scholarly journals Characterization of translocation contact sites involved in the import of mitochondrial proteins.

1987 ◽  
Vol 105 (1) ◽  
pp. 235-246 ◽  
Author(s):  
M Schwaiger ◽  
V Herzog ◽  
W Neupert
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Proteins ◽  
Mitochondrial Matrix ◽  
Contact Sites ◽  
Close Proximity ◽  
Precursor Proteins ◽  
Stable Manner ◽  
One Step ◽  
Distinct Membrane

Import of proteins into the mitochondrial matrix requires translocation across two membranes. Translocational intermediates of mitochondrial proteins, which span the outer and inner membrane simultaneously and thus suggest that translocation occurs in one step, have recently been described (Schleyer, M., and W. Neupert, 1985, Cell, 43:339-350). In this study we present evidence that distinct membrane areas are involved in the translocation process. Mitochondria that had lost most of their outer membrane by digitonin treatment (mitoplasts) still had the ability to import proteins. Import depended on proteinaceous structures of the residual outer membrane and on a factor that is located between the outer and inner membranes and that could be extracted with detergent plus salt. Translocational intermediates, which had been preformed before fractionation, remained with the mitoplasts under conditions where most of the outer membrane was subsequently removed. Submitochondrial vesicles were isolated in which translocational intermediates were enriched. Immunocytochemical studies also suggested that the translocational intermediates are located in areas where outer and inner membranes are in close proximity. We conclude that the membrane-potential-dependent import of precursor proteins involves translocation contact sites where the two membranes are closely apposed and are linked in a stable manner.

scholarly journals The C-terminal domain of Fcj1 is required for formation of crista junctions and interacts with the TOB/SAM complex in mitochondria

2012 ◽  
Vol 23 (11) ◽  
pp. 2143-2155 ◽  
Author(s):  
Christian Körner ◽  
Miguel Barrera ◽  
Jovana Dukanovic ◽  
Katharina Eydt ◽  
Max Harner ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Inner Membrane ◽  
Functional Roles ◽  
Architectural Elements ◽  
Contact Sites ◽  
Oligomer Formation ◽  
Terminal Domain ◽  
Moderate Reduction ◽  
Close Proximity ◽  
Inner Membrane Protein

Crista junctions (CJs) are tubular invaginations of the inner membrane of mitochondria that connect the inner boundary with the cristae membrane. These architectural elements are critical for mitochondrial function. The yeast inner membrane protein Fcj1, called mitofilin in mammals, was reported to be preferentially located at CJs and crucial for their formation. Here we investigate the functional roles of individual domains of Fcj1. The most conserved part of Fcj1, the C-terminal domain, is essential for Fcj1 function. In its absence, formation of CJ is strongly impaired and irregular, and stacked cristae are present. This domain interacts with full-length Fcj1, suggesting a role in oligomer formation. It also interacts with Tob55 of the translocase of outer membrane β-barrel proteins (TOB)/sorting and assembly machinery (SAM) complex, which is required for the insertion of β-barrel proteins into the outer membrane. The association of the TOB/SAM complex with contact sites depends on the presence of Fcj1. The biogenesis of β-barrel proteins is not significantly affected in the absence of Fcj1. However, down-regulation of the TOB/SAM complex leads to altered cristae morphology and a moderate reduction in the number of CJs. We propose that the C-terminal domain of Fcj1 is critical for the interaction of Fcj1 with the TOB/SAM complex and thereby for stabilizing CJs in close proximity to the outer membrane. These results assign novel functions to both the C-terminal domain of Fcj1 and the TOB/SAM complex.

scholarly journals Solid-phase inclusion as a mechanism for regulating unfolded proteins in the mitochondrial matrix

2020 ◽  
Vol 6 (32) ◽  
pp. eabc7288
Author(s):  
Linhao Ruan ◽  
Joshua T. McNamara ◽  
Xi Zhang ◽  
Alexander Chih-Chieh Chang ◽  
Jin Zhu ◽  
...  
Keyword(s):  
Solid Phase ◽  
Tricarboxylic Acid Cycle ◽  
Yeast Cells ◽  
Mitochondrial Proteins ◽  
Mitochondrial Matrix ◽  
Unfolded Proteins ◽  
Daughter Cells ◽  
Contact Sites ◽  
Age Dependent ◽  
Mother And Daughter

Proteostasis declines with age, characterized by the accumulation of unfolded or damaged proteins. Recent studies suggest that proteins constituting pathological inclusions in neurodegenerative diseases also enter and accumulate in mitochondria. How unfolded proteins are managed within mitochondria remains unclear. Here, we found that excessive unfolded proteins in the mitochondrial matrix of yeast cells are consolidated into solid-phase inclusions, which we term deposits of unfolded mitochondrial proteins (DUMP). Formation of DUMP occurs in mitochondria near endoplasmic reticulum–mitochondria contact sites and is regulated by mitochondrial proteins controlling the production of cytidine 5′-diphosphate–diacylglycerol. DUMP formation is age dependent but accelerated by exogenous unfolded proteins. Many enzymes of the tricarboxylic acid cycle were enriched in DUMP. During yeast cell division, DUMP formation is necessary for asymmetric inheritance of damaged mitochondrial proteins between mother and daughter cells. We provide evidence that DUMP-like structures may be induced by excessive unfolded proteins in human cells.

scholarly journals Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites.

1989 ◽  
Vol 109 (4) ◽  
pp. 1421-1428 ◽  
Author(s):  
J Rassow ◽  
B Guiard ◽  
U Wienhues ◽  
V Herzog ◽  
F U Hartl ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Outer Membrane ◽  
Dihydrofolate Reductase ◽  
Protein Translocation ◽  
Close Contact ◽  
Membrane Surface ◽  
Mitochondrial Protein ◽  
Contact Sites ◽  
Precursor Proteins ◽  
The Matrix

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.

scholarly journals Cooperation of translocase complexes in mitochondrial protein import

2007 ◽  
Vol 179 (4) ◽  
pp. 585-591 ◽  
Author(s):  
Stephan Kutik ◽  
Bernard Guiard ◽  
Helmut E. Meyer ◽  
Nils Wiedemann ◽  
Nikolaus Pfanner
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Protein Complexes ◽  
Mitochondrial Protein ◽  
Mitochondrial Proteins ◽  
Intermembrane Space ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
Precursor Proteins ◽  
Preprotein Translocation

Most mitochondrial proteins are synthesized in the cytosol and imported into one of the four mitochondrial compartments: outer membrane, intermembrane space, inner membrane, and matrix. Each compartment contains protein complexes that interact with precursor proteins and promote their transport. These translocase complexes do not act as independent units but cooperate with each other and further membrane complexes in a dynamic manner. We propose that a regulated coupling of translocases is important for the coordination of preprotein translocation and efficient sorting to intramitochondrial compartments.

Structure of contact sites between the outer and inner mitochondrial membranes investigated by HVEM tomography

1996 ◽  
Vol 54 ◽  
pp. 966-967
Author(s):  
C.A. Mannella ◽  
K.F. Buttle ◽  
K.A. O‘Farrell ◽  
A. Leith ◽  
M. Marko
Keyword(s):  
Liver Mitochondrion ◽  
Adenine Nucleotide ◽  
Protein Complexes ◽  
Mitochondrial Membranes ◽  
Electron Microscopic ◽  
Contact Sites ◽  
Transmission Electron ◽  
Precursor Proteins ◽  
Membrane Contacts ◽  
And Function

Early transmission electron microscopy of plastic-embedded, thin-sectioned mitochondria indicated that there are numerous junctions between the outer and inner membranes of this organelle. More recent studies have suggested that the mitochondrial membrane contacts may be the site of protein complexes engaged in specialized functions, e.g., import of mitochondrial precursor proteins, adenine nucleotide channeling, and even intermembrane signalling. It has been suggested that the intermembrane contacts may be sites of membrane fusion involving non-bilayer lipid domains in the two membranes. However, despite growing interest in the nature and function of intramitochondrial contact sites, little is known about their structure.We are using electron microscopic tomography with the Albany HVEM to determine the internal organization of mitochondria. We have reconstructed a 0.6-μm section through an isolated, plasticembedded rat-liver mitochondrion by combining 123 projections collected by tilting (+/- 70°) around two perpendicular tilt axes. The resulting 3-D image has confirmed the basic inner-membrane organization inferred from lower-resolution reconstructions obtained from single-axis tomography.

Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Lipid Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Multi Drug Resistance ◽  
Gram Negative Bacteria ◽  
Membrane Channels ◽  
Gram Negative ◽  
Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from <i>Escherichia coli</i>, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (<i>k<sub>on</sub></i>) and lower dissociation (<i>k<sub>off</sub></i>) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

scholarly journals Exploiting the Amazing Diversity of Natural Source-Derived Polysaccharides: Modern Procedures of Isolation, Engineering, and Optimization of Antiviral Activities

Polymers ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 136
Author(s):  
Bimalendu Ray ◽  
Martin Schütz ◽  
Shuvam Mukherjee ◽  
Subrata Jana ◽  
Sayani Ray ◽  
...  
Keyword(s):  
Structural Characteristics ◽  
Antiviral Drug ◽  
Natural Source ◽  
Target Interaction ◽  
Charge Densities ◽  
Linkage Pattern ◽  
Naturally Occurring ◽  
Antiviral Activities ◽  
One Step

Naturally occurring polysaccharide sulfates are highly diverse, owning variations in the backbone structure, linkage pattern and stereochemistry, branching diversity, sulfate content and positions of sulfate group(s). These structural characteristics bring about diverse sulfated polymers with dissimilar negative charge densities and structure–activity relationships. Herein, we start with a short discussion of techniques needed for extraction, purification, chemical sulfation, and structural characterization of polysaccharides. Processes of isolation and sulfation of plant-derived polysaccharides are challenging and usually involve two steps. In this context, we describe an integrated extraction-sulfation procedure that produces polysaccharide sulfates from natural products in one step, thereby generating additional pharmacological activities. Finally, we provide examples of the spectrum of natural source-derived polysaccharides possessing specific features of bioactivity, in particular focusing on current aspects of antiviral drug development and drug–target interaction. Thus, the review presents a detailed view on chemically engineered polysaccharides, especially sulfated derivatives, and underlines their promising biomedical perspectives.

New horizons in mitochondrial contact site research

2020 ◽  
Vol 401 (6-7) ◽  
pp. 793-809
Author(s):  
Naama Zung ◽  
Maya Schuldiner
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Mechanisms ◽  
Contact Site ◽  
New Horizons ◽  
Contact Sites ◽  
Future Goals ◽  
Close Proximity ◽  
Recent Developments ◽  
Site Field ◽  
First Contact

AbstractContact sites, areas where two organelles are held in close proximity through the action of molecular tethers, enable non-vesicular communication between compartments. Mitochondria have been center stage in the contact site field since the discovery of the first contact between mitochondria and the endoplasmic reticulum (ER) over 60 years ago. However, only now, in the last decade, has there been a burst of discoveries regarding contact site biology in general and mitochondrial contacts specifically. The number and types of characterized contacts increased dramatically, new molecular mechanisms enabling contact formation were discovered, additional unexpected functions for contacts were shown, and their roles in cellular and organismal physiology were emphasized. Here, we focus on mitochondria as we highlight the most recent developments, future goals and unresolved questions in the field.

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