Fig. 4. Di-leucine-like and acidic cluster constitute the second basolateral sorting signal. (A) Confocal microscopy images of VSV-AQP4 mutants bearing increasing deletions of DNRSQ, VETE and LIL in the DNRSQVETEDLILKPGVVHVI sequence (Δ272–286, Δ272–290 and Δ272–294, respectively). (B) Simultaneous (ETEDLIL-7×A) but not serial alanine substitution of the acidic cluster (ETED-AAA) and the leucine like motif (LIL-AAA) was necessary to target AQP4 to the apical membranes. Two different focal planes taken at the level of the apical and basolateral membranes of the same cells are shown in (A) and (B). In mutants shown in (A) and (B), the tyrosine-based basolateral sorting motif was removed by deletion (Δ272–281) and GSY-AAA substitution, respectively. Apical versus basolateral repartition was quantified by biotinylation and western blotting (see legend to Figure 2).