Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy.

PARKIN, an E3 ligase mutated in familial Parkinson's disease, promotes mitophagy by ubiquitinating mitochondrial proteins for efficient engagement of the autophagy machinery. Specifically, PARKIN-synthesized ubiquitin chains represent targets for the PINK1 kinase generating phosphoS65-ubiquitin (pUb), which constitutes the mitophagy signal. Physiological regulation of PARKIN abundance, however, and the impact on pUb accumulation are poorly understood. Using cells designed to discover physiological regulators of PARKIN abundance, we performed a pooled genome-wide CRISPR/Cas9 knockout screen. Testing identified genes individually resulted in a list of 53 positive and negative regulators. A transcriptional repressor network including THAP11 was identified and negatively regulates endogenous PARKIN abundance. RNAseq analysis revealed the PARKIN-encoding locus as a prime THAP11 target, and THAP11 CRISPR knockout in multiple cell types enhanced pUb accumulation. Thus, our work demonstrates the critical role of PARKIN abundance, identifies regulating genes, and reveals a link between transcriptional repression and mitophagy, which is also apparent in human induced pluripotent stem cell-derived neurons, a disease-relevant cell type.

Stendomycin selectively inhibits TIM23-dependent mitochondrial protein import.

Tim17 and Tim23 are the main subunits of the TIM23 complex, one of the two major essential mitochondrial inner-membrane protein translocon machineries (TIMs). No chemical probes that specifically inhibit TIM23-dependent protein import were known to exist. Here we show that the natural product stendomycin, produced by Streptomyces hygroscopicus, is a potent and specific inhibitor of the TIM23 complex in yeast and mammalian cells. Furthermore, stendomycin-mediated blockage of the TIM23 complex does not alter normal processing of the major regulatory mitophagy kinase PINK1, but TIM23 is required to stabilize PINK1 on the outside of mitochondria to initiate mitophagy upon membrane depolarization.

ORA1, a zebrafish olfactory receptor ancestral to all mammalian V1R genes, recognizes 4-hydroxyphenylacetic acid, a putative reproductive pheromone.

The teleost v1r-related ora genes are a small, highly conserved olfactory receptor gene family of only six genes, whose direct orthologues can be identified in lineages as far as that of cartilaginous fish. However, no ligands for fish olfactory receptor class A related genes (ORA) had been uncovered so far. Here we have deorphanized the ORA1 receptor using heterologous expression and calcium imaging. We report that zebrafish ORA1 recognizes with high specificity and sensitivity 4-hydroxyphenylacetic acid. The carboxyl group of this compound is required in a particular distance from the aromatic ring, whereas the hydroxyl group in the para-position is not essential, but strongly enhances the binding efficacy. Low concentrations of 4-hydroxyphenylacetic acid elicit increases in oviposition frequency in zebrafish mating pairs. This effect is abolished by naris closure. We hypothesize that 4-hydroxyphenylacetic acid might function as a pheromone for reproductive behavior in zebrafish. ORA1 is ancestral to mammalian V1Rs, and its putative function as pheromone receptor is reminiscent of the role of several mammalian V1Rs as pheromone receptors.

Regulation of mitochondrial phospholipids by Ups1/PRELI-like proteins depends on proteolysis and Mdm35.

The mitochondrial phospholipid metabolism critically depends on members of the conserved Ups1/PRELI-like protein family in the intermembrane space. Ups1 and Ups2 (also termed Gep1) were shown to regulate the accumulation of cardiolipin (CL) and phosphatidylethanolamine (PE), respectively, in a lipid-specific but coordinated manner. It remained enigmatic, however, how the relative abundance of both phospholipids in mitochondrial membranes is adjusted on the molecular level. Here, we describe a novel regulatory circuit determining the accumulation of Ups1 and Ups2 in the intermembrane space. Ups1 and Ups2 are intrinsically unstable proteins, which are degraded by distinct mitochondrial peptidases. The turnover of Ups2 is mediated by the i-AAA protease Yme1, whereas Ups1 is degraded by both Yme1 and the metallopeptidase Atp23. We identified Mdm35, a member of the twin Cx(9)C protein family, as a novel interaction partner of Ups1 and Ups2. Binding to Mdm35 ensures import and protects both proteins against proteolysis. Homologues to all components of this pathway are present in higher eukaryotes, suggesting that the regulation of mitochondrial CL and PE levels is conserved in evolution.

The mitochondrial amidoxime-reducing component (mARC1) is a novel signal-anchored protein of the outer mitochondrial membrane.

The mitochondrial amidoxime-reducing component (mARC) was recently discovered as the fifth eukaryotic molybdenum cofactor-containing enzyme. The human genome encodes two mARC proteins, mARC1 and mARC2, sharing significant homologies with respect to sequence and function. Whereas mARC2 was identified as a mitochondrial enzyme, the subcellular localization of mARC1 has remained uncharacterized, although the similarity of both proteins suggested identical subcellular localizations. In addition, neither mARC1 nor mARC2 could be attributed unambiguously to one of the four mitochondrial subcompartments. Accordingly, mechanisms triggering the subcellular distribution of both enzymes have been unexplored so far. Here, we shed light on the subcellular localization of mARC1 and demonstrate that it is integrated into the outer mitochondrial membrane. The C-terminal catalytic domain of the protein remains exposed to the cytosol and confers an N((in))-C((out)) membrane orientation of mARC1. This localization is triggered by the N terminus of the enzyme, being composed of a weak N-terminal mitochondrial targeting signal and a downstream transmembrane helix. We demonstrate the transmembrane domain of mARC1 to be sufficient for mitochondrial targeting and the N-terminal targeting signal to function as a supportive receptor for the outer mitochondrial membrane. According to its localization and targeting mechanism, we classify mARC1 as a novel signal-anchored mitochondrial protein. During mitochondrial import, mARC1 is not processed, and membrane integration proceeds membrane potential independently but requires external ATP, which finally results in the assembly of mARC1 into high oligomeric protein complexes.

TRIAP1/PRELI complexes prevent apoptosis by mediating intramitochondrial transport of phosphatidic acid.

Cardiolipin (CL), a mitochondria-specific glycerophospholipid, is required for diverse mitochondrial processes and orchestrates the function of various death-inducing proteins during apoptosis. Here, we identify a complex of the p53-regulated protein TRIAP1 (p53CSV) and PRELI in the mitochondrial intermembrane space (IMS), which ensures the accumulation of CL in mitochondria. TRIAP1/PRELI complexes exert lipid transfer activity in vitro and supply phosphatidic acid (PA) for CL synthesis in the inner membrane. Loss of TRIAP1 or PRELI impairs the accumulation of CL, facilitates the release of cytochrome c, and renders cells vulnerable to apoptosis upon intrinsic and extrinsic stimulation. Survival of TRIAP1- and PRELI-deficient cells is conferred by an excess of exogenously provided phosphatidylglycerol. Our results reveal a p53-dependent cell-survival pathway and highlight the importance of the CL content of mitochondrial membranes in apoptosis.

The genetic interactome of prohibitins: coordinated control of cardiolipin and phosphatidylethanolamine by conserved regulators in mitochondria.

Prohibitin ring complexes in the mitochondrial inner membrane regulate cell proliferation as well as the dynamics and function of mitochondria. Although prohibitins are essential in higher eukaryotes, prohibitin-deficient yeast cells are viable and exhibit a reduced replicative life span. Here, we define the genetic interactome of prohibitins in yeast using synthetic genetic arrays, and identify 35 genetic interactors of prohibitins (GEP genes) required for cell survival in the absence of prohibitins. Proteins encoded by these genes include members of a conserved protein family, Ups1 and Gep1, which affect the processing of the dynamin-like GTPase Mgm1 and thereby modulate cristae morphogenesis. We show that Ups1 and Gep1 regulate the levels of cardiolipin and phosphatidylethanolamine in mitochondria in a lipid-specific but coordinated manner. Lipid profiling by mass spectrometry of GEP-deficient mitochondria reveals a critical role of cardiolipin and phosphatidylethanolamine for survival of prohibitin-deficient cells. We propose that prohibitins control inner membrane organization and integrity by acting as protein and lipid scaffolds.