Fig. 5.

Ubiquitination of AQP2 increases its lysosomal degradation. (A) Cryosections of fixed MDCK cells expressing AQP2-K270R (1, 2) or AQP2-Ub (3) were immunolabeled for AQP2 (10-nm gold particles). Endosomes are divided into three types: I = early endosome, II = late endosome, and III = late endosome/lysosome (1). Overview of the apical part of an MDCK cell showing all three types of endosomes. The arrows point to labeled dense vesicles that mediate either endocytosis or recycling (2). Detail of an early endosome with AQP2-K270R label at the limiting membrane (3). Detail of late endosomes/lysosomes with the majority of label associated with internal membranes. (Scale bars, 200 nm.) (B) Protein synthesis in MDCK-AQP2 or MDCK-AQP2-K270R was inhibited (cycloheximide), and TPA was added in the absence or presence of chloroquine for the indicated times. Subsequently, cells were lysed and immunoblotted for AQP2. (C) MDCK cells expressing wt-AQP2 or AQP2-Ub were treated with chloroquine for the indicated times. Subsequently, cells were lysed and immunoblotted for AQP2.