Generation of a Yeast Cell Model Potentially Useful to Identify the Mammalian Mitochondrial N-Acetylglutamate Transporter.

The human mitochondrial carrier family (MCF) consists of 53 members. Approximately one-fifth of them are still orphans of a function. Most mitochondrial transporters have been functionally characterized by reconstituting the bacterially expressed protein into liposomes and transport assays with radiolabeled compounds. The efficacy of this experimental approach is constrained to the commercial availability of the radiolabeled substrate to be used in the transport assays. A striking example is that of N-acetylglutamate (NAG), an essential regulator of the carbamoyl synthetase I activity and the entire urea cycle. Mammals cannot modulate mitochondrial NAG synthesis but can regulate the levels of NAG in the matrix by exporting it to the cytosol, where it is degraded. The mitochondrial NAG transporter is still unknown. Here, we report the generation of a yeast cell model suitable for identifying the putative mammalian mitochondrial NAG transporter. In yeast, the arginine biosynthesis starts in the mitochondria from NAG which is converted to ornithine that, once transported into cytosol, is metabolized to arginine. The deletion of ARG8 makes yeast cells unable to grow in the absence of arginine since they cannot synthetize ornithine but can still produce NAG. To make yeast cells dependent on a mitochondrial NAG exporter, we moved most of the yeast mitochondrial biosynthetic pathway to the cytosol by expressing four E. coli enzymes, argB-E, able to convert cytosolic NAG to ornithine. Although argB-E rescued the arginine auxotrophy of arg8∆ strain very poorly, the expression of the bacterial NAG synthase (argA), which would mimic the function of a putative NAG transporter increasing the cytosolic levels of NAG, fully rescued the growth defect of arg8∆ strain in the absence of arginine, demonstrating the potential suitability of the model generated.

Citrate Regulates the Saccharomyces cerevisiae Mitochondrial GDP/GTP Carrier (Ggc1p) by Triggering Unidirectional Transport of GTP.

The yeast mitochondrial transport of GTP and GDP is mediated by Ggc1p, a member of the mitochondrial carrier family. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria in exchange for GDP generated in the matrix. ggc1Δ cells exhibit lower levels of GTP and increased levels of GDP in mitochondria, are unable to grow on nonfermentable substrates and lose mtDNA. Because in yeast, succinyl-CoA ligase produces ATP instead of GTP, and the mitochondrial nucleoside diphosphate kinase is localized in the intermembrane space, Ggc1p is the only supplier of mitochondrial GTP required for the maturation of proteins containing Fe-S clusters, such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. In this work, it was demonstrated that citrate is a regulator of purified and reconstituted Ggc1p by trans-activating unidirectional transport of GTP across the proteoliposomal membrane. It was also shown that the binding site of Ggc1p for citrate is different from the binding site for the substrate GTP. It is proposed that the citrate-induced GTP uniport (CIGU) mediated by Ggc1p is involved in the homeostasis of the guanine nucleotide pool in the mitochondrial matrix.

A dual role of YAP in driving TGFß-mediated endothelial-to-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT) is the biological process through which endothelial cells transdifferentiate into mesenchymal cells. During embryo development, EndMT regulates endocardial cushion formation via TGFß/BMP signaling. In adults, EndMT is mainly activated during pathological conditions. Hence, it is necessary to characterize molecular regulators cooperating with TGFß signaling in driving EndMT, to identify potential novel therapeutic targets to treat these pathologies. Here, we studied YAP, a transcriptional co-regulator involved in several biological processes, including epithelial-to-mesenchymal transition (EMT). As EndMT is the endothelial-specific form of EMT, and YAP (herein referring to YAP1) and TGFß signaling cross-talk in other contexts, we hypothesized that YAP contributes to EndMT by modulating TGFß signaling. We demonstrate that YAP is required to trigger TGFß-induced EndMT response, specifically contributing to SMAD3-driven EndMT early gene transcription. We provide novel evidence that YAP acts as SMAD3 transcriptional co-factor and prevents GSK3ß-mediated SMAD3 phosphorylation, thus protecting SMAD3 from degradation. YAP is therefore emerging as a possible candidate target to inhibit pathological TGFß-induced EndMT at early stages.