Role of CIC-5 in Renal Endocytosis Is Unique among CIC Exchangers and Does Not Require PY-motif-dependent Ubiquitylation

Abstract

Inactivation of the mainly endosomal 2Cl؊ /H ؉ -exchanger ClC-5 severely impairs endocytosis in renal proximal tubules and underlies the human kidney stone disorder Dent's disease. In heterologous expression systems, interaction of the E3 ubiquitin ligases WWP2 and Nedd4-2 with a "PY-motif" in the cytoplasmic C terminus of ClC-5 stimulates its internalization from the plasma membrane and may influence receptor-mediated endocytosis. We asked whether this interaction is relevant in vivo and generated mice in which the PY-motif was destroyed by a point mutation. Unlike ClC-5 knock-out mice, these knock-in mice displayed neither low molecular weight proteinuria nor hyperphosphaturia, and both receptor-mediated and fluidphase endocytosis were normal. The abundances and localizations of the endocytic receptor megalin and of the Na ؉ -coupled phosphate transporter NaPi-2a (Npt2) were not changed, either. To explore whether the discrepancy in results from heterologous expression studies might be due to heteromerization of ClC-5 with ClC-3 or ClC-4 in vivo, we studied knock-in mice additionally deleted for those related transporters. Disruption of neither ClC-3 nor ClC-4 led to proteinuria or impaired proximal tubular endocytosis by itself, nor in combination with the PY-mutant of ClC-5. Endocytosis of cells lacking ClC-5 was not impaired further when ClC-3 or ClC-4 was additionally deleted. We conclude that ClC-5 is unique among CLC proteins in being crucial for proximal tubular endocytosis and that PY-motif-dependent ubiquitylation of ClC-5 is dispensable for this role.

mTOR Regulates Endocytosis and Nutrient Transport in Proximal Tubular Cells

“…16 Morphologic analysis revealed a strong reduction of fluid-phase endocytosis in Rap DTubule and RapRic DTubule animals, whereas no change was observed in Ric DTubule mice (Figure 2A). Similarly, confocal microscopy analysis showed a reduced receptor-mediated endocytosis in Rap…”

Short-Term Functional Adaptation of Aquaporin-1 Surface Expression in the Proximal Tubule, a Component of Glomerulotubular Balance

“…44 Immunoprecipitation AQP1-OKCs were resuspended in coimmunoprecipitation buffer(25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40) supplemented with protease inhibitor cocktail (Roche Diagnostics), 1 mg/ml ubiquitin aldehyde, and 5 mM N-ethylmaleimide to limit protein degradation and deubiquitination as well as phosphatase inhibitors (100 mM NaF, 10 mM diNa-pyrophosphate, and 1 mM Na-orthovanadate). Insoluble material was removed, and 500 mg protein per sample was incubated with 4 mg rabbit IgG (The Jackson Laboratory) or polyclonal AQP1 at 4°C added with protein G Dynabeads (Invitrogen) for 4 hours.…”

Sorting Motifs of the Endosomal/Lysosomal CLC Chloride Transporters

“…For ClC-4, which has mostly been reported to localize to endosomes (4,27) but also to the endoplasmic reticulum (ER) (28), an N-terminal amino acid stretch is reportedly involved in ER retention when expressed heterologously (28). The predominantly endosomal ClC-5 (4, 27, 29 -32), a small portion of which is endogenously found also in the plasma membrane (30,33), bears a C-terminal-located PY motif (34). Although not required for in vivo ClC-5 function (33), studies in Xenopus oocytes and cultured opossum kidney cells revealed that ubiquitin ligases bind this motif and ubiquitylate ClC-5, stimulating its internalization from the cell surface (34,35).…”

“…The predominantly endosomal ClC-5 (4, 27, 29 -32), a small portion of which is endogenously found also in the plasma membrane (30,33), bears a C-terminal-located PY motif (34). Although not required for in vivo ClC-5 function (33), studies in Xenopus oocytes and cultured opossum kidney cells revealed that ubiquitin ligases bind this motif and ubiquitylate ClC-5, stimulating its internalization from the cell surface (34,35). Although endogenous ClC-6 localizes to late endosomes (36), heterologously expressed ClC-6 has been found on early and recycling endosomes (37).…”