The mitochondrion is the major energy-producing organelle of the cell and coordinates key activities such as signaling, apoptosis, and phospholipid synthesis. These processes are essential for survival and pathogenesis of Candida albicans, the most common fungal pathogen in humans. C. albicans respiration occurs via three distinct pathways compared to vertebrates and is associated with virulence properties such as morphogenesis and cell wall synthesis. Our long-term research goal is to understand the cellular and molecular mechanisms that govern C. albicans survival in response to environmental stress and antifungal drugs. We previously discovered that gene classes involved mitochondrial functions are highly transcribed in response to osmotic and cell wall stress, and the gene SLP3 (stomatin like protein 3) was significantly upregulated. SLP3 is a member of the conserved SPFH (Stomatin, Prohibitin, Flotillin, HflK/HflC) protein superfamily. In eukaryotes, SPFH proteins are required for essential mitochondrial processes such as respiration, mitophagy, and apoptosis and mediate pathogenicity in several parasites. Our objective in this proposal is to determine the role of the C. albicans SPFH protein family in mitochondrial function. We were the first to demonstrate that Slp3p overproduction disrupted mitochondrial membrane potential and triggered apoptotic-like death specifically following prolonged exposure to oxidative stress. However, the molecular function Slp3p remains unknown. Thus, our central hypothesis is that C. albicans SPFH proteins form membrane complexes to coordinate mitochondrial function. This hypothesis is based upon observations with mammalian SPFH proteins and our published and preliminary findings. Human SPFH complexes appear as punctate foci when viewed using fluorescence microscopy. We found that Slp3p form puncta at the plasma membrane. Further, we identified a mitochondrial targeting signal in Slp2p, Phb1p and Phb2p, and our Slp2p-GFP fusion protein formed mitochondrial puncta. We will utilize a high-throughput molecular genetic and cellular approach that is cost-effective and time-saving to determine SPFH protein localization, protein complex composition, cellular function, and role in C. albicans infection. We will create SPFH-GFP fusion proteins and use fluorescence microscopy to determine cellular localization. We will construct SPFH-epitope-tagged strains and perform Co-IP and LC-MS/MS analyses to identify putative SPFH protein binding partners. We will create SPFH mutants via CRISPR-Cas9 genome editing and phenotypically characterize mutant strains in growth and cellular assays. We will examine SPFH function in C. albicans pathogenesis using a novel invertebrate infection system. Our findings will address a poorly understood area in C. albicans biology and provide a model for studying SPFH proteins in pathogenic fungi. This proposal is innovative as we will characterize the SPFH family in the context of a critical, ye...