Formation, Folding and Trafficking of the ABCG5 ABCG8 Sterol Transporter

Grants and Contracts Details

Description

Sitosterolemia is a genetic disorder caused by mutations in ABCG5 (G5) or ABCG8 (G8) that results in hyperabsorption of dietary sterols and a failure to excrete sterols into bile. In humans and mouse models of this disease, a key feature is diet responsiveness to cholesterol. In addition, mice deficient in G5 and G8 fail to excrete cholesterol into bile. Thus, G5 and G8 playa critical role in reducing the absorption of dietary sterols and the excretion of both dietary and endogenously synthesized cholesterol. G5 and G8 are ATP binding cassette half-transporters that must dimerize in order to traffic beyond the endoplasmic reticulum (ER) and function. The G5/G8 heterodimer is localized on the apical surface of enterocytes (lumenal) and hepatocytes (canalicular) in polarized cells and in mice. The transport of the G5G8 complex to the cell surface can be divided into a three step process: 1) dimer formation, 2) escape from the ER quality control machinery and sorting into COPII vesicles, and 3) trafficking to the apical surface. The molecular determinants for G5/G8 heterodimer formation and trafficking are not known. The specific aims of this proposal are to 1) determine the motif(s) required for dimerization of G5 and G8, 2) determine the residues within G5 and G8 monitored by the ER quality control system and COPII sorting machinery, and 3) determine the motif that confers apical targeting of the G5/G8 sterol transporter. Chimeras between G2 and G8 will be used to broadly define domains within G8 that are necessary for dimerization, trafficking and apical targeting of the G5/G8 sterol transporter. Additional chimeras and site-directed mutagenesis studies will be utilized to further define these domains. Chimeras and mutants will be expressed in CHO cells and analyzed by endoglycosidases, immunoprecipitation, sedimentation gradients, immunofluorescence microscopy, cell fractionation and native gel electrophoresis in order to determine the domains within this complex that mediate the proper folding and trafficking of this sterol transporter to the apical surface.
StatusFinished
Effective start/end date1/1/0512/31/05

Funding

  • American Heart Association: $65,000.00

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