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Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, the ubiquitin kinase PINK1 phosphorylates ubiquitin to activate the ubiquitin ligase parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors. Using genome editing to knockout five autophagy receptors in HeLa cells, here we show that two receptors previously linked to xenophagy, NDP52 and optineurin, are the primary receptors for PINK1- and parkin-mediated mitophagy. PINK1 recruits NDP52 and optineurin, but not p62, to mitochondria to activate mitophagy directly, independently of parkin. Once recruited to mitochondria, NDP52 and optineurin recruit the autophagy factors ULK1, DFCP1 and WIPI1 to focal spots proximal to mitochondria, revealing a function for these autophagy receptors upstream of LC3. This supports a new model in which PINK1-generated phospho-ubiquitin serves as the autophagy signal on mitochondria, and parkin then acts to amplify this signal. This work also suggests direct and broader roles for ubiquitin phosphorylation in other autophagy pathways., Selective autophagy clears intracellular pathogens and mediates cellular quality control by engulfing cargo into autophagosomes and delivering it to lysosomes for degradation. Autophagy receptors bind ubiquitinated cargo and LC3-coated phagophores [...]

Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step--specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys. doi/10.1083/jcb.200905134

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2006.03.006 Byline: Carine de Marcos-Lousa (a), Dionisia P Sideris (a)(b), Kostas Tokatlidis (a)(c) Abstract: Most of the mitochondrial inner-membrane proteins are generated without a presequence and their targeting depends on inadequately defined internal segments. Despite the numerous components of the import machinery identified by proteomics, the properties of hydrophobic import substrates remain poorly understood. Recent studies support several principles for these membrane proteins: first, they become organized into partially assembled forms within the translocon; second, they present noncontiguous targeting signals; and third, they induce conformational changes in translocase subunits, thereby mediating 'assembly on demand' of the import machinery. It is possible that the energy needed for these proteins to pass across the outer membrane, to travel through the intermembrane space and to target the inner-membrane surface is provided by conformational changes involving import components that seem to have natively unfolded structures. Such structural malleability might render some of the translocase subunits more adept at driving the protein import process. Author Affiliation: (a) Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology (IMBB-FORTH), PO Box 1385, Heraklion 71110, Crete, Greece (b) Department of Biology, University of Crete, PO Box 2208, Heraklion 71409, Crete, Greece (c) Department of Chemistry, University of Crete, PO Box 1470, Heraklion 71409, Crete, Greece

The endoplasmic reticulum (ER) provides an environment optimized for oxidative protein folding through the action of Ero1p, which generates disulfide bonds, and Pdi1p, which receives disulfide bonds from Ero1p and transfers them to substrate proteins. Feedback regulation of Ero1p through reduction and oxidation of regulatory bonds within Ero1p is essential for maintaining the proper redox balance in the ER. In this paper, we show that Pdi1p is the key regulator of Ero1p activity. Reduced Pdi1p resulted in the activation of Ero1p by direct reduction of Ero1p regulatory bonds. Conversely, upon depletion of thiol substrates and accumulation of oxidized Pdi1p, Ero1p was inactivated by both autonomous oxidation and Pdi1pmediated oxidation of Ero1p regulatory bonds. Pdi1p responded to the availability of free thiols and the relative levels of reduced and oxidized glutathione in the ER to control Ero1p activity and ensure that cells generate the minimum number of disulfide bonds needed for efficient oxidative protein folding. [ABSTRACT FROM AUTHOR]

Summary: A new fluorescent Zn2+ indicator, namely, ICPBCZin was synthesized and the spectral profile of its free and Zn2+ bound forms was studied. The newly synthesized zinc indicator incorporates as chromophore the chromeno [3′,2′:3,4]pyrido[1,2a] [1,3]benzimidazole moiety and belongs to the dicarboxylate-type of zinc probes. The compound is excited with visible light, exhibits high selectivity for zinc in the presence of calcium and other common biological ions, and its Zn2+ dissociation constant is 4.0nM. Fluorescence spectra studies of ICPBCZin indicated a clear shift in its emission wavelength maxima upon Zn2+ binding, as it belongs to the class of Photoinduced Charge Transfer (PCT) indicators, along with changes in fluorescence intensity that enable the compound to be used as a ratiometric, visible-excitable Zn2+ probe. [Copyright &y& Elsevier]

The MIA40 pathway is a novel import pathway in mitochondria specific for cysteine-rich proteins of the intermembrane space (IMS). The newly synthesised precursors are trapped in the IMS by a disulfide relay mechanism that involves introduction of disulfides from the sulfhydryl oxidase Erv1 to the redox-regulated import receptor Mia40 and then on to the substrate. This thiol-disulfide exchange mechanism is essential for the import and oxidative folding of the incoming cysteine-rich substrate proteins. In this chapter we will describe the experimental methods that have been developed in order to study and characterise disulfide-trapped intermediates in yeast mitochondria.