A dynamic model of the mitochondrial protein import machinery.
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Biological Transport↗
Biomedical subjects
J Rassow
Publications and source records attributed to J Rassow.
Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space.
Cytochrome b2 reaches the intermembrane space of mitochondria by transport into the matrix followed by export across the inner membrane. While in the matrix, the protein interacts with hsp60, which arrests its folding prior to export. The bacterial-type export sequence in pre-cytochrome b2 functions by inhibiting the ATP-dependent release of the protein from hsp60. Release for export apparently requires, in addition to ATP, the interaction of the signal sequence with a component of the export machinery in the inner membrane. Export can occur before import is complete provided that a critical length of the polypeptide chain has been translocated into the matrix. Thus, hsp60 combines two activities: catalysis of folding of proteins destined for the matrix, and maintaining proteins in an unfolded state to facilitate their channeling between the machineries for import and export across the inner membrane. Anti-folding signals such as the hydrophobic export sequence in cytochrome b2 may act as switches between these two activities.
Base Sequence↗
Targeting of a chemically pure preprotein to mitochondria does not require the addition of a cytosolic signal recognition factor.
To analyze the role of cytosolic cofactors in mitochondrial protein targeting, we prepared a chemically pure mitochondrial preprotein. When diluted out of 7 M urea, this precursor protein was efficiently imported into mitochondria without the addition of cytosolic cofactors. Extensive prewashing of mitochondria (up to 2 M KCl) did not reduce its import. Import of the purified precursor showed the characteristics of authentic mitochondrial import including use of the receptor MOM19, requirement for a membrane potential, and proteolytic processing. When the precursor was preincubated at a low concentration of urea, cytosolic cofactors were needed to preserve its import competence. We conclude that targeting of this preprotein via the mitochondrial master receptor MOM19 does not require a cytosolic signal recognition factor; cytosolic cofactors apparently have chaperone-like functions in mitochondrial protein uptake. Moreover, we found that a cleavable presequence was sufficient to direct protein import via MOM19. Together with the cofactor-independent function of MOM19, it is thus conceivable that MOM19 functions as mitochondrial presequence receptor.
Animals↗
Mapping of the protein import machinery in the mitochondrial outer membrane by crosslinking of translocation intermediates.
Mitochondria contain a complex machinery for the import of nuclear-encoded proteins. Receptor proteins exposed on the outer membrane surface are required for the specific binding of precursor proteins to mitochondria, either by binding of cytosolic signal recognition factors or by direct recognition of the precursor polypeptides. Subsequently, the precursors are inserted into the outer membrane at the general insertion site GIP (general insertion protein). Here we report the analysis of receptors and GIP by crosslinking of translocation intermediates and by coimmunoprecipitation. Surface-accumulated precursors were crosslinked to the receptors MOM19 and MOM72, suggesting a direct interaction of preproteins with surface receptors. We identified three novel mitochondrial outer membrane proteins, MOM7, MOM8, and MOM30 that, together with the previously identified MOM38, seem to form the GIP site and are present in the mitochondrial receptor complex.
Animals↗
[The special aspects in the planning and use of an electron linear accelerator facility for intraoperative radiotherapy (IORT)].
Limiting conditions for the planning of the installation of an electron linear accelerator in an operating theatre are described taking into account the radiotherapeutical and surgical requirements based on the example of the Essen facility for intraoperative radiotherapy (IORT) with electrons. Special features of the facility like a non-contact electron applicator system with television monitoring, table-top trolley, which is used also during irradiation, are explained in detail. Measured isodose curves for all tubes and all electron energies serve for the radiotherapist as criteria for decision on tube size and electron energy selection during surgical operation. The influence of misalignments of the tube on dose distributions is investigated. Limit values of alignment tolerances are deduced. Daily constancy tests for monitor calibration, electron energy, and tube alignment system are to be performed for quality assurance. Tables of the planned and realized treatments and of a representative time schedule of an IORT give a survey on the indications and the necessary time effort and staff. Completion of the set of tubes as well as optimization of the tube fixation accessory and of the table-top trolley are main subjects for future developments.
Anesthesiology↗
Mitochondrial preproteins en route from the outer membrane to the inner membrane are exposed to the intermembrane space.
Mitochondrial precursor proteins are known to be imported at sites of close contact between mitochondrial outer and inner membranes. We have identified translocation intermediates exposed to the intermembrane space, including the precursor of the ADP/ATP carrier accumulated at the general insertion site GIP, and the precursor of F1-ATPase subunit beta accumulated on its import pathway at low levels of ATP. These results suggest that mitochondrial contact sites are not sealed structures, but that polypeptides pass (at least partly) through the intermembrane space on their route from the outer membrane to the inner membrane.
Animals↗
Biogenesis of the mitochondrial phosphate carrier.
The mitochondrial phosphate carrier (PiC) is a member of the family of inner-membrane carrier proteins which are generally synthesized without a cleavable presequence. Surprisingly, the cDNA sequences of bovine and rat PiC suggested the existence of an amino-terminal extension sequence in the precursor of PiC. By expressing PiC in vitro, we found that PiC is indeed synthesized as a larger precursor. This precursor was imported and proteolytically processed by mitochondria, whereby the correct amino-terminus of the mature protein was generated. Import of PiC showed the characteristics of mitochondrial protein uptake, such as dependence on ATP and a membrane potential and involvement of contact sites between mitochondrial outer and inner membranes. The precursor imported in vitro was correctly assembled into the functional form, demonstrating that the authentic import and assembly pathway of PiC was reconstituted when starting with the presequence-carrying precursor. These results are discussed in connection with the recently postulated role of PiC as an import receptor located in the outer membrane.
Amino Acid Sequence↗
Dosimetry and fluence measurements with a new irradiation arrangement for neutron capture therapy of tumours in mice.
Incorporation of 10B in human tumours treated with fast neutrons would increase the local dose in the tumour. In tissue the neutrons are thermalized mainly causing the neutron capture reaction 10B (n,a) 7Li. The dose enhancement can be calculated using the thermal neutron fluence measured by activation of gold foils in a phantom. At a phantom depth of 5 cm a dose enhancement of (0.056 +/- 0.0028)% for each microgram of 10B per gram of tissue was determined in the tumour. A therapeutic gain by this dose enhancement in fast neutron therapy should be examined for a solid tumour in a mouse. The feasibility for neutron capture therapy of tumours in mice depends on the distribution of the tumour dose and on the effective shielding of the bodies of the mice. Therefore, a special holding device for simultaneous irradiation of four tumours was developed. The dose distribution in the tumours and in the surrounding bodies of mice was measured with TLD-300 in a special mouse phantom.
Animals↗
Analysis of mitochondrial protein import using translocation intermediates and specific antibodies.
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Animals↗
Polypeptides traverse the mitochondrial envelope in an extended state.
Most mitochondrial proteins are synthesized as precursors in the cytosol and imported through contact sites between outer and inner mitochondrial membranes. The molecular mechanism of membrane translocation of precursor proteins is largely unclear. For this report, various hybrid proteins between portions of the precursor of cytochrome b2 and the entire dihydrofolate reductase (DHFR) were accumulated in mitochondrial contact sites. We unexpectedly found that about 50 amino acid residues of the polypeptide chain in transit were sufficient to span both membranes. This suggests a linear translocation of the polypeptide chain and presents evidence for a high degree of unfolding of polypeptides traversing the mitochondrial membranes.
Biological Transport↗
Energy requirements for unfolding and membrane translocation of precursor proteins during import into mitochondria.
ATP is involved in conferring transport competence to numerous mitochondrial precursor proteins in the cytosol. Unfolded precursor proteins were found not to require ATP for import into mitochondria, suggesting a role of ATP in the unfolding of precursors. Here we report the unexpected finding that a hybrid protein containing the tightly folded passenger protein dihydrofolate reductase becomes unfolded and specifically translocated across the mitochondrial membranes independently of added ATP. Moreover, interaction of the precursor with the mitochondrial receptor components does not require ATP. The results suggest that ATP is not involved in the actual process of unfolding during membrane translocation of precursors. ATP rather appears to be necessary for preventing the formation of improper structures of precursors in the cytosol and for folding of imported polypeptides on (and release from) chaperone-like molecules in the mitochondrial matrix.
Adenosine Triphosphate↗
Nucleotide sequence of a full-length cDNA coding for the mitochondrial precursor protein of the beta-subunit of F1-ATPase from Neurospora crassa.
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Amino Acid Sequence↗
Contact sites between inner and outer membranes: structure and role in protein translocation into the mitochondria.
Contact sites between both mitochondrial membranes play a predominant role in the transport of nuclear-coded precursor proteins into mitochondria. The characterization of contact sites was greatly advanced by the reversible accumulation of precursor proteins in transit (translocation intermediates). It was found that the sites are saturable, apparently contain proteinaceous components and mediate extensive unfolding of the polypeptide chain in translocation. Some components of mitochondrial contact sites are currently being identified.
Animals↗
Stress proteins and mitochondrial protein import.
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Adenosine Triphosphate↗
Treatment planning for neutron therapy of high-grade astrocytomas and pencil gliomas.
If astrocytomas of grade III and IV and pencil gliomas of the spinal cord would be good indications for neutron therapy, high accuracy and homogeneity of the target absorbed dose and the planned spatial dose distribution would be pre-conditions for acceptable low complication rates. Therefore, results of treatment planning calculations have been compared with in vivo measurements and found to be in good agreement. Special care is necessary if the percentage of the gamma-ray component is spatially varying and additionally for fixation of the patient and combination of radiation fields in the case of pencil gliomas. Examples are given how to avoid overdosage and to hold the inhomogeneity even at the borderline of adjacent fields within 10%.
Brain Neoplasms↗
[Neutron flow measurements in the d(14) + Be neutron radiation field from the cyclotron in Essen].
It exists the possibility in the neutron therapy of specific tumours to increase the local energy dose in the tumour by treatment with fast neutrons under use of B-10. Thermal neutrons cause the neutron capture reaction B-10(n, alpha)Li-7 in tissue. A sufficiently high number of thermic neutrons is necessary combined with a possibly selective concentration of B-10 in the tumour as target volume for the clinical application. Gold foils were activated in a water phantom at several depths for the quantitative determination of the thermal neutron fluence. A result of this measurements is a maximal thermal neutron fluence of 1.44 X 10(10) cm-2 at depth of 5 cm with applied total energy dose of 0.8 Gy.
Fast Neutrons↗
[Neutron dosimetry in a model of the body's inhomogeneities using the TLD-300 detector].
The modification of the absorbed dose distribution due to the presence of inhomogeneities in the human body requires thorough research within the scope of radiation treatment planning for tumour therapy with fast neutrons. In this work the effect of air and lung cavities and the depth dose distribution in the near and distant vicinity of the inhomogeneity is studied. By means of thermoluminescence detectors TLD-300 the total absorbed dose is measured in mixed neutron-photon radiation fields in the used polystyrene phantom with embedded inhomogeneity. Lung tissue is simulated by cork. Differences in the concentration of hydrogen between inhomogeneity and surrounding soft tissue lead to new build-up effects. These cause an increase behind and a decrease of the total dose beside and in front of the inhomogeneity. In case of the cork inhomogeneity an absorbed dose increase of maximally 20% in comparison to the absorbed dose in the homogeneous phantom was found. With air as inhomogeneity the increase of absorbed dose reaches a maximum of 30% compared to the homogeneous phantom.
Air↗
Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites.
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.
Amino Acid Sequence↗