Intracellular trafficking of splice variants of the zinc transporter SLC30A5 in response to zinc

Zinc is an essential component of the diet and plays a role if a vast array of functions in cells and tissues. There is evidence that sub-clinical zinc deficiency may be important with respect to aging, immune function and neonatal health. Zinc can also be toxic if over accumulated. Therefore organisms, including humans require efficient mechanisms to take up zinc from their diet or extracellular environment. Thus, precise regulatory mechanisms are also required to control this uptake to maintain adequate zinc status while preventing its over accumulation. Despite the importance of zinc, we still lack understanding of the molecular basis for zinc homeostasis within in cells and tissues. Zinc homeostasis in mammals is primarily regulated through the control of zinc absorption in the intestine and the loss of zinc from the body through secretion into the intestine. Movement of zinc into and out of cells is mediated by zinc transporter proteins, one of which is called SLC30A5. There are at least two forms of this zinc transporter (SLC30A5a and SLC30A5b) which differ slightly in the amino acids that make them up. Both forms maybe important for zinc uptake from the diet when situated at the membrane of the intestinal cell across which nutrient absorption occurs. However under certain conditions SLC30A5 maybe located within the cell. It is therefore a possibility that the different forms of SLC30A5 may demonstrate a zinc induced movement to avoid zinc toxicity by reducing the amount of zinc entering the cell. This project will investigate how the two different forms of SLC30A5 move within the cell, between the absorptive membrane and other compartments within the cell, in response to changes in zinc availability. To achieve these aims, the two different forms of SLC30A5 will be expressed with the addition of a fluorescent tag in a cell line model of the human intestine. Understanding how SLC30A5 responds to changes in the amount of zinc in the diet will give insight into the mechanisms through which zinc balance is maintained.

Despite the importance of zinc in fundamental cellular processes and in health, mechanisms of zinc homeostasis remain ill defined. We hypothesise that the zinc tranporter SLC305A plays a role in such mechanisms and that two previously identifed splice variants fulfil different roles through their different functional properties and different subcellular distributions in respect to zinc. We will examine the subcellular localisation of two specific splice variants at different concentrations of zinc by stably transfecting plasmid constructs from which one or other of the SLC30A5 splice variants is expressed with a C-terminal GFP tag into Caco-2 cells grown under differing zinc conditions and will investigate the effect of re-localisation on intracellular zinc concentration and distribution using zinc sensitive fluorescent dyes. We will also investigate the role of the different N- and C- termini of the two SLC30A5 splice variants in the trafficking of this transporter by creating chimeric GFP constructs. We will also identify proteins that interact with regions of SLC30A5 shown to affect trafficking usings a mammalian 2 hybrid screen.