Elzbieta Glaser

The amyloid β-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae

The amyloid β-peptide (Aβ) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by Aβ and accumulation of Aβ has been detected in mitochondria. Because Aβ is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial Aβ uptake. Our results from rat mitochondria show that Aβ is transported into mitochondria via the translocase of the outer membrane (TOM) machinery. The import was insensitive to valinomycin, indicating that it is independent of the mitochondrial membrane potential. Subfractionation studies following the import experiments revealed Aβ association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of Aβ to mitochondrial cristae. A similar distribution pattern of Aβ in mitochondria was shown by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for Aβ in mitochondria and demonstrate both in vitro and in vivo that Aβ is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied Aβ can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of Aβ, a peptide considered to be of major significance in Alzheimers disease.

Interaction of plant mitochondrial and chloroplast signal peptides with the Hsp70 molecular chaperone

Most mitochondrial and chloroplast proteins are synthesized on cytosolic polyribosomes as precursor proteins, with an N-terminal signal sequence that targets the precursor to the correct organelle. In mitochondria, the chaperone Hsp70 functions as a molecular motor, pulling the precursor across the mitochondrial membranes; 97.0% of plant mitochondrial presequences contain an Hsp70 binding site. In chloroplasts, the outer envelope, intermembrane space and a stromal Hsp70 are thought to participate in protein import; 82.5% of chloroplast transit peptides have an Hsp70 binding site. The interaction of signal peptides with Hsp70 during the import process is supported by biochemical and bioinformatic studies.

Modulation of the endoplasmic reticulum–mitochondria interface in Alzheimer’s disease and related models

It is well-established that subcompartments of endoplasmic reticulum (ER) are in physical contact with the mitochondria. These lipid raft-like regions of ER are referred to as mitochondria-associated ER membranes (MAMs), and they play an important role in, for example, lipid synthesis, calcium homeostasis, and apoptotic signaling. Perturbation of MAM function has previously been suggested in Alzheimer’s disease (AD) as shown in fibroblasts from AD patients and a neuroblastoma cell line containing familial presenilin-2 AD mutation. The effect of AD pathogenesis on the ER–mitochondria interplay in the brain has so far remained unknown. Here, we studied ER–mitochondria contacts in human AD brain and related AD mouse and neuronal cell models. We found uniform distribution of MAM in neurons. Phosphofurin acidic cluster sorting protein-2 and σ1 receptor, two MAM-associated proteins, were shown to be essential for neuronal survival, because siRNA knockdown resulted in degeneration. Up-regulated MAM-associated proteins were found in the AD brain and amyloid precursor protein (APP)Swe/Lon mouse model, in which up-regulation was observed before the appearance of plaques. By studying an ER–mitochondria bridging complex, inositol-1,4,5-triphosphate receptor–voltage-dependent anion channel, we revealed that nanomolar concentrations of amyloid β-peptide increased inositol-1,4,5-triphosphate receptor and voltage-dependent anion channel protein expression and elevated the number of ER–mitochondria contact points and mitochondrial calcium concentrations. Our data suggest an important role of ER–mitochondria contacts and cross-talk in AD pathology.

Mitochondrial protein import in plants. Signals, sorting, targeting, processing and regulation.

Mitochondrial biogenesis requires a coordinated expression of both the nuclear and the organellar genomes and specific intracellular protein trafficking, processing and assembly machinery. Most mitochondrial proteins are synthesised as precursor proteins containing an N-terminal extension which functions as a targeting signal, which is proteolytically cleaved off after import into mitochondria. We review our present knowledge on components and mechanisms involved in the mitochondrial protein import process in plants. This encompasses properties of targeting peptides, sorting of precursor proteins between mitochondria and chloroplasts, signal recognition, mechanism of translocation across the mitochondrial membranes and the role of cytosolic and organellar molecular chaperones in this process. The mitochondrial protein processing in plants is catalysed by the mitochondrial processing peptidase (MPP), which in contrast to other sources, is integrated into the bc1 complex of the respiratory chain. This is the most studied component of the plant import machinery characterised to date. What are the biochemical consequences of the integration of the MPP into an oligomeric protein complex and how are several hundred presequences of precursor proteins with no sequence similarities and no consensus for cleavage, specifically cleaved off by MPP? Finally we will address the emerging area of the control of protein import into mitochondria.

Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP.

Recently we have identified the novel mitochondrial peptidase responsible for degrading presequences and other short unstructured peptides in mitochondria, the presequence peptidase, which we named PreP peptidasome. In the present study we have identified and characterized the human PreP homologue, hPreP, in brain mitochondria, and we show its capacity to degrade the amyloid β-protein (Aβ). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zinc-binding motif. We show that hPreP is localized to the mitochondrial matrix. In situ immuno-inactivation studies in human brain mitochondria using anti-hPreP antibodies showed complete inhibition of proteolytic activity against Aβ. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu78 in the inverted zinc-binding motif replaced by Gln. In vitro studies using recombinant hPreP and liquid chromatography nanospray tandem mass spectrometry revealed novel cleavage specificities against Aβ-(1-42), Aβ-(1-40), and Aβ Arctic, a protein that causes increased protofibril formation an early onset familial variant of Alzheimer disease. In contrast to insulin degrading enzyme, which is a functional analogue of hPreP, hPreP does not degrade insulin but does degrade insulin B-chain. Molecular modeling of hPreP based on the crystal structure at 2.1 Å resolution of AtPreP allowed us to identify Cys90 and Cys527 that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial Aβ by hPreP may potentially be of importance in the pathology of Alzheimer disease.

Isolation and Identification of a Novel Mitochondrial Metalloprotease (PreP) That Degrades Targeting Presequences in Plants

Most of the nuclear encoded mitochondrial precursor proteins contain an N-terminal extension called the presequence that carries targeting information and that is cleaved off after import into mitochondria. The presequences are amphiphilic, positively charged, membrane-interacting peptides with a propensity to form α-helices. Here we have investigated the proteolysis of the presequences that have been cleaved off inside mitochondria. A presequence derived from the overexpressed F1β subunit of the ATP synthase and specific synthetic fluorescent peptides (Pep Tag Protease assay) have been shown to undergo rapid degradation catalyzed by a matrix located protease. We have developed a three-step chromatographic procedure including affinity and anion exchange chromatography for isolation of the protease from potato tuber mitochondria. Two-dimensional gel electrophoresis of the isolated proteolytically active fraction followed by electrospray ionization-mass spectrometry/mass spectrometry and data base searches allowed identification of the presequence peptide-degrading protease inArabidopsis thaliana data base as a novel mitochondrial metalloendoprotease with a molecular mass of 105 kDa. The identified metalloprotease contains an inverted zinc-binding motif and belongs to the pitrilysin family.

Nuclear Photosynthetic Gene Expression Is Synergistically Modulated by Rates of Protein Synthesis in Chloroplasts and Mitochondria

Arabidopsis thaliana mutants prors1-1 and -2 were identified on the basis of a decrease in effective photosystem II quantum yield. Mutations were localized to the 5′-untranslated region of the nuclear gene PROLYL-tRNA SYNTHETASE1 (PRORS1), which acts in both plastids and mitochondria. In prors1-1 and -2, PRORS1 expression is reduced, along with protein synthesis in both organelles. PRORS1 null alleles (prors1-3 and -4) result in embryo sac and embryo development arrest. In mutants with the leaky prors1-1 and -2 alleles, transcription of nuclear genes for proteins involved in photosynthetic light reactions is downregulated, whereas genes for other chloroplast proteins are upregulated. Downregulation of nuclear photosynthetic genes is not associated with a marked increase in the level of reactive oxygen species in leaves and persists in the dark, suggesting that the transcriptional response is light and photooxidative stress independent. The mrpl11 and prpl11 mutants are impaired in the mitochondrial and plastid ribosomal L11 proteins, respectively. The prpl11 mrpl11 double mutant, but neither of the single mutants, resulted in strong downregulation of nuclear photosynthetic genes, like that seen in leaky mutants for PRORS1, implying that, when organellar translation is perturbed, signals derived from both types of organelles cooperate in the regulation of nuclear photosynthetic gene expression.

Processing peptidases in mitochondria and chloroplasts

Most of the mitochondrial and chloroplastic proteins are nuclear encoded and synthesized in the cytosol as precursor proteins with N-terminal extensions called targeting peptides. Targeting peptides function as organellar import signals, they are recognized by the import receptors and route precursors through the protein translocons across the organellar membranes. After the fulfilled function, targeting peptides are proteolytically cleaved off inside the organelles by different processing peptidases. The processing of mitochondrial precursors is catalyzed in the matrix by the Mitochondrial Processing Peptidase, MPP, the Mitochondrial Intermediate Peptidase, MIP (recently called Octapeptidyl aminopeptidase 1, Oct1) and the Intermediate cleaving peptidase of 55kDa, Icp55. Furthermore, different inner membrane peptidases (Inner Membrane Proteases, IMPs, Atp23, rhomboids and AAA proteases) catalyze additional processing functions, resulting in intra-mitochondrial sorting of proteins, the targeting to the intermembrane space or in the assembly of proteins into inner membrane complexes. Chloroplast targeting peptides are cleaved off in the stroma by the Stromal Processing Peptidase, SPP. If the protein is further translocated to the thylakoid lumen, an additional thylakoid-transfer sequence is removed by the Thylakoidal Processing Peptidase, TPP. Proper function of the D1 protein of Photosystem II reaction center requires its C-terminal processing by Carboxy-terminal processing protease, CtpA. Both in mitochondria and in chloroplasts, the cleaved targeting peptides are finally degraded by the Presequence Protease, PreP. The organellar proteases involved in precursor processing and targeting peptide degradation constitute themselves a quality control system ensuring the correct maturation and localization of proteins as well as assembly of protein complexes, contributing to sustenance of organelle functions. Dysfunctions of several mitochondrial processing proteases have been shown to be associated with human diseases. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

The voltage-dependent anion channel, a major component of the tRNA import machinery in plant mitochondria

In plants, as in most eukaryotic cells, import of nuclear-encoded cytosolic tRNAs is an essential process for mitochondrial biogenesis. Despite its broad occurrence, the mechanisms governing RNA transport into mitochondria are far less understood than protein import. This article demonstrates by Northwestern and gel-shift experiments that the plant mitochondrial voltage-dependent anion channel (VDAC) protein interacts with tRNA in vitro. It shows also that this porin, known to play a key role in metabolite transport, is a major component of the channel involved in the tRNA translocation step through the plant mitochondrial outer membrane, as supported by inhibition of tRNA import into isolated mitochondria by VDAC antibodies and Ruthenium red. However VDAC is not a tRNA receptor on the outer membrane. Rather, two major components from the TOM (translocase of the outer mitochondrial membrane) complex, namely TOM20 and TOM40, are important for tRNA binding at the surface of mitochondria, suggesting that they are also involved in tRNA import. Finally, we show that proteins and tRNAs are translocated into plant mitochondria by different pathways. Together, these findings identify unexpected components of the tRNA import machinery and suggest that the plant tRNA import pathway has evolved by recruiting multifunctional proteins.

Integration of the Mitochondrial-Processing Peptidase into the Cytochrome bc1 Complex in Plants

The plant mitochondrial cytochrome bc1 complex, like nonplant mitochondrial complexes,consists of cytochromes b and c1, the Rieske iron–sulfur protein, two Core proteins, and fivelow-molecular mass subunits. However, in contrast to nonplant sources, the two Core proteinsare identical to subunits of the general mitochondrial processing peptidase (MPP). The MPPis a fascinating enzyme that catalyzes the specific cleavage of the diverse presequence peptidesfrom hundreds of the nuclear-encoded mitochondrial precursor proteins that are synthesizedin the cytosol and imported into the mitochondrion. Integration of the MPP into the bc1complex renders the bc1 complex in plants bifunctional, being involved both in electrontransport and in protein processing. Despite the integration of MPP into the bc1 complex,electron transfer as well as translocation of the precursor through the import channel areindependent of the protein-processing activity. Recognition of the processing site by MPPoccurs via the recognition of higher-order structural elements in combination with charge andcleavage-site properties. Elucidation of the three-dimensional (3-D) structure of the mammaliancytochrome bc1 complex is highly useful for understanding of the mechanism of action of MPP.