N-glycosylation of the Xenopus laevis ClC-5 protein plays a role in cell surface expression, affecting transport activity at the plasma membrane.
Mutations in the gene encoding ClC-5 lead to X-linked hypercalciuric nephrolithiasis (XLHN), characterized by proteinuria, hypercalciuria, and phosphaturia. In renal proximal tubule cells, ClC-5 was identified as an important player in endocytosis, which ensures reabsorption of filtered protein. However, the recent finding that ClC-5 is a Cl(-)/H(+) antiporter and not a Cl(-) channel as long thought points to the lack of understanding of its functional role. Also, little biochemical data are available about ClC-5 and its post-translational modifications have not been investigated. Here, we examined the role of N-glycosylation of xClC-5 in the Xenopus oocyte expression system by comparing wild-type (WT) xClC-5 and N-glycosylation site mutants. We found that xClC-5 is N-glycosylated on asparagines 169 and 470, which are the only N-glycosylated sites. xClC-5 mutants have an increased susceptibility to polyubiquitination and proteasomal degradation; however, without a notable impact on the expression level. Using a cross-linking reagent, we showed that xClC-5 assembles into protein complexes, independent of its N-glycosylation. Voltage-clamp measurements showed a reduced conductance in the presence of tunicamycin and with xClC-5 N-glycosylation site mutants. Using immunocytochemistry, we localized xClC-5 mainly in intracellular compartments, and found that its cell surface pool is reduced in the absence of N-glycans. We further examined the plasma membrane retrieval of WT and mutant xClC-5 in the presence of Brefeldin A (BFA), and found that the non-glycosylated mutant was retrieved more than five times faster than the WT protein. We conclude that N-glycosylation enhances cell surface expression of xClC-5, increasing its plasma membrane transport activity.
Schmieder
Sandra
S
Laboratoire de Physiologie Cellulaire et Moléculaire Des Systèmes Intégrés, Université de Nice-Sophia Antipolis/CNRS, UMR 6548, Nice, France.
Bogliolo
Stéphanie
S
Ehrenfeld
Jordi
J
D000818:Animals; D001216:Asparagine / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018118:Chloride Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D005260:Female; D006031:Glycosylation / Q000187:drug effects; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D046911:Macromolecular Substances / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011134:Polysaccharides / Q000378:metabolism / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D013997:Time Factors; D029867:Xenopus Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
C097503:CLC-5 chloride channel; D018118:Chloride Channels; D002712:Chlorides; D046911:Macromolecular Substances; D011134:Polysaccharides; D011500:Protein Synthesis Inhibitors; D029867:Xenopus Proteins; D001216:Asparagine; D046988:Proteasome Endopeptidase Complex