Sorting Motifs of the Endosomal/Lysosomal CLC Chloride Transporters

Abstract

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, ␥-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 ␤-subunit, Ostm1, was determined by that of ClC-7. Ostm1 was not capable of redirecting ClC-7 to lysosomes.

Common Gating of Both CLC Transporter Subunits Underlies Voltage-dependent Activation of the 2Cl−/1H+ Exchanger ClC-7/Ostm1

“…This mechanism suggests that the block of ion transport is confined to subunits carrying the td mutation. Although not indicated in the following text, all ClC-7 subunits additionally carried the L23A,L24A and L68A,L69A mutations in N-terminal AP-and GGA-binding sites, respectively, which lead to partial plasma membrane localization of ClC-7 (14,35). Furthermore, all constructs were co-expressed with the essential ␤-subunit Ostm1 (12,14).…”

Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking

“…Multiple partial carboxyl-terminal truncations and deletions constitutively locked hClC-2 in a closed conformation, without any effect on surface membrane insertion (50). For CLC anion/ proton antiporters that normally reside in intracellular cell organelles crucial roles for correct trafficking and sorting have been assigned to the carboxyl terminus (52)(53)(54)(55). The chloride/ proton antiporter hClC-7 is the only other member of the CLC family with an obligatory subunit, Ostm1, which is needed for proper function and subcellular localization (56).…”

Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions

“…For ClC-3a removal of a dileucine motif sequence (LLDLLDE (S1) Fig. 4A) allows surface membrane insertion and functional analysis of the protein (8,16,34). ClC-3b contains the same sequence motif, however, its removal did not result in surface membrane insertion (data not shown).…”