Methods to evaluate Zinc transport into and out of the secretory and endosomal-lysosomal compartments in DT40 cells.

Zinc plays crucial roles in diverse biological processes. Recently, in addition to zinc mobilization into and out of the cell, zinc mobilization into and out of intracellular organelles, including the secretory and endosomal-lysosomal compartments, has received growing interest. In vertebrate cells, the Zrt/Irt-like proteins (ZIPs) and Zn transporters (ZnTs) are the two major families of zinc transport proteins involved in zinc mobilization across cellular membranes. Importantly, nearly half of them are localized to subcellular compartments. Thus, to elucidate the numerous zinc-related cellular events, understanding those ZIP and ZnT functions is critical. This chapter describes advanced methods used in our laboratory to examine zinc mobilization by them. Specifically, genetic and molecular approaches using chicken DT40 cells deficient in multiple ZIPs and ZnTs are described. Moreover, procedures to evaluate zinc-related phenotypes caused by the impairment of zinc mobilization into and out of the secretory and endosomal-lysosomal compartments are also described. These methods should be useful in characterizing the roles of zinc in diverse cellular events including endosomal signaling.