Elucidating the mechanism of intracellular trafficking of Organic Anion Transport Proteins (OATP)

Abstract

Previous studies identified a family of organic anion transporting protein (OATPs) involved in the clearance of endogenous compounds and xenobiotics from the circulation. Several OATP family members have a C-terminal PDZ binding consensus sequence and the sequence in rat oatp l a l binds the PDZ domain of PDZK 1. Experiments exploring the interaction of oatp l al with PDZK1 in vivo were examined using mouse models with the Oatplal homologue that contains the same PDZ binding sequence KTKL. In PDZK1 knockout mice and transfected cells lacking PDZK1 expression, Oatpla1 does not reach the plasma membrane and instead accumulates in intracellular vesicles. A corresponding decrease in the clearance of sulfobromophthalein (BSP) a compound that is transported by Oatplal was seen for PDZK1 knockout mice. Taken together, PDZK1 is required for cell surface localization of Oatplal where it actively transports organic anions.;We want to test the hypothesis that trafficking of Oatplal-associated vesicles along intact microtubules to and from the cell surface is regulated by PDZK1 recruitment of specific motor proteins. Immunofluoresent stained vesicles prepared from wild-type mouse liver had 50% of Oatplal-associated vesicles colocalizing with PDZK1. Kinesin-1, a plus -end directed motor, was largely associated with Oatplal-containing vesicles from wild-type mice, whereas Oatplalcontaining vesicles from PDZK1 knockout mice associates mostly with the minus-end directed motor, dynein. Quantification of motility on directionally marked microtubules following addition of 50 microM ATP showed that wild type vesicles moved equally towards the plus and minus ends while PDZK1 knockout vesicles moved predominantly towards the minus end, consistent with net movement towards the cell interior. These studies reveal a novel mechanism by which PDZK1 regulates intracellular trafficking of oatplal by recruiting specific motors to oatp 1 al -containing vesicles. In the absence of PDZK 1, oatp la 1-containing vesicles can't recruit kinesin-1 and associate with dynein as a predominant minus-end directed motor. Whether this is a result of direct interaction of the oatp 1 al cytoplasmic domain with dynein or with a dyneincontaining protein complex remains to be established.