Determining the Molecular Mechanisms of PACS-1-mediated Protein Sorting
Abstract
Membrane trafficking events are required to direct proteins to their precise subcellular locations. The cellular Phosphofurin Acidic Cluster Sorting protein – 1 (PACS-1) has emerged as a protein of interest in controlling the localization of a multitude of cellular and viral proteins. Specifically, PACS-1 is hijacked by type-1 Human Immunodeficiency Virus (HIV-1) to contribute to immune evasion in addition to regulating neuroendocrine hormone storage and release. To accomplish this, PACS-1 connects the cytoplasmic tail of cellular receptors to the heterotetrameric adaptor proteins (APs) to form a functional trafficking unit. Throughout this dissertation, I explored the role of PACS-1 and AP-1 to drive the localization of unique cargo proteins; the type-1 major histocompatibility complex (MHC-I) and the adrenocorticotropic hormone (ACTH). I have utilized the intracellular protein-protein interaction reporter assay: bimolecular fluorescence complementation (BiFC), in combination with super-resolution microscopy to uncover the membrane trafficking route undertaken by the HIV-viral accessory protein Nef and the cellular receptor MHC-I. Additionally, I have generated a tool termed “viral BiFC” to study virus:host interactions in cells. These studies revealed a mechanism by which Nef re-routes MHC-I from the cell surface, toward the trans-Golgi Network (TGN) by hijacking both early and recycling endosomes. Interestingly, Nef requires PACS-1 to permit the transport of MHC-I from the endosomes to the TGN, a process which is also dependent on AP-1 recruitment. Moreover, the role of PACS-1 extends beyond its implication in HIV-1 infection. The regulation of neuroendocrine cell hormone storage and secretion is a tightly regulated process requiring coordinated trafficking of multiple proteins. Thus, I hypothesized that PACS-1 would promote proper storage of ACTH within specialized storage granules. These studies identified a key role for PACS-1 and AP-1 in directing ACTH to the storage granule. Undoubtedly, the function of PACS-1 is important in not only the immune evasive capabilities of HIV-1 Nef, but also in the regulation of hormone secretion. By understanding how cargo molecules are targeted throughout the cell by PACS-1, we can begin to unravel the molecular details of viral pathogenesis and cellular homeostasis.