Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARK2/Parkin mutations cause autosomal recessive forms of Parkinson's disease. Upon a loss of mitochondrial membrane potential (ΔΨ m ) in human cells, cytosolic Parkin has been reported to be recruited to mitochondria, which is followed by a stimulation of mitochondrial autophagy. Here, we show that the relocation of Parkin to mitochondria induced by a collapse of ΔΨ m relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal ΔΨ m . We also show that once at the mitochondria, Parkin is in close proximity to PINK1, but we find no evidence that Parkin catalyzes PINK1 ubiquitination or that PINK1 phosphorylates Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into mitochondrial aggregates and/or large perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin, together with PINK1, modulates mitochondrial trafficking, especially to the perinuclear region, a subcellular area associated with autophagy. Thus by impairing this process, mutations in either Parkin or PINK1 may alter mitochondrial turnover which, in turn, may cause the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinson's disease.autophagy | Parkinson's disease | phosphatase and tensin homolog-induced putative kinase 1 T he common neurodegenerative disorder Parkinson's disease (PD) occasionally can be inherited (1, 2). Parkinson disease 6/ phosphatase and tensin homolog (PTEN)-induced putative kinase-1 (PARK6/PINK1) is among the gene products associated with familial PD (2, 3). This 581-amino acid polypeptide is localized to the mitochondria and has only a single recognized functional domain, a serine/threonine kinase with a high degree of homology to that of the Ca 2+ /calmodulin kinase family. Overexpression of WT PINK1 rescues abnormal mitochondrial morphology that has been described in Drosophila carrying Pink1 mutations (4, 5), a finding that supports the notion that the mutated allele gives rise to a loss-of-function phenotype. Loss-offunction mutations in the gene encoding PARK2/Parkin (an E3 ubiquitin ligase) also can cause an autosomal recessive form of familial PD (2, 6). Parkin is thought to operate within the same molecular pathway as PINK1 to modulate mitochondrial dynamics (4, 5, 7). This possibility is intriguing, because Parkin has been reported to be essentially cytosolic (8, 9). However, we have shown that PINK1 spans the outer mitochondrial membrane, with its kinase domain facing the cytoplasm (10). These details of PINK1 topology are relevant to the reported Parkin/PINK1 genetic interaction because they place the only known functional domain of PINK1 in the same subcellular compartment as Parkin.However, the role played by Parkin, PINK1, or both in mitochondrial dynamics is still uncertain. Perhaps, the beginning of an answer to th...

Proton pumping respiratory complex I is a major player in mitochondrial energy conversion. Yet little is known about the molecular mechanism of this large membrane protein complex. Understanding the details of ubiquinone reduction will be prerequisite for elucidating this mechanism. Based on a recently published partial structure of the bacterial enzyme, we scanned the proposed ubiquinone binding cavity of complex I by sitedirected mutagenesis in the strictly aerobic yeast Yarrowia lipolytica. The observed changes in catalytic activity and inhibitor sensitivity followed a consistent pattern and allowed us to define three functionally important regions near the ubiquinone-reducing iron-sulfur cluster N2. We identified a likely entry path for the substrate ubiquinone and defined a region involved in inhibitor binding within the cavity. Finally, we were able to highlight a functionally critical structural motif in the active site that consisted of Tyr-144 in the 49-kDa subunit, surrounded by three conserved hydrophobic residues.Respiratory chain NADH:ubiquinone oxidoreductase (complex I) is a large membrane protein complex that catalyzes electron transfer from NADH to ubiquinone and thereby pumps protons across the inner mitochondrial or bacterial plasma membrane (1). Electron microscopy revealed that complex I is L-shaped (2-9) and is composed of a hydrophobic arm embedded in the membrane and a peripheral arm protruding into the mitochondrial matrix or the bacterial cytosol. The peripheral arm contains all known redox centers, one FMN and eight or nine iron-sulfur clusters. Based on sequence comparisons (10, 11), mutational analysis (12-15), and photoaffinity labeling studies (16), we have proposed previously (14, 17) that the PSST and the 49-kDa subunit that are homologous to the small and large subunit of [NiFe] hydrogenases form part of the quinone reducing catalytic core of complex I (note that the bovine nomenclature will be used for the central subunits of complex I throughout). Recently the crystal structure of the peripheral domain of complex I from Thermus thermophilus has been solved at 3.3 Å resolution (18). This structure (Fig. 1) shows a wire of iron-sulfur clusters connecting the NADH-binding site near FMN with a broad cavity formed by the PSST and the 49-kDa subunit that should comprise the active site for ubiquinone reduction and the binding region for the large number of inhibitors that have been found for complex I (19).Because reduction of ubiquinone is likely to be a key event in the energy-coupling mechanism of complex I (1, 20), the quinone-binding site in the PSST and the 49-kDa subunit next to iron sulfur-cluster N2 is of particular interest. To identify the domains essential for catalytic activity and inhibitor binding, we introduced a set of point mutations in the PSST and the 49-kDa subunits of complex I from our model organism, the strictly aerobic yeast Yarrowia lipolytica. Positions for point mutations were chosen by analyzing the T. thermophilus structure so that they would probe all...

Background:The Parkinson-related kinase Pink1 is implicated in mitochondrial quality control. Results: Integration of multiple targeting signals results in the localization of Pink1 and its processing product to the surface of the outer membrane. Conclusion: Pink1 follows a unique import pathway that allows probing the functional integrity of mitochondria. Significance: Determining the Pink1 targeting mode provides the basis for defining its molecular function.

Numerous hydrophobic and amphipathic compounds including several detergents are known to inhibit the ubiquinone reductase reaction of respiratory chain complex I (proton pumping NADH:ubiquinone oxidoreductase). Guided by the X-ray structure of the peripheral arm of complex I from Thermus thermophilus we have generated a large collection of site-directed mutants in the yeast Yarrowia lipolytica targeting the proposed ubiquinone and inhibitor binding pocket of this huge multiprotein complex at the interface of the 49-kDa and PSST subunits. We could identify a number of residues where mutations changed I(50) values for representatives from all three groups of hydrophobic inhibitors. Many mutations around the domain of the 49-kDa subunit that is homologous to the [NiFe] centre binding region of hydrogenase conferred resistance to DQA (class I/type A) and rotenone (class II/type B) indicating a wider overlap of the binding sites for these two types of inhibitors. In contrast, a region near iron-sulfur cluster N2, where the binding of the n-alkyl-polyoxyethylene-ether detergent C(12)E(8) (type C) was exclusively affected, appeared comparably well separated. Taken together, our data provide structure-based support for the presence of distinct but overlapping binding sites for hydrophobic inhibitors possibly extending into the ubiquinone reduction site of mitochondrial complex I.

Proton pumping NADH:ubiquinone oxidoreductase (complex I) is the largest and remains by far the least understood enzyme complex of the respiratory chain. It consists of a peripheral arm harbouring all known redox active prosthetic groups and a membrane arm with a yet unknown number of proton translocation sites. The ubiquinone reduction site close to iron-sulfur cluster N2 at the interface of the 49-kDa and PSST subunits has been mapped by extensive site directed mutagenesis. Independent lines of evidence identified electron transfer events during reduction of ubiquinone to be associated with the potential drop that generates the full driving force for proton translocation with a 4 H+/2e− stoichiometry. Electron microscopic analysis of immuno-labelled native enzyme and of a subcomplex lacking the electron input module indicated a distance of 35–60 Å of cluster N2 to the membrane surface. Resolution of the membrane arm into subcomplexes showed that even the distal part harbours subunits that are prime candidates to participate in proton translocation because they are homologous to sodium/proton antiporters and contain conserved charged residues in predicted transmembrane helices. The mechanism of redox linked proton translocation by complex I is largely unknown but has to include steps where energy is transmitted over extremely long distances. In this review we compile the available structural information on complex I and discuss implications for complex I function.