Expression of the Ciona intestinalis alternative oxidase (AOX) in Drosophila complements defects in mitochondrial oxidative phosphorylation.

Defects in mitochondrial OXPHOS are associated with diverse and mostly intractable human disorders. The single-subunit alternative oxidase (AOX) found in many eukaryotes, but not in arthropods or vertebrates, offers a potential bypass of the OXPHOS cytochrome chain under conditions of pathological OXPHOS inhibition. We have engineered Ciona intestinalis AOX for conditional expression in Drosophila melanogaster. Ubiquitous AOX expression produced no detrimental phenotype in wild-type flies. However, mitochondrial suspensions from AOX-expressing flies exhibited a significant cyanide-resistant substrate oxidation, and the flies were partially resistant to both cyanide and antimycin. AOX expression was able to complement the semilethality of partial knockdown of both cyclope (COXVIc) and the complex IV assembly factor Surf1. It also rescued the locomotor defect and excess mitochondrial ROS production of flies mutated in dj-1beta, a Drosophila homolog of the human Parkinson's disease gene DJ1. AOX appears to offer promise as a wide-spectrum therapeutic tool in OXPHOS disorders.

Native proteomic analysis of protein complexes in murine intestinal brush border membranes.

Intestinal epithelial cell protrusions referred as microvilli or brush border membranes (BBMs) are specialized in the digestion, uptake, and transport of nutrients, trace elements and vitamins from intestinal lumen into the circulation. Disorders of intestinal absorption are common in human pathology and include serious defects such as malabsorption. A detailed description of native digestive protein complexes in BBMs is therefore essential for understanding the physiology and pathology of digestion and absorption. In this study, we employed blue native PAGE (BN-PAGE) technique to separate protein complexes from purified mouse intestinal BBMs. We found 23 distinct protein complexes, which were cut off from the gel, and their protein composition was determined by LC-MS/MS. A total of 55 individual proteins were identified including peptidases, enzymes of carbohydrate metabolism, membrane transporters, cytoskeletal proteins, chaperones, and regulatory enzymes. From the identified proteins, 50% represent molecules with at least one predicted transmembrane domain as predicted by SOSUI software. To the best of our knowledge, this work is the first attempt aimed to characterize the native membrane proteome of intestinal BBM. As demonstrated here, BN-PAGE is a powerful tool for the separation of not only mitochondrial, but also membrane hydrophobic proteins in general. In addition, BN-PAGE technique preserves metal-protein interactions, as shown by the presence of 65Zn in metalloprotein complexes, isolated from zinc-radiolabeled BBMs.

Identification of heme binding protein complexes in murine erythroleukemic cells: study by a novel two-dimensional native separation -- liquid chromatography and electrophoresis.

In the current postgenomic era there is a growing interest in analysis of protein complexes in their native state. Here we present a novel two-dimensional separation technique for assessment of native protein complexes. The method combines native chromatography with native electrophoresis. The approach was used to study heme-binding protein complexes in murine erythroleukemia cells. The cells were metabolically labeled with [(59)Fe]-heme and cellular lysates were separated by anion-exchange chromatography. Fractions containing the (59)Fe isotope were collected, concentrated and further separated by native gel electrophoresis. A total of 13 radioactive protein bands were detected and analyzed by liquid chromatography-tandem mass spectrometry. Thirty-three individual proteins were identified and attributed to four novel multiprotein complexes representing four different 'snapshots' of cellular events involved in hemoglobin biosynthesis.

Native electrophoretic separation and femtomolar detection of 65Zn-containing proteins by storage phosphorimaging.

Over 300 zinc-containing proteins have been described and over 1000 genes in the human genome encode proteins with zinc finger domains. Despite the important role of zinc in mammalian physiology, understanding its cellular homeostasis remains limited. In this study, we demonstrate the utility of radioactive zinc detection using nondenaturating, native electrophoresis. We tested 65Zn-labeled enterocyte and enterocyte brush border membrane proteins and 65Zn-metallothionein. The radioactive decay of 65Zn is efficiently captured by storage phosphorimaging screen, enabling detection of 65Zn-labeled metalloproteins in subpicogram range. This powerful technique has a wide potential for studies of zinc absorption, incorporation of zinc into apoproteins and transcription factors that require zinc, zinc turnover in metallothionein, and other aspects of cellular zinc metabolism.