| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* MRC Clinical Sciences Centre, Faculty of Medicine, Hammersmith Hospital Campus, Imperial College, London, UK; and
Gene Targeting Unit, Department of Cellular and Molecular Neurosciences, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Charing Cross Campus, Imperial College, London, UK
1Correspondence: MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Rd., London W12 0NN, UK. E-mail: a.sardini{at}csc.mrc.ac.uk
SPECIFIC AIMS
A FEW STUDIES OF the function and subcellular location of the human ClC-4 (hClC-4) protein, a chloride/proton exchanger, have appeared recently. ClC-4 has been reported to colocalize with CFTR on the plasma membrane, with ClC-5 in endosomes, where it might contribute to their acidification, or with the copper pump ATP7B in the trans-Golgi network (TGN) to facilitate transport of copper by generation of a countercurrent. We studied the tissue distribution of the hClC-4 and its subcellular location. The motif responsible for its localization was investigated by use of chimeric proteins between hClC-4 and the highly homologous vesicular hClC-3 as well as the unrelated plasma membrane protein Ly49E.
PRINCIPAL FINDINGS
1. ClC4 is mainly expressed in excitable tissues where predominantly localizes in the endoplasmic reticulum
By Western blotting analysis of mouse tissue homogenates, we showed that ClC-4 is mainly expressed in excitable tissues such as the skeletal muscle and the nervous system. To identify its subcellular location, membrane fractions of mouse brain homogenates were obtained and analyzed by Western blotting: endogenous ClC-4 was predominantly found in fractions enriched for the ER marker sarco/endoplasmic reticulum calcium ATPase (SERCA) and negative for markers of Golgi membranes, lysosomes, and endosomes. These results suggest that ClC-4 is mainly located in the ER of cells of the nervous system, which was confirmed when HEK293 cells were transfected with hClC-4 conjugated at its C-terminus with green fluorescent protein (GFP). hClC-4-GFP showed an ER location, remarkably different from the vesicular location exhibited by the highly homologous protein hClC-3. This location was confirmed by colocalization with SERCA and with the ER tracker blue-white DPX, a fluorescent probe that binds specifically to ER lipids. A similar subcellular distribution pattern was observed when the C-terminal GFP tag was substituted with the much smaller Flag tag, thereby ruling out that the ER localization of ClC-4 was merely a mislocalization due to the presence of the GFP tag. The location of hClC-4-GFP was also analyzed by transient expression in skeletal muscles, a tissue that supports expression of endogenous ClC-4. Murine anterior tibial skeletal muscles were transfected by plasmid electrotransfer. Analysis by confocal microscopy of cryosections of transfected muscles revealed a sarcoplasmic reticulum (SR) location of hClC-4-GFP (Fig. 1
A) that was confirmed by the absence of colocalization with actine myosin fibers stained with phalloidin (Fig. 1B
) but by colocalization with SERCA as well and ryanodine receptor staining, both markers of SR (Fig. 1C, D
).
2. ClC-4 is located to the ER by an N-terminal motif
To identify the sequence motif responsible for ER location, the amino acid sequence of ClC-4 was analyzed for known ER retention/retrieval motives, concentrating on (but not limited to) those motives absent from the highly homologous CLC proteins, ClC-3 and ClC-5, which are not retained in the ER but are localized in vesicular intracellular compartments. GFP-tagged hClC-4 proteins were generated by exchanging either amino acids of hClC-4 with the corresponding segments of hClC-3 or the unrelated type II plasma membrane protein Ly49E, or by deleting the N- and/or C-terminal tail of hClC-4. The subcellular distribution of these altered hClC-4-GFP proteins following transient transfection into HEK293 cells was analyzed by confocal microscopy. Residence in the ER was evaluated by colocalization with SERCA. Exchange of the C-terminus of hClC-3 for the C terminus of hClC-4 did not change the vesicular location of hClC-3, showing that the putative ER retention signals in the C terminus of hClC-4 were not responsible for its ER localization. Two chimeras were created by exchanging between hClC-4 and hClC-3 the first 71 amino acids at the N-terminus. These two complementary chimeras provide control that excludes the possibility that ER targeting is a consequence of the retention, by the quality control mechanism, of misfolded protein in the ER. If the chimeras are not simply retained in the ER, one chimeric protein should be localized to the vesicular compartment and its mirror image pair should be localized to the ER. This was confirmed since hClC-4 substituted at its N-terminus with the corresponding N-terminal sequence of hClC-3 showed a vesicular location and its mirror pair, namely, hClC-3 substituted at the N-terminus with the equivalent sequence of hClC-4 was localized to the ER. This finding clearly shows that the first N-terminal 71 amino acids of ClC-4 contain a motif responsible for ER localization. By the creation of N-terminal truncations of hClC-4, we ascertained that the motif contained in the N-terminus of hClC-4 is sufficient for ER localization, and we refined the stretch of amino acids to residues 14 to 63 where the motif is contained. To confirm that this stretch of amino acids contains an ER localization motif, this segment was transferred to an unrelated plasma membrane protein, Ly49E, a type II protein consisting of a single transmembrane 
-helix, a short cytoplasmic N-terminus, and an extracellular C-terminus. The chimeric protein so created showed a sensible reduction on plasma membrane localization and was identified as retained in the ER.
CONCLUSIONS AND SIGNIFICANCE
hClC-4 is expressed mainly in excitable tissues such as skeletal muscle and nervous system. Identification of endogenous expression of ClC-4 in ER-enriched membrane derived from mouse brain homogenate suggests that its subcellular location is predominantly the ER/SR, as ascertained by transfection of GFP tagged hClC-4 into human epithelial cells and in murine skeletal muscle, where it colocalizes with the ER marker SERCA. Its location is different from that of the highly homologous hClC-3-GFP, which localizes in a vesicular compartment (Fig. 2
A). Using chimeras between hClC-4 and hClC-3, we have shown that the N-terminus of ClC-4, and not its C-terminus, is responsible for ER localization (Fig. 2B
). hClC-4 truncations refined the stretch of amino acids to residues 14–63 as responsible for ER localization. Analysis of this sequence identified a common motif present in the ER resident protein Sec63, although modification by single point mutations of this motif did not achieve a change in location of hClC-4. In conclusion, we have shown that the amino acid segment, residues 14–63, at the N-terminus of ClC-4 contains a novel motif necessary and sufficient to confer ER location not only to homologous protein as ClC-3, but also to unrelated plasma membrane proteins such as Ly49E. What could be the physiological role of ClC-4 in the ER/SR membranes? Excitable cells such as neurons and skeletal muscle fibers mobilize calcium from the SR/ER to the cytosol during activity. The cellular homeostatic control restores the cytosol calcium concentration by extruding calcium from the cell or by pumping it back into the SR/ER compartments via SERCA activity. By providing an influx of chloride anions into and an efflux of protons from the SR/ER, ClC-4 might help dissipate the electrical gradient generated by the SERCA activity and facilitate calcium reuptake into the SR/ER (Fig. 2C
). The role of ClC-4 could be accompanied by a concomitant H+ and K+ efflux from the SR/ER, since both conductances are known to be present in SR/ER membrane. If ClC-4 plays such a role in the ion homeostasis of the SR/ER, a screening for mutations in ClC-4 could prove useful in identifying the etiology of diseases of excitable tissues such as skeletal muscle diseases and epilepsy.
|
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5588fje
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
