The frequency of fungal infections is rising while the treatment options are dwindling with an increasing number of drug-resistant fungal species. Ergosterol, the predominant sterol in fungal plasma membranes, is a common target for anti-fungal compounds. Sterol synthesis is an oxygen-dependent process, but during anaerobic conditions, the average cell growth is supported by the uptake of exogenous sterols, for example, serum cholesterol from the host organisms by fungal pathogens. In Saccharomyces cerevisiae, two ABC transporters, Aus1p and Pdr11p, are responsible for the uptake of exogenous sterol to help assist the normal growth of budding yeast. Localisation studies on Aus1p in Candia albicans, an opportunistic fungus, have shown this transporter to localise in a plasma membrane domain enriched with sterol and sphingolipids. Such domains have sizes ranging in nanometres, making them visible only through advanced and super-resolution microscopic methods. Here, we attempt to create a toolbox that would assist in the live-cell localisation of such domain-residing proteins in Saccharomyces cerevisiae with the application of (a) conventional fluorescent proteins and (b) SNAP-tag, a self-labelling enzyme tag.

Target proteins were tagged using conventional fluorescent proteins using two: (a) chromosomal tagging and (b) centromeric plasmid-based expression system. The fusion proteins, thus generated, were studied for their localisation and functionality in the living yeast cells. We successfully generated three backbone centromeric plasmids and one expression vector carrying an Aus1p-eGFP fusion protein. Our results demonstrate that centromeric plasmids are a suitable alternative to genomic integration for localisation studies. The utility of SNAP-tag in budding yeast cells was tested under in vitro conditions using whole cell lysates, in living cells with the assistance of electroporation and prolonged incubation and in chemically fixed yeast cells. Our results indicate that SNAP-tag can be effective for in vitro studies in budding yeast; however, in living cells and chemically fixed cells, the application of SNAP-tag is influenced by its position of localisation in the yeast cell and the choice of SNAP dye. Further steps to optimise this technology in budding yeast cells are required for their practical application in super-resolution microscopic techniques.

Through this study, we attempt to generate a toolbox that can help identify the presence of membrane domains in budding yeast under anaerobic conditions while using Aus1p and Pdr11p as the membrane targets. Studying these proteins enables us to determine their exact domain of localisation in the membrane and potentially their interacting partners, which could serve as new targets for developing new and improved anti-fungal compounds.