The fur gene of Escherichia coli is involved in all iron-regulated transcriptions hitherto studied. The nucleotide sequence of an 868 basepair fragment containing the fur gene was determined. There was only a single longer reading frame. The amino acid sequence derived from the nucleotide sequence comprised 148 amino acids that together made a polypeptide of 16,795 daltons. The amino acid sequence was confirmed by determination of the amino acid composition, the carboxy-terminal lysine residue and the internal Lys-Lys and Lys-Arg sequences of the isolated Fur protein. The nucleotide sequence contains typical initiation and termination sites for transcription and translation.

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.

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