Abstract
Mutations in the human kidney anion exchanger 1 (kAE1) membrane glycoprotein cause impaired urine acidification resulting in distal renal tubular acidosis (dRTA). Dominant and recessive dRTA kAE1 mutants exhibit distinct trafficking defects with retention in the endoplasmic reticulum (ER), Golgi, or mislocalization to the apical membrane in polarized epithelial cells. We examined the interaction of kAE1 with the quality control system responsible for the folding of membrane glycoproteins and the retention and degradation of misfolded mutants. Using small molecule inhibitors to disrupt chaperone interactions, two functional, dominant kAE1 mutants (R589H and R901stop), retained in the ER and targeted to the proteasome for degradation by ubiquitination, were rescued to the basolateral membrane of Madin-Darby canine kidney cells. In contrast, the Golgi-localized, recessive G701D and the severely misfolded, ER-retained dominant Southeast Asian ovalocytosis (SAO) mutants were not rescued. These results show that functional dRTA mutants are retained in the ER due to their interaction with molecular chaperones, particularly calnexin, and that disruption of these interactions can promote their escape from the ER and cell surface rescue.
Highlights
Expression of kidney anion exchanger 1 (kAE1) in Madin-Darby canine kidney (MDCK) Cells—To examine the role chaperones play in the intracellular retention of kAE1 mutants, WT kAE1, kSAO, and three different kAE1 distal renal tubular acidosis (dRTA) mutants (R589H, G701D, and R901stop) were expressed in MDCK cells
Three dominant mutations in kAE1 resulted in retention in the endoplasmic reticulum (ER) in nonpolarized MDCK cells, whereas the recessive G701D is localized to the Golgi [10, 14, 15, 27]
Previous immunofluorescence studies of the kAE1 mutants kSAO, R589H, and R901stop co-localize these mutants to the ER, suggesting that the ER chaperone calnexin may play a role in folding of kAE1, a membrane glycoprotein [14, 15]
Summary
A heightened interaction of the high mannose-contain- Surface of MDCK Cells—Confocal microscopy was used to ing mutant glycoproteins kSAO, R589H, G701D, and R901stop examine the effect of calnexin binding inhibition by CST on the with calnexin was seen by co-immunoprecipitation indicative localization of kAE1 and dRTA mutants (Fig. 3). Biotinylation experiments were performed on the dominant dRTA mutants expressed in MDCK cells in the presence of CST; no R589H or R901stop kAE1 was detectable in the bound biotinylated fraction (Fig. 5B, R901stop, data not shown).
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