IDENTIFICATION AND ANALYSIS OF GOLGI TRANSPORTERS THAT PROVIDE HOMEOSTASIS FOR GLYCOSYLATION AND OTHER GOLGI PROCESSES
Snyder, Nathan A.
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The Golgi apparatus of eukaryotes is a critical organizing center that is responsible for protein and lipid maturation, sorting, and trafficking. Because of the diverse population of proteins and lipids being modified at any given time, the Golgi is host to a large collection of resident enzymes and transporters that are required to achieve the necessary modifications. Many of the Golgi resident proteins, accounting for a large portion of the entire genome, are involved in the process and regulation of glycosylation, the process by which complex oligosaccharides are assembled on proteins and lipids. These oligosaccharides assist in folding, stability, and trafficking of the substrate molecule. The complex nature of the reactions and mechanisms taking place within the Golgi require careful maintenance of the homeostatic balance of all substrates, cofactors, and products, because failure of any single component could result in downstream consequences that compound to become very dangerous for the cell. The mechanisms and transporters of the Golgi apparatus are reviewed in Chapter 1. This dissertation aims to elucidate the roles of several transporters involved in Golgi homeostasis, which have roles in regulating the pH as well as concentrations of Ca2+, Mn2+, and inorganic phosphate (Pi), and identify a new method that may be used to screen antifungals for essential protein targets, including those affiliated with Golgi processes. In the second chapter, I identify a new highly conserved class of reversible H+/Ca2+ exchanger in the Golgi of yeast, Gdt1. Through genetic analysis, I demonstrate that Gdt1 functions alongside the secretory pathway Ca2+ ATPase, Pmr1, to supply Ca2+ to the Golgi apparatus and detoxify high levels of Ca2+ in the cytoplasm. I further show that this activity requires the establishment of a pH gradient, generated by the V-ATPase between the Golgi and cytoplasm. Interestingly, abolishment of this pH gradient by deletion of the V-ATPase resulted in Gdt1 instead removing Ca2+, supplied by Pmr1, from the Golgi, demonstrating the reversibility of the H+/Ca2+ exchange. I also identify and characterize another Golgi transporter of yeast, Erd1, which functions to recycle Pi from the Golgi to the cytoplasm, as it is otherwise lost to the environment through the secretory pathway. This Pi was produced from the breakdown of the GDP byproduct of glycosylation into Pi and GMP, which is recycled by Vrg4 for GDP-mannose, needed for further glycosylation reactions. In Chapter 3, I investigate the mammalian homolog of Gdt1, TMEM165, and its role in lactation in mice. I present evidence that TMEM165, the expression of which is highly upregulated in the mammary gland during lactation, supplies the Golgi with Mn2+, required for activity of lactose synthase, along with Ca2+, secreted into milk as a component of casein micelles, while removing H+, released by lactose synthesis and glycosylation, to prevent acidification of the Golgi. A mouse model, deficient for TMEM165 in the mammary gland, exhibited decreased secretion of lactose into milk, which reduced the osmotic potential of the milk, decreasing its volume, and concentrating the protein and nutritional metals it contains. This ultimately decreased the nutritional quality of the milk, resulting in low weight gain in pups nursed by these mothers. Chapter 4 develops and examines a new technology that can be used to study essential genes in yeast. A collection of yeast strains containing genes expressing proteins tagged with the auxin-inducible degron (AID) was generated. The AID tag enables proteins to be rapidly ubiquitylated and targeted for degradation after the addition of the drug auxin. I analyze the ability of this system to be used in the screening of tagged yeast strains for sensitivity or resistance to various drugs and inhibitors. Using co-varied concentrations of auxin and SDZ 90-215, I demonstrated that a yeast strain containing AID-tagged Vrg4 displayed synergistic sensitivity to the combination of drugs. Further analysis demonstrated that SDZ 90-215 inhibits the activity of Vrg4, blocking the transport of GDP-mannose into the Golgi of yeast, preventing glycosylation of proteins and lipids. This inhibition ultimately kills the yeast cell, allowing SDZ 90-215 to function as a fungicide. The final chapter provides a summary of the implications of the major findings in this work and provides insight into future directions that research into these topics may take.