# Unveiling Specificity, Redundancy, and Promiscuity of Five Saccharomyces cerevisiae Mitochondrial Carriers

## TL;DR

This study explores the transport capabilities of five yeast mitochondrial proteins, revealing they handle a wider range of substances than previously known.

## Contribution

The study identifies broader substrate specificities and functional redundancy in yeast mitochondrial transporters using in vivo assays and simulations.

## Key findings

- Pet9 transports multiple organic acids and amino acids.
- Yhm2 uptakes nine amino acids and fumaric acid.
- Crc1 shows promiscuous transport activity.

## Abstract

The transport of metabolites across biological membranes is vital for normal cellular functions, including nutrient uptake, homeostasis, and toxin efflux. In eukaryotes, mitochondrial transporters in the inner mitochondrial membrane (IMM) play a pivotal role in energy production, metabolism, and the biosynthesis of a wide range of compounds. While functional assignments exist for over half of the mitochondrial transporters, emerging high-throughput methodologies underscore the need for reassessment and expansion of the current knowledge, particularly as evidence suggesting functional redundancy and substrate promiscuity has emerged. In this study, we investigated the substrate specificity of five yeast mitochondrial transporters—Crc1 (YOR100c), Ctp1 (YBR291c), Oac1 (YKL120w), Pet9 (YBL030c), and Yhm2 (YMR241w)—via heterologous gene expression in Xenopus laevis oocytes and liquid chromatography-mass spectrometry (LC-MS)-based transport assays. We used two substrate mixtures: a 17-compound organic acid mix and a 13C-labeled yeast metabolite extract. Our results revealed broader substrate specificities than previously reported, as partially supported by substrate docking simulations. Pet9 transported several organic acids and amino acids, while Yhm2 showed uptake of nine amino acids and fumaric acid. Additional promiscuous transport activity was observed for Crc1, indicating that these proteins may have more extensive metabolic roles than previously known. This study advances the understanding of yeast mitochondrial transporter function, demonstrating redundancy and broad substrate specificity among mitochondrial carriers. It highlights the importance of utilizing in vivo heterologous systems and physiologically relevant substrate mixtures to elucidate transporter functionality.