Date of Award

12-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Interdisciplinary Program

Research Advisor

John D. Schuetz, Ph.D.

Committee

Linda Hendershot, Ph.D. James I. Morgan, Ph.D. Anjaparavanda P. Naren, Ph.D. Jie Zheng, Ph.D.

Abstract

The ATP-binding cassette (ABC) transporters play an important role as a barrier to protect cells from the accumulation of toxic xenobiotics and metabolites due to their ability to translocate a wide array of compounds across lipid bilayers. However, many ABC transporters, especially the ones localized in the intracellular organelles, are involved in critical biological processes such as antigen presentation. The core unit of ABC transporters contains two functional domains: the membrane spanning domain (MSD) and the nucleotide binding domain. The full transporters contain two of these units in tandem in a single polypeptide, whereas the half transporters only contain one and must homo- or hetero-dimerize in order to exert their functions. A half transporter ABCB6 has been shown to localize in mitochondria and suggested to play a role in iron homeostasis; however, its function remained elusive. Therefore, we aimed to characterize several aspects of ABCB6: identification of substrates, physiological role, transport mechanism and intracellular trafficking. In this study, we show that ABCB6 is a homodimeric mitochondrial outer membrane protein. Furthermore, we identified ABCB6 as a porphyrin transporter that facilitates heme biosynthesis, in which the intermediates must be shuttled between mitochondria and the cytosol. Translocation of substrates by ABC transporters occurs in an ATP-dependent manner, although the role of ATP binding and hydrolysis in the transport process remains controversial. Taking advantage of its ability to bind hemin conjugated to agarose beads, we demonstrate that the ATP binding at the NBD is sufficient to induce a conformational change to a low affinity state in ABCB6. In an attempt to understand how ABCB6 trafficks intracellularly, we identified a post-translational modification that indicates ER to Golgi trafficking during its maturation. Moreover, we identified a novel N-terminal disulfide bond that plays an important role in the ER exit of ABCB6. This disulfide bond motif is found in other ABC family members and the loss of the conserved cysteine residue in ABCC8/SUR1 is the genetic basis for hyperinsulinemic hypoglycemia. Because ER redox status appears to play an important role in the trafficking of these proteins, expression patterns of ABC transporters may be altered in pathophysiological conditions such as diabetes where microsomal redox status is shifted.

DOI

10.21007/etd.cghs.2008.0100

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