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Aspartyl-aminopeptidase regulates albumin endocytosis by interaction with ClC-5

A. Lee,1 D. Hryciw,1 S. Wilk,2 E. Wilk,2 V. Valova,3 P. Robinson3 and P. Poronnik,1 1School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia, 2Department of Pharmacology, Mount Sinai School of Medicine, New York 10029, USA and 3Cell Signalling Unit, Children's Medical Research Institute, Wentworthville, NSW 2145, Australia.

ClC-5 is a voltage gated chloride channel that is essential for the constitutive reuptake of urinary albumin in the renal proximal tubule. Studies by our group and others have demonstrated that ClC-5 has a key role in mediating the assembly of the albumin endocytic complex that contains ClC-5, the albumin receptor megalin and several other interacting proteins. In order to identify novel proteins that interact with the endocytic complex, we adopted a proteomics approach to isolate proteins that associated with the C-terminus of ClC-5. Rat kidney lysates were incubated with a glutathione S-transferase (GST) fusion protein expressing the C-terminus of ClC-5 and pull-down assays performed to isolate interacting proteins. Bound proteins were resolved on SDS-PAGE and candidate bands were excised and prepared for mass-spectrometry. One protein that was very prominent in the samples was identified as aspartyl-aminopeptidase (EC, a recently characterised cytosolic protein that catalyses the release of N-terminal aspartate/glutamate residues from target peptides. The binding of aspartyl-aminopeptidase to ClC-5 was confirmed both in vitro by GST pull-down and in vivo by co-immunoprecipitation. Opossum kidney (OK) cells are the standard model for renal albumin endocytosis. Confocal immunofluorescence revealed that exposure of OK cells to albumin resulted in a recruitment of aspartyl-aminopeptidase to the apical domain of the cells where it co-localized with ClC-5. Over-expression of aspartyl-aminopeptidase in OK cells increased albumin uptake (∼20% above control), which was accompanied by increased cell-surface levels of ClC-5. In contrast, over-expression of a catalytically-inactive form of aspartyl-aminopeptidase prevented both stimulation of albumin uptake and increase in cell-surface levels of ClC-5. These data show that aspartyl-aminopeptidase is a novel binding partner of ClC-5 and plays a key role in albumin uptake.