Phafin2 is a member of the Phafin protein family. Phafins are modular with an N-terminal PH (Pleckstrin Homology) domain followed by a central FYVE (Fab1, YOTB, Vac1, and EEA1) domain. Both the Phafin2 PH and FYVE domains bind phosphatidylinositol 3-phosphate [PtdIns(3)P], a phosphoinositide mainly found in endosomal and lysosomal membranes. Phafin2 acts as a PtdIns(3)P effector for endosomal cargo trafficking, macropinocytosis, apoptosis, and autophagy. The PtdIns(3)P binding activity is critical to the localization of Phafin2 on a specific membrane and, subsequently, helps the recruitment of other binding partners to the same membrane surface. However, there are no studies on the structural basis of PtdIns(3)P binding, the PtdIns(3)P-binding properties of each domain, and the apparent redundancy of two PtdIns(3)P binding domains in Phafin proteins. In the present dissertation, different biochemical and biophysical techniques were utilized to investigate the structural features of Phafin2 and its lipid interactions. This dissertation shows that Phafin2 is a moderately elongated monomer with a predicted α/β structure and ~40% random coil content. Phafin2 binds lipid bilayer-embedded PtdIns(3)P with high affinity; its PH and FYVE domains display distinct PtdIns(3)P-binding properties. Unlike the PH domain, the Phafin2 FYVE domain binds both membrane-embedded PtdIns(3)P and water-soluble dibutanoyl PtdIns(3)P with similar affinity. An intramolecular autoinhibition mechanism is found in Phafin2, in which a conserved C-terminal aspartic acid-rich (polyD) motif inhibits the binding of Phafin2 PH domain to PtdIns(3)P. The polyD motif specifically interacts with the Phafin2 PH domain. Using negative-stain Transmission Electron Microscopy, Phafin2 was found to cause membrane tubulation in a PtdIns(3)P-dependent manner. In conclusion, this study provides the structural and functional basis of Phafin2 lipid interactions and evidence of an intramolecular autoinhibition mechanism for PtdIns(3)P binding to the Phafin2 PH domain, which is mediated by the C-terminal polyD. The distinct PtdIns(3)P binding properties of the Phafin2 PH and FYVE domains may indicate that these two domains have different functions. Considering that the Phafin2 PH domain's PtdIns(3)P binding is intramolecularly regulated, cells may employ a unique mechanism to release the Phafin2 PH domain from the conserved C-terminal motif and control the functions of Phafin2 in PtdIns(3)P- and PH domain-dependent signaling pathways. / Doctor of Philosophy / Living cells need to absorb extracellular materials to sustain their growth and achieve cellular homeostasis. When cells require an uptake of liquids, they employ pinocytosis ("cell drinking"); when cells uptake solid particles, they use phagocytosis ("cell eating"); and when cells are in nutrient starvation status, they exploit an evolutionarily conserved process to survive known as autophagy ("self-eating"). Cells coordinate these activities through complex biochemical signaling systems. In each of these activities, a specific pathway is used to transfer the extracellular materials into the intracellular compartments and regulate the intracellular communications. Protein-lipid interactions are critical to these signaling pathways. This study focuses on the interactions between Phafin2 and phosphatidylinositol 3-phosphate [PtdIns(3)P]. Phafin2 is a cytoplasmic protein involved in autophagy, and PtdIns(3)P is a transient lipid signaling molecule localized to a specific organelle. After cells trigger autophagic events, Phafin2 protein molecules are associated with PtdIns(3)P. Subsequently, Phafin2 will recruit other protein binding partners. In this research project, biochemical and biophysical approaches were employed to study the structural features and PtdIns(3)P binding properties of Phafin2. Phafin2 was found to have two distinct PtdIns(3)P-binding domains; however, one of them is intramolecularly regulated. The results of this study help us to understand why Phafin2 displays two PtdIns(3)P-binding domains with different properties and how this is regulated, information that might be instrumental to understanding the roles of Phafin2 in physiological and disease scenarios.

The parasite Plasmodium falciparum causes the most virulent form of human malaria and is responsible for the vast majority of malaria-related deaths. During the asexual intraerythrocytic stage, the parasite must transport newly synthesized proteins and endocytosed cargo to a variety of organelles, many of which are formed de novo and have no human equivalent. This process in mammalian cells would utilize an endosomal protein trafficking system, but no endosomal structures or proteins have been described in the parasite. Prior work on the parasite genome indicated that several proteins, which could potentially coordinate an endosomal network, were encoded in the genome and expressed during the asexual parasite stages. In this study, we have localized and attempted to further characterize these proteins in the context of the endosomal system. Two well-conserved protein components of the late endosome, the retromer cargo-selective complex and Rab7, were found on a previously un-described inherited structure adjacent to the parasite Golgi apparatus and in close opposition to nascent rhoptries (specialized secretory organelles required for invasion). The retromer cargo-selective complex was also in close proximity to its putative cargo, a P. falciparum homolog of the sortilin family of protein sorting receptors, PfSortilin. Another protein, PfFCP, the sole FYVE domain-containing protein in the P. falciparum genome, was localized to the membrane of a specialized acidic organelle, known as the food vacuole, where the parasite catabolizes the majority of its host cell hemoglobin. We analyzed the effects of a PfFCP dominant negative mutant and found that it altered food vacuole morphology and trafficking. A previous report localized the early endosome phosphoinositide, phosphatidylinositol 3-phosphate, to the food vacuole membrane, and in conjunction with our studies on PfFCP, this has raised doubts about the food vacuole as a lysosome equivalent in the parasite. The combination of both early and late endosome protein homologs in the parasite, and their potential function, has led to a new model of protein trafficking within the parasite that includes the food vacuole as a terminal early endosome and the apical organelles as lysosome equivalents. / Ph. D.