Basolateral sorting of the Mg2+ transporter CNNM4 requires interaction with AP-1A and AP-1B

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 μ1B, a cargo-recognition subunit of AP-1B, did not affect basolateral localization, but simultaneous knockdown of the μ1A 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 μ1A and μ1B. These results imply that CNNM4 is sorted to the basolateral membrane by the complementary function of AP-1A and AP-1B.