ClC-7 requires Ostm1 as a β-subunit to support bone resorption and lysosomal function

Lange, Philipp F.; Wartosch, Lena; Jentsch, Thomas J.; Fuhrmann, Jens C.

doi:10.1038/nature04535

Cited by 309 publications

(435 citation statements)

References 26 publications

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“…ClC-7 is required for ER export of its ␤-subunit, Ostm1, but ClC-7 is targeted to lysosomes even in the absence of Ostm1 (38). As we found weak binding of APs to the ClC-7 ␤-subunit Ostm1 (Figs.…”

Section: Internalization Of Heterologously Expressed Clc-5 Is Largelysupporting

confidence: 53%

“…A basic amino acid stretch in ClC-6 seems to be involved in the recruitment of ClC-6 into detergent-resistant membranes that may play a role in its subcellular localization (37). For ClC-7 or its ␤-subunit Ostm1 (38), no motifs responsible for their lysosomal localization (38 -40) have been reported so far. ClC-7 is targeted to lysosomes in the absence of Ostm1, whereas Ostm1 requires ClC-7 to be exported from the ER (38).…”

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confidence: 99%

“…For ClC-7 or its ␤-subunit Ostm1 (38), no motifs responsible for their lysosomal localization (38 -40) have been reported so far. ClC-7 is targeted to lysosomes in the absence of Ostm1, whereas Ostm1 requires ClC-7 to be exported from the ER (38). The aim of this study was to systematically identify the cytosolic sorting motifs of all endosomal/lysosomal CLCs and to investigate their role in subcellular sorting.…”

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confidence: 99%

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Sorting Motifs of the Endosomal/Lysosomal CLC Chloride Transporters

Stauber

Jentsch

2010

Journal of Biological Chemistry

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102

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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.

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Section: Internalization Of Heterologously Expressed Clc-5 Is Largelysupporting

confidence: 53%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Sorting Motifs of the Endosomal/Lysosomal CLC Chloride Transporters

Stauber

Jentsch

2010

Journal of Biological Chemistry

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102

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“…The anti-Ac45 antibody was precleared by absorption with bacterial cell lysates and purified by protein-agarose beads (30). Antibodies against the a3 subunit of the V-type H ϩ -ATPase were raised in guinea pigs and were kindly provided by Dr. Jens C. Fuhrmann (Zentrum fur Molekulare Neurobiologie Hamburg, ZMNH, Universitat Hamburg, Falkenried, Hamburg, Germany) (31). Rabbit polyclonal anti-d2 antibodies were raised against GST-d2 peptide antigen produced in our lab and affinity-purified.…”

Section: Methodsmentioning

confidence: 99%

Cytoplasmic Terminus of Vacuolar Type Proton Pump Accessory Subunit Ac45 Is Required for Proper Interaction with V0 Domain Subunits and Efficient Osteoclastic Bone Resorption

Feng

Cheng

Pavlos

et al. 2008

Journal of Biological Chemistry

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Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H؉)-ATPases (V-ATPases)polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V 1 and V 0 . The peripheral cytoplasmically oriented V 1 domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V 0 domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V 0 domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c؆, and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c؆, and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V 0 domain and efficient osteoclastic bone resorption.Osteoclasts are terminally differentiated multinucleated cells derived from the hematopoietic lineage that specialize in the solubilization of mineralized bone material (1). Upon attachment to the bone surface, the osteoclast undergoes a series of cytoskeletal reorganization and polarization events that culminate in the formation of a unique apical membrane known as the ruffled border. The ruffled border is the "resorptive organelle" of the osteoclast and is formed by the polarized targeting and fusion of acidified intracellular vesicles with the plasma membrane (2-4). These transport vesicles courier acidifying machineries as well as osteolytic enzymes such as, TRACP, cathepsin K, and matrix metalloproteinases (2, 5-7) to the ruffled border membrane domain where each cargo plays a discrete role in the resorptive function. Vacuolar H ϩ -adenosine triphosphatases (V-ATPases) 5 that line the ruffled border have long been established to play a vital role in the resorptive process. V-ATPases function to pump H ϩ into the underlying resorptive lacunae. This elevation in protons results in a highly acidified microenvironment that solubilizes the mineralized component of bone while providing optimal conditions for the degradation of the exposed organic phase by collagenolytic enzymes such cathepsin K (2, 7).The importance of V-ATPase in osteoclastic bone resorption is exemplified by studies employing specific inhibitors and gene knock-out strategies to impair V-ATPase functions (8 -11). Furthermore, mutations in the human TCIRG1 g...

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“…17 It resides mainly in the ruffled border of osteoclasts and in the late endosomes and lysosomes, where it resides with its b-subunit OSTM1. 18 Mice deficient for the CLCN7 gene develop osteopetrosis in which the osteoclasts lack the ruffled border and the acidification ability and are unable to resorb bone. 3 Lack of ClC-7 in the brain and kidney induces accumulation of lysosomal storage material.…”

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confidence: 99%

Differentially expressed genes in autosomal dominant osteopetrosis type II osteoclasts reveal known and novel pathways for osteoclast biology

Coudert

Fattore

Baulard

et al. 2014

Laboratory Investigation

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Autosomal dominant osteopetrosis type II (ADO II) is a rare, heritable bone disorder characterized by a high bone mass and insufficient osteoclast activity. Mutations in the CLCN7 gene have been reported to cause ADO II. To gain novel insights into the pathways dysregulated in ADOII osteoclasts, we identified changes in gene expression in osteoclasts from patients with a heterozygous mutation of CLCN7. To do this, we carried out a transcriptomic study comparing gene expression in the osteoclasts of patients with ADO II and healthy donors. Our data show that, according to our selection criteria, 182 genes were differentially expressed in osteoclasts from patients and controls. From the 18 displaying the highest change in microarray, we confirmed differential expression for seven by qPCR. Although two of them have previously been found to be expressed in osteoclasts (ITGB5 and SERPINE2), the other five (CES1 (carboxyl esterase 1), UCHL1 (ubiquitin carboxy-terminal esterase L1, also known as ubiquitin thiolesterase), WARS (tryptophanyl-tRNA synthetase), GBP4 (guanylate-binding protein 4), and PRF1) are not yet known to have a role in this cell type. At the protein level, we confirmed elevated expression of ITGB5 and reduced expression of WARS, PRF1, and SERPINE2. Transfection of ClC-7 harboring the G215R mutation into osteoclasts resulted in an increased ITGB5 and reduced PRF1 expression of borderline significance. Finally, we observed that the ADO II patients presented a normal or increased serum level of bone formation markers, demonstrating a coupling between dysfunctional osteoclasts and osteoblasts. Sphingosine kinase 1 mRNA was expressed at the same level in ADO II and control osteoclasts. In conclusion, these data suggest that in addition to an acidification dysfunction caused by the CLCN7 mutation, a change in ITGB5, PRF1, WARS, and SERPINE2 expression could be part of the osteoclastic phenotype of ADO II.

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ClC-7 requires Ostm1 as a β-subunit to support bone resorption and lysosomal function

Cited by 309 publications

References 26 publications

Sorting Motifs of the Endosomal/Lysosomal CLC Chloride Transporters

Sorting Motifs of the Endosomal/Lysosomal CLC Chloride Transporters

Cytoplasmic Terminus of Vacuolar Type Proton Pump Accessory Subunit Ac45 Is Required for Proper Interaction with V0 Domain Subunits and Efficient Osteoclastic Bone Resorption

Differentially expressed genes in autosomal dominant osteopetrosis type II osteoclasts reveal known and novel pathways for osteoclast biology

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