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

A genome-wide phenotypic CRISPR/Cas9 screen identifies novel regulators of PARK2 level and mitophagy priming

PARKIN, an E3 ligasemutated 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 mitoph- agy 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 abun- dance, 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 net- work including THAP11 was identified and negatively regulates en- dogenous 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 repres- sion andmitophagy,which is also apparent in human induced plurip- otent stem cell-derived neurons, a disease-relevant cell type. PARKI

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IAB-93-2200-00 AK 619 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

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

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Available from UB Bonn(5)-4-95-1400 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

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

Ups1 and Ups2 regulate mitochondrial phospholipid metabolism in the mitochondrial intermembrane space. This study shows that Ups1 and Ups2 are intrinsically unstable proteins and are degraded by distinct mitochondrial peptidases. Mdm35 binds to Ups1 and Ups2 and prevents their degradation by mitochondrial proteases

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