The supernatant of a 10-min centrifugation at 10,000 × g was supplemented with 1% final Triton X-100. For tissue lysate preparation, two brains or four kidneys of WT C57Bl/6 mice were homogenized in 10 ml of HEPES-buffered saline with 0.5 mg/ml AEBSF, protease inhibitor mixture (Complete®), and 1 m m Na 3VO 4. Pulldown assays from mouse brain or kidney lysate were performed equivalently, with PBS replaced by HEPES-buffered saline. After sedimenting the Sepharose beads, the eluate was separated by SDS-PAGE and probed by immunoblot. After 4 washes with PBS, supplemented with 0.1% (w/v) Triton X-100, 0.5 mg/ml AEBSF, and protease inhibitor mixture (Complete®), bound protein was eluted by incubation in SDS sample buffer at 55 ☌ for 15 min. The N terminus of ClC-7 sufficed to target ClC-0 to lysosomes, and combined disruption of the first two motifs in 7 LL23/24AA,L元6/37AA-0–0 resulted in cell surface localization.įor a single pulldown experiment, 1.0–1.5 ml of lysate of a 10-cm dish of confluent HeLa cells in PBS supplemented with 1% (w/v) Triton X-100, 0.5 mg/ml AEBSF (Pefabloc SC), protease inhibitor mixture (Complete®), and 1 m m Na 3VO 4 was centrifuged at 10,000 × g for 10 min, and the supernatant was incubated with roughly 50 μg of GST fusion protein coupled to Sepharose for 2 h under constant agitation at 4 ☌. B, sorting determinants in the ClC-7 N terminus investigated in HeLa cells transfected with a chimeric protein (7-0-0) in which the N terminus of the plasma membrane channel ClC-0 was replaced by that of rClC-7 ( top panel) or by ClC-7 N termini carrying two combinations of mutations in the EGAPLL 24, EETPLL 37, and DDELL 67 dileucine motifs (EETPLL 37 present in rat, but not humans). The 0-7-0 chimera shows weak plasma membrane expression in addition to strong ER-like staining. ClC-7 colocalizes with LAMP-1, whereas ClC-0 shows plasma membrane expression. A, subcellular localization of rClC-7 ( top), a chimera of rClC-7 with N- and C-terminal domains replaced by those of ClC-0 (0-7-0, below), and WT ClC-0 ( bottom) after transient transfection of HeLa cells, in comparison to LAMP-1 as marker for late endosomes and lysosomes. Ostm1 was not capable of redirecting ClC-7 to lysosomes.įIGURE 5 Lysosomal sorting determined by the ClC-7 N terminus. The localization of its β-subunit, Ostm1, was determined by that of ClC-7. However, ClC-7 could be partially shifted from lysosomes to the plasma membrane by combined mutation of N-terminal sorting motifs. Surprisingly, some vesicular CLCs retained their localization after disruption of interaction sites. The impact of the identified binding sites on the subcellular localization of CLC transporters was determined by heterologous expression of mutants. 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 resulting interaction pattern fitted well to the known subcellular localizations of the CLCs. 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. Although their partially overlapping subcellular distribution has been studied extensively, little is known about their targeting mechanism. 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. The CLC protein family contains plasma membrane chloride channels and the intracellular chloride-proton exchangers ClC-3–7. Glycobiology and Extracellular Matrices.
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