Abstract Zinc is a ubiquitous biological metal found in about 10% of the eukaryotic proteome. The spatiotemporal zinc dynamics provides crucial cellular signaling opportunities, but also challenges intracellular zinc homeostasis with broad disease implications. Zinc transporters play a central role in regulating cellular zinc balance and subcellular zinc distributions. Current biochemistry of zinc transporters is largely based on purified proteins in test-tubes. However, the molecular functions of zinc transporters in mammalian cells are cell context dependent, vary with subcellular locations and regulated by pathophysiologic stimuli. At present, we have no knowledge of in-cell functions of endogenous zinc transporters at the molecular level. To fill in this knowledge vacuum, we will use an islet-specific zinc transporter ZnT8 and its host cells, the insulin-producing pancreatic β-cells as a mammalian model system to address two questions of the greatest importance to zinc biology: (i) how ZnT8-mediated zinc transport is compartmentalized at the correct subcellular locations to perform intended tasks, (ii) how the biosynthetic burden of ZnT8 is managed by the protein quality control network that ensures clearance of terminally damaged ZnT8 under cell stress. The second question is not directly related to zinc transport, but has a profound impact on β-cell biology with implications in pathogenesis of type-2 diabetes. Our prior studies have uncovered functional coupling of ZnT8 and insulin in two critical cellular processes, glucose-stimulated insulin secretion and inflammation-induced unfolded protein response in the endoplasmic reticulum. The proposed research will characterize subcellular locations and protein levels of endogenous ZnT8 in response to metabolic and inflammatory stress (Aim-1), identify regulators of ZnT8 subcellular functions, and elucidate their mechanism of actions (Aim-2). The proposed research will reveal unprecedented molecular details of ZnT8 subcellular functions in molecular and cellular processes that impact cell biology of host cells, leading to a new paradigm of in-cell molecular functions of zinc transporters in mammalian cells.