Ancient conserved domain protein/cyclin M (CNNM) 4 is an evolutionarily conserved Mg2+ transporter that localizes at the basolateral membrane of the intestinal epithelia. Here, we show the complementary importance of clathrin adaptor protein (AP) complexes AP-1A and AP-1B in basolateral sorting of CNNM4. We first confirmed the basolateral localization of both endogenous and ectopically expressed CNNM4 in Madin-Darby Canine Kidney cells, which form highly polarized epithelia in culture. Single knockdown of mu 1B, a cargo-recognition subunit of AP-1B, did not affect basolateral localization, but simultaneous knockdown of the mu 1A subunit of AP-1A abrogated localization. Mutational analyses showed the importance of three conserved dileucine motifs in CNNM4 for both basolateral sorting and interaction with mu 1A and mu 1B. These results imply that CNNM4 is sorted to the basolateral membrane by the complementary function of AP-1A and AP-1B. (C) 2014 Elsevier Inc. All rights reserved.