Sorting motifs of the endosomal/lysosomal CLC chloride transporters

The CLC protein family contains plasma membrane chloride channels and the intracellular chloride-proton exchangers ClC-3-7. The latter proteins mainly reside on the various compartments of the endosomal-lysosomal system where they are involved in the luminal acidification or chloride accumulation. Although their partially overlapping subcellular distribution has been studied extensively, little is known about their targeting mechanism. In a comprehensive study we now performed pulldown experiments to systematically map the differential binding of adaptor proteins of the endosomal sorting machinery (adaptor proteins and GGAs (Golgi-localized, γ-ear containing, Arf binding)) as well as clathrin to the cytosolic regions of the intracellular CLCs. The resulting interaction pattern fitted well to the known subcellular localizations of the CLCs. By mutating potential sorting motifs, we could locate almost all binding sites, including one already known for ClC-3 and several new motifs for ClC-5, -6, and -7. The impact of the identified binding sites on the subcellular localization of CLC transporters was determined by heterologous expression of mutants. Surprisingly, some vesicular CLCs retained their localization after disruption of interaction sites. However, ClC-7 could be partially shifted from lysosomes to the plasma membrane by combined mutation of N-terminal sorting motifs. The localization of its β-subunit, Ostm1, was determined by that of ClC-7. Ostm1 was not capable of redirecting ClC-7 to lysosomes.