ABCR is a member of the ABCA subclass of ATP binding cassette transporters that is responsible for Stargardt macular disease and implicated in retinal transport across photoreceptor disc membranes. It consists of a single polypeptide chain arranged in two tandem halves, each having a multi-spanning membrane domain followed by a nucleotide binding domain. To delineate between several proposed membrane topological models, we have identified the exocytoplasmic (extracellular/lumen) N-linked glycosylation sites on ABCR. Using trypsin digestion, site-directed mutagenesis, concanavalin A binding, and endoglycosidase digestion, we show that ABCR contains eight glycosylation sites. Four sites reside in a 600-amino acid exocytoplasmic domain of the N-terminal half between the first transmembrane segment H1 and the first multi-spanning membrane domain, and four sites are in a 275-amino acid domain of the C half between transmembrane segment H7 and the second multi-spanning membrane domain. This leads to a model in which each half has a transmembrane segment followed by a large exocytoplasmic domain, a multi-spanning membrane domain, and a nucleotide binding domain. Other ABCA transporters, including ABC1 linked to Tangier disease, are proposed to have a similar membrane topology based on sequence similarity to ABCR. Studies also suggest that the N and C halves of ABCR are linked through disulfide bonds.ABCR, formerly known as the rim protein, is an ATP binding cassette (ABC) 1 transporter found in vertebrate retinal photoreceptor cells (1-3). It is localized along the rim region of photoreceptor rod outer segment disc membranes (1, 4, 5) and more recently has been found in human foveal and peripheral cone outer segments (6). Several studies have implicated ABCR in the retinoid cycle, possibly functioning as a retinal extruder or retinal-phosphatidylethanolamine flippase to facilitate the removal of all-trans-retinal from disc membranes following the photobleaching of rhodopsin (7-9).Mutations in the ABCA4 gene encoding ABCR have been linked to Stargardt disease, a relatively common juvenile macular dystrophy characterized by a decrease in visual acuity, progressive bilateral atrophy of the central retina, and the accumulation of yellow deposits within the retinal pigment epithelial cell layer (2, 10 -13). Mutations in ABCA4 have also been linked to related disease variants, including late-onset fundus flavimaculatus (14), cone-rod dystrophy (15), retinitis pigmentosa-like dystrophy (16, 17), and age-related macular degeneration (18), although the latter remains controversial (19).ABCR is a member of the ABCA subclass of ABC transporters (20). Like most other mammalian ABC transporters, members of this subclass consist of a single long polypeptide chain organized into two tandemly arranged halves. Each half contains a membrane-spanning domain (MSD) followed by a cytoplasmic nucleotide binding domain (NBD). The ABCA subclass is distinguished from other ABC transporter subclasses by the presence of a large domain between the...

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. It couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. One FMN and up to nine iron-sulfur (FeS) clusters participate in the redox reaction. So far, complex I has been described mainly by means of EPR- and UV-vis spectroscopy. Here, we report for the first time an infrared spectroscopic characterization of complex I. Electrochemically induced FT-IR difference spectra of complex I from Escherichia coli and of the NADH dehydrogenase fragment of this complex were obtained for critical potential steps. The spectral contributions of the FMN in both preparations were derived from a comparison using model compounds and turned out to be unexpectedly small. Furthermore, the FT-IR difference spectra reveal that the redox transitions of the FMN and of the FeS clusters induce strong reorganizations of the polypeptide backbone. Additional signals in the spectra of complex I reflect contributions induced by the redox transition of the high-potential FeS cluster N2 which is not present in the NADH dehydrogenase fragment. Part of these signals are attributed to the reorganization of protonated/deprotonated Asp or Glu side chains. On the basis of these data we discuss the role of N2 for proton translocation of complex I.

The proton-pumping NADH:ubiquinone oxidoreductase of Escherichia coli is composed of 14 different subunits and contains one FMN and up to nine iron-sulfur clusters as prosthetic groups. By use of salt treatment, the complex can be split into an NADH dehydrogenase fragment, a connecting fragment and a membrane fragment. The water-soluble NADH dehydrogenase fragment has a molecular mass of approximately 170,000 Da and consists of the subunits NuoE, F, and G. The fragment harbors the FMN and probably six iron-sulfur clusters, four of them being observable by EPR spectroscopy. Here, we report that the fully assembled fragment can be overproduced in E. coli when the genes nuoE, F, and G were simultaneously overexpressed with the genes nuoB, C, and D. Furthermore, riboflavin, sodium sulfide, and ferric ammonium citrate have to be added to the culture medium. The fragment was purified from the cytoplasm by means of ammonium sulfate fractionation and chromatographic steps. The preparation contains one noncovalently bound FMN per molecule. Two binuclear (N1b and N1c) and two tetranuclear (N3 and N4) iron-sulfur clusters were detected by EPR in the NADH reduced preparation with spectral characteristics identical with those of the corresponding clusters in complex I. The preparation fulfills all prerequisites for crystallization of the fragment.

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Complex I of Escherichia coli can be split into three fragments. One of these fragments, the soluble NADH dehydrogenase fragment, represents the electron input part of complex I. It comprises the subunits NuoE, F and G and harbors one flavin mononucleotide and up to six iron-sulfur clusters. Here, we report the one-step purification of this fragment by means of affinity chromatography on StrepTactin. This was achieved by fusing the Strep-tag II peptide to the Cterminus of NuoF or NuoG. Fusion of this peptide to the Nterminus of either NuoE or NuoF disturbed the assembly of the NADH dehydrogenase fragment.z 1999 Federation of European Biochemical Societies.