Figure 6.

Basolateral membrane sorting of Kir 2.3 occurs by a mechanism that is independent of the μIb clathrin adaptin subunit. (A) Confocal image of Kir 2.3 stably transfected in LLC-PK1 cells, demonstrating basolateral expression in a cell line that is deficient in the μIb clathrin adaptor subunit. (Upper) X–Y plane is shown. (Lower) Z-plane reconstruction is shown. (B) Yeast two-hybrid interaction study. Yeast were cotransfected with vectors expressing (i) a recombinant LexA protein fused to the Kir 2.3 sorting signal (final 27 aa), a triple repeat of a tyrosine-based sorting signal (YRRL), or the Kir 2.3 sorting signal without the PDZ ligand (442x); (ii) a recombinant AD protein fused to the μIb subunit; and (iii) a LacZ reporter gene under Lex A operator control. Yeast were plated on 5-bromo-4-chloro-3-indolyl β-d-galactoside plates containing galactose (+) or glucose (−) to induce or repress the expression of the AD protein. Yeast that turned blue were scored positively.