Citation: Gorgoglione, R.; Seccia, R.; Ahmed, A.; Vozza, A.; Capobianco, L.; Lodi, A.; Marra, F.; Paradies, E.; Palmieri, L.; Coppola, V.; et al. Generation of a Yeast Cell Model Potentially Useful to Identify the Mammalian Mitochondrial N-Acetylglutamate Transporter. Biomolecules 2023, 13, 808. https:// doi.org/10.3390/biom13050808 Academic Editor: Frank Krause Received: 24 April 2023 Revised: 7 May 2023 Accepted: 8 May 2023 Published: 10 May 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomolecules Article Generation of a Yeast Cell Model Potentially Useful to Identify the Mammalian Mitochondrial N -Acetylglutamate Transporter Ruggiero Gorgoglione 1,† , Roberta Seccia 1,† , Amer Ahmed 1 , Angelo Vozza 1 , Loredana Capobianco 2 , Alessia Lodi 3,4 , Federica Marra 5 , Eleonora Paradies 6 , Luigi Palmieri 1 , Vincenzo Coppola 7 , Vincenza Dolce 5, * and Giuseppe Fiermonte 1, * 1 Department of Bioscience, Biotechnology and Environment, University of Bari, 70125 Bari, Italy 2 Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy 3 Department of Nutritional Sciences, College of Natural Sciences, The University of Texas at Austin, Austin, TX 78712, USA 4 Dell Pediatric Research Institute, Dell Medical School, The University of Texas at Austin, Austin, TX 78723, USA 5 Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, Italy 6 CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), 70125 Bari, Italy 7 Department of Cancer Biology and Genetics, College of Medicine, The Ohio State University and Arthur G. James Comprehensive Cancer Center, Columbus, OH 43210, USA * Correspondence: vincenza.dolce@unical.it (V.D.); giuseppe.fiermonte@uniba.it (G.F.); Tel.: +39-0984-493119 (V.D.);+39-080-5442741 (G.F.) † These authors contributed equally to this work. Abstract: 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 arg8Δ 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 arg8Δ strain in the absence of arginine, demonstrating the potential suitability of the model generated. Keywords: N-acetylglutamate; urea cycle; mitochondrial carriers; yeast cell model 1. Introduction Mitochondrial carriers (MCs) are a family of eukaryotic intracellular transport proteins, primarily localized in the inner mitochondrial membrane, characterized by a tripartite struc- ture consisting of three homologous domains, each composed of approximately 100 amino Biomolecules 2023, 13, 808. https://doi.org/10.3390/biom13050808 https://www.mdpi.com/journal/biomolecules