The ABC (ATP-Binding-Cassette)-type protein BSEP (ABCB11) is essential for bile formation. BSEP resides primarily in the canalicular membrane of hepatocytes and mediates ATP-dependent transport of conjugated bile acids from cytoplasm to bile. Correct targeting and recycling to the apical membrane are fundamental to BSEP function and regulation. Mutations that cause BSEP retention in the endoplasmic reticulum produce Progressive Familial Intrahepatic Cholestasis type II. Additionally, BSEP removal from the canalicular membrane is associated with drug and sepsis induced intrahepatic cholestasis. BSEP mobilization and targeting to the apical membrane are mediated by association with factors that connect the transporter to sorting and trafficking networks. We previously identified proteins that interact with BSEP in the apical domain and regulate its plasma membrane delivery and endocytosis. However, there is no information regarding proteins that bind BSEP and regulate its progress through the early secretory pathway or exit from the Trans Golgi Network, which represents the central sorting hub of the vesicular transport network. We have identified two proteins, a clathrin adaptor and a putative endoplasmic reticulum sorting receptor, which interact with BSEP and influence its apical membrane expression. The goal of the proposed research is to investigate the role these proteins play in regulating BSEP trafficking. Experiments in Aim 1 will clarify which aspect of the BSEP trafficking itinerary in polarized cells and hepatocytes is controlled by interaction with the clathrin adaptor.
Aim 2 focuses on determining the extent to which accessory proteins, which form a macromolecular complex with BSEP and the clathrin adaptor, participate in BSEP delivery to the plasma membrane.
Specific Aim 3 will investigate the relevance of an endoplasmic reticulum resident protein, which directly interacts with BSEP, to transporter maturation and vesicular trafficking to the Golgi. We will also ascertain whether overexpression of the interacting protein improves trafficking of BSEP PFIC II mutants. Inherited or acquired defects that compromise BSEP expression in the canalicular membrane are likely causes of intrahepatic cholestasis. Therefore, elucidating pathways that govern transfer and recruitment of ABC-transporters to the apical membrane, and identification of proteins which control these processes, will provide critical insight into mechanisms underlying cholestasis and suggest targets for drug design. ? ? ?
| Chan, Wayne; Calderon, German; Swift, Amy L et al. (2005) Myosin II regulatory light chain is required for trafficking of bile salt export protein to the apical membrane in Madin-Darby canine kidney cells. J Biol Chem 280:23741-7 |
| Ortiz, Daniel F; Moseley, James; Calderon, German et al. (2004) Identification of HAX-1 as a protein that binds bile salt export protein and regulates its abundance in the apical membrane of Madin-Darby canine kidney cells. J Biol Chem 279:32761-70 |
