Opt1 imports CoA precursors as glutathione mixed disulfides.

Pantothenate is a key vitamin for the intracellular biosynthesis of the essential molecule coenzyme A (CoA). Pantothenate can be biosynthesized or is taken up by cells via plasma membrane transporters. In the cell, pantothenate, ATP, and cysteine are required to synthesize CoA via five enzymatic steps. This canonical CoA biosynthesis route is well-studied in various organisms. Alternative routes that begin with the uptake of pantetheine (PanSH) or 4'-phosphopantetheine (PPanSH) as initial CoA precursors also exist. These alternative routes are vital for numerous unicellular organisms and are of interest for treating human diseases caused by defects in the canonical CoA biosynthesis pathway. In contrast to the uptake mechanisms for pantothenate, the cellular uptake mechanisms for PanSH and/or PPanSH are unresolved. Through a combination of in vivo experiments, yeast genetics, and the use of chemically traceable compounds, we uncovered a non-canonical CoA biosynthesis pathway. We demonstrate that extracellularly, PanSH and PPanSH form mixed disulfides with glutathione, followed by uptake via the oligopeptide transporter Opt1. Once PanSH or PPanSH are imported, they are converted into CoA. Via this route, several proteins essential for the canonical pantothenate-cysteine-dependent CoA biosynthesis pathway become dispensable. Additionally, we show that yeast strains cultured on PanSH or PPanSH have a growth advantage under conditions of decreased cysteine biosynthesis. The identified non-canonical CoA biosynthesis route provides a framework to treat CoA-linked diseases and to manipulate the growth of pathogenic or beneficial organisms that grow on PanSH or PPanSH.