are a conserved feature of Hig1 family members. Based on our findings, we propose a model where the Hig1 proteins regulate the COX enzyme activity through Cox3 and associated Cox12 protein, in a manner that may be influenced by the neighboring AAC proteins.