Statement of the purpose – Cell wall and membrane–embedded proteins comprise a very special class of molecular components. They are usually involved in processes such as adhesion, signal transduction, and metabolism. Moreover, some of them have even been associated to disease. It is for this reason that these proteins, and also their cytosolic interacting partners, constitute major targets in drug discovery for pharmacotherapy. Despite being often difficult to study because of their hydrophobicity, methods have been developed to study membrane proteins and their interactions in vivo. In Saccharomyces cerevisiae, the membrane sensor Mtl1p has been shown to have a role in the cellular response to oxidative stress induced by exposure to H2O2, and to glucose starvation. The purpose of our research is the identification of protein partners whose interactions with the cytosolic domain of Mtl1p are relevant to its function in order to test them as targets for therapeutic drug discovery in pathogenic fungi. Methods – Our method is based in the integrated Membrane Yeast Two Hybrid (iMYTH) system for the detection of interactions between two proteins of interest. The membrane protein Mtl1p was employed as the bait, while all cytosolic and some membrane protein partners of Mtl1p that were captured by this assay (referred to as the prey) were subjected to a confirmatory test. Summary of results – We have preliminarily identified 25 novel prey proteins that interacted with the cytosolic domain of the Mtl1p bait and were classified as potentially relevant to its function. Conclusions – Our results demonstrate that novel interactions with cytosolic partners of trans-membrane proteins can be identified using this experimental approach. Acknowledgments - This research is supported by the University of Puerto Rico, Medical Sciences Campus, and NIH awards RCMI G12MD007600, RISE R25GM061838, and INBRE P20GM103475.