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J Rassow

Publications and source records attributed to J Rassow.

At least 37 records · Page 2Linked to original sources
PubMed1999

The J-related segment of tim44 is essential for cell viability: a mutant Tim44 remains in the mitochondrial import site, but inefficiently recruits mtHsp70 and impairs protein translocation.

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Delta18 in addition to the endogenous wild-type Tim44. Tim44Delta18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Delta18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Delta18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.

Amino Acid Sequence↗
PubMed1999

The preprotein translocase of the mitochondrial inner membrane: function and evolution.

Growing mitochondria acquire most of their proteins by the uptake of mitochondrial preproteins from the cytosol. To mediate this protein import, both mitochondrial membranes contain independent protein transport systems: the Tom machinery in the outer membrane and the Tim machinery in the inner membrane. Transport of proteins across the inner membrane and sorting to the different inner mitochondrial compartments is mediated by several protein complexes which have been identified in the past years. A complex containing the integral membrane proteins Tim17 and Tim23 constitutes the import channel for preproteins containing amino-terminal hydrophilic presequences. This complex is associated with Tim44 which serves as an adaptor protein for the binding of mtHsp70 to the membrane. mtHsp70, a 70 kDa heat shock protein of the mitochondrial matrix, drives the ATP-dependent import reaction of the processed preprotein after cleavage of the presequence. Preproteins containing internal targeting information are imported by a separate import machinery, which consists of the intermembrane-space proteins Tim9, Tim10, and Tim12, and the inner membrane proteins Tim22 and Tim54. The proteins Tim17, Tim22, and Tim23 have in common a similar topology in the membrane and a homologous amino acid sequence. Moreover, they show a sequence similarity to OEP16, a channel-forming amino acid transporter in the outer envelope of chloroplasts, and to LivH, a component of a prokaryotic amino acid permease, defining a new PRAT-family of preprotein and amino acid transporters.

Adenosine Triphosphatases↗
PubMed1999

Organisation and management of the first clinical trial of BNCT in Europe (EORTC protocol 11961).EORTC BNCT study group.

Boron Neutron Capture Therapy is based on the ability of the isotope 10B to capture thermal neutrons and to disintegrate instantaneously producing high LET particles. The only neutron beam available in Europe for such a treatment is based at the European High Flux Reactor HFR at Petten (The Netherlands). The European Commission, owners of the reactor, decided that the potential benefit of the facility should be opened to all European citizens and therefore insisted on a multinational approach to perform the first clinical trial in Europe on BNCT. This precondition had to be respected as well as the national laws and regulations. Together with the Dutch authorities actions were undertaken to overcome the obvious legal problems. Furthermore, the clinical trial at Petten takes place in a nuclear research reactor, which apart from being conducted in a non-hospital environment, is per se known to be dangerous. It was therefore of the utmost importance that special attention is given to safety, beyond normal rules, and to the training of staff. In itself, the trial is an unusual Phase I study, introducing a new drug with a new irradiation modality, with really an unknown dose-effect relationship. This trial must follow optimal procedures, which underscore the quality and qualified manner of performance.

Boron Neutron Capture Therapy↗
PubMed1999

Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway.

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23-Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22-Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23-Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.

Amino Acid Sequence↗
PubMed1998

The yeast mitochondrial intermembrane space: purification and analysis of two distinct fractions.

We have developed a protocol for the sequential release of the intermembrane space (IMS) content of Saccharomyces cerevisiae mitochondria. Two distinct fractions were obtained: a soluble IMS with cytochrome b2 as key marker and a salt-extractable IMS with cytochrome c as key marker. The identity of several proteins was determined by amino-terminal amino acid sequencing. The IMS fractions were devoid of contaminations from cytosol and mitochondrial outer and inner membranes. By subtraction analysis, the protein profiles of soluble and salt-extractable IMS fractions were depleted of contaminating bands derived from matrix proteins. The fractionation method will provide the basis for the further analysis of IMS proteins and characterization of their functions in bioenergetics, mitochondrial biogenesis, and regulatory processes.

Cell Fractionation↗
PubMed1998

Separation of structural and dynamic functions of the mitochondrial translocase: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins.

The essential gene TIM44 encodes a subunit of the inner mitochondrial membrane preprotein translocase that forms a complex with the matrix heat-shock protein Hsp70. The specific role of Tim44 in protein import has not yet been defined because of the lack of means to block its function. Here we report on a Saccharomyces cerevisiae mutant allele of TIM44 that allows selective and efficient inactivation of Tim44 in organello. Surprisingly, the mutant mitochondria are still able to import preproteins. The import rate is only reduced by approximately 30% compared with wild-type as long as the preproteins do not carry stably folded domains. Moreover, the number of import sites is not reduced. However, the mutant mitochondria are strongly impaired in pulling folded domains of preproteins close to the outer membrane and in promoting their unfolding. Our results demonstrate that Tim44 is not an essential structural component of the import channel, but is crucial for import of folded domains. We suggest that the concerted action of Tim44 and mtHsp70 drives unfolding of preproteins and accelerates translocation of loosely folded preproteins. While mtHsp70 is essential for import of both tightly and loosly folded preproteins, Tim44 plays a more specialized role in translocation of tightly folded domains.

Adenosine Triphosphatases↗
PubMed1998

The import route of ADP/ATP carrier into mitochondria separates from the general import pathway of cleavable preproteins at the trans side of the outer membrane.

The ADP/ATP carrier (AAC) of the mitochondrial inner membrane is synthesized in the cytosol without a cleavable presequence. The preprotein preferentially binds to the mitochondrial surface receptor Tom70 and joins the import pathway of presequence-carrying preproteins at the cis side of the outer membrane. Little is known about the translocation of the AAC across the outer membrane and where its import route separates from that of cleavable preproteins. Here we have characterized a translocation intermediate of AAC during transfer across the outer membrane. The major portion of the preprotein is exposed to the intermembrane space, while a short segment is still accessible to externally added protease. This intermediate can be quantitatively chased to the fully imported form in the inner membrane. Its accumulation depends on Tom7, but not on the intermembrane space domain of Tom22 in contrast to cleavable preproteins. Moreover, opening of the intermembrane space inhibits the import of AAC, but not that of cleavable preproteins into mitoplasts. We conclude that the import route of AAC diverges from the general import pathway of cleavable preproteins already at the trans side of the outer membrane.

Carrier Proteins↗
PubMed1997

The sorting route of cytochrome b2 branches from the general mitochondrial import pathway at the preprotein translocase of the inner membrane.

Cytochrome b2 is synthesized in the cytosol with a bipartite presequence. The first part of the presequence targets the protein to mitochondria and mediates translocation into the mitochondrial matrix compartment; the second part contains the sorting signal that is required for delivery of the protein to the intermembrane space. The localization of the structures that recognize the sorting signal is unclear. Here we show that upon import in vivo, the sorting signal of cytochrome b2 causes an early divergence from the general matrix import pathway and thereby prevents translocation of a folded C-terminal domain into mitochondria. By co-immunoprecipitations we find that translocation intermediates of cytochrome b2 are associated with Tim23, a component of the inner membrane protein import machinery. Cytochrome b2 constructs with an alteration in the sorting signal are mistargeted to the matrix of wild-type mitochondria. In mitochondria containing a mutant form of Tim23, however, the translocation of the altered sorting signal across the inner membrane is inhibited, and cytochrome b2 is correctly sorted to the intermembrane space. We suggest that the sorting signal of cytochrome b2 is recognized within the inner membrane in close vicinity to Tim23.

Biological Transport↗
PubMed1997

The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44.

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70-Tim44 driving system. By blue native electrophoresis, we identify an approximately 90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence-containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.

Amino Acid Sequence↗
PubMed1997

The chaperonin cycle cannot substitute for prolyl isomerase activity, but GroEL alone promotes productive folding of a cyclophilin-sensitive substrate to a cyclophilin-resistant form.

The chaperonin GroEL and the peptidyl-prolyl cis-trans isomerase cyclophilin are major representatives of two distinct cellular systems that help proteins to adopt their native three-dimensional structure: molecular chaperones and folding catalysts. Little is known about whether and how these proteins cooperate in protein folding. In this study, we have examined the action of GroEL and cyclophilin on a substrate protein in two distinct prolyl isomerization states. Our results indicate that: (i) GroEL binds the same substrate in different prolyl isomerization states. (ii) GroEL-ES does not promote prolyl isomerizations, but even retards isomerizations. (iii) Cyclophilin cannot promote the correct isomerization of prolyl bonds of a GroEL-bound substrate, but acts sequentially after release of the substrate from GroEL. (iv) A denatured substrate with all-native prolyl bonds is delayed in folding by cyclophilin due to isomerization to non-native prolyl bonds; a substrate that has proceeded in folding beyond a stage where it can be bound by GroEL is still sensitive to cyclophilin. (v) If a denatured cyclophilin-sensitive substrate is first bound to GroEL, however, productive folding to a cyclophilin-resistant form can be promoted, even without GroES. We conclude that GroEL and cyclophilin act sequentially and exert complementary functions in protein folding.

Amino Acid Isomerases↗
PubMed1997

Multiple interactions of components mediating preprotein translocation across the inner mitochondrial membrane.

The protein transport machinery of the inner mitochondrial membrane contains three essential Tim proteins. Tim17 and Tim23 are thought to build a preprotein translocation channel, while Tim44 transiently interacts with the matrix heat shock protein Hsp70 to form an ATP-driven import motor. For this report we characterized the biogenesis and interactions of Tim proteins. (i) Import of the precursor of Tim44 into the inner membrane requires mtHsp70, whereas import and inner membrane integration of the precursors of Tim17 and Tim23 are independent of functional mtHsp70. (ii) Tim17 efficiently associates with Tim23 and mtHsp70, but only weakly with Tim44. (iii) Depletion of Tim44 does not affect the co-precipitation of Tim17 with antibodies directed against mtHsp70. (iv) Tim23 associates with both Tim44 and Tim17, suggesting the presence of two Tim23 pools in the inner membrane, a Tim44-Tim23-containing sub-complex and a Tim23-Tim17-containing sub-complex. (v) The association of mtHsp70 with the Tim23-Tim17 sub-complex is ATP sensitive and can be distinguished from the mtHsp70-Tim44 interaction by the differential influence of an amino acid substitution in mtHsp70. (vi) Genetic evidence, suppression of the protein import defect of a tim17 yeast mutant by overexpression of mtHsp70 and synthetic lethality of conditional mutants in the genes of Tim17 and mtHsp70, supports a functional interaction of mtHsp70 with Tim17. We conclude that the protein transport machinery of the mitochondrial inner membrane consists of dynamically interacting sub-complexes, each of which transiently binds mtHsp70.

Adenosine Triphosphate↗
PubMed1996

Covalent attachment of FAD derivatives to a fusion protein consisting of 6-hydroxy-D-nicotine oxidase and a mitochondrial presequence. Folding, enzyme activity, and import of the modified protein into yeast mitochondria.

Autoflavinylation of 6-hydroxy-D-nicotine oxidase (6-HDNO) was successfully employed to modify the protein covalently with FAD derivatives. The model compounds N6-(2-aminoethyl)-FAD and N6-(6-carboxyhexyl)-FAD were spontaneously bound to a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa F0-ATPase subunit 9 (Su9) attached to 6-HDNO. When translated in the rabbit reticulocyte lysate, Su9-6-HDNO was in the trypsin-sensitive apoenzyme form; when translated in the presence of flavins it adopted a trypsin-resistant conformation characteristic of the 6-HDNO holoenzyme. With flavin derivatives, Su9-6-HDNO exhibited approximately 50% of the 6-HDNO activity observed with FAD. The covalently modified Su9-6-HDNO was imported into Saccharomyces cerevisiae mitochondria with an efficiency equal to that of the apoenzyme. Apparently the increase in size and charge of the FAD moiety did not hamper translocation across the mitochondrial membranes. Yeast mutant ssc1-2 mitochondria deficient in mtHsp70 unfoldase activity imported the flavinylated Su9-6-HDNO protein. In mutant ssc1-3 mitochondria deficient in both mtHsp70 unfoldase and translocase activity Su9-6-HDNO was trapped as translocation intermediate; the Su9 presequence was passed to the matrix where it was proteolytically cleaved by the mitochondrial processing peptidase; (MPP); the translocation-arrested 6-HDNO moiety adopted a trypsin-sensitive conformation. Our results indicate that unfolding of the FAD-stabilized flavin-binding domain of 6-HDNO in passage through the mitochondrial general insertion pore does not require the activity of mtHsp70.

Animals↗
PubMed1996

The preprotein translocase of the inner mitochondrial membrane: evolutionary conservation of targeting and assembly of Tim17.

The preprotein translocase of the inner mitochondrial membrane has only been described in Saccharomyces cerevisiae to date. We report that the essential subunit Tim17 is highly conserved in evolution. The targeting and assembly of yeast Tim17 as well as that of human and Drosophila melanogaster Tim17 were characterized with isolated yeast mitochondria. Targeting signals in the mature protein direct the Tim17 precursors to the receptor Tom70 on the mitochondrial surface. In a membrane potential-dependent step the precursors insert into the inner membrane, adopt a characteristic topology and assemble with Tim23. The mechanisms of targeting and assembly were indistinguishable between the Tim17s from distinct organisms, indicating a high evolutionary conservation.

Adenosine Triphosphatases↗
PubMed1996

Differential requirement for the mitochondrial Hsp70-Tim44 complex in unfolding and translocation of preproteins.

The mitochondrial heat shock protein Hsp70 is essential for import of nuclear-encoded proteins, involved in both unfolding and membrane translocation of preproteins. mtHsp70 interacts reversibly with Tim44 of the mitochondrial inner membrane, yet the role of this interaction is unknown. We analysed this role by using two yeast mutants of mtHsp70 that differentially influenced its interaction with Tim44. One mutant mtHsp70 (Ssc1-2p) efficiently bound preproteins, but did not show a detectable complex formation with Tim44; the mitochondria imported loosely folded preproteins with wild-type kinetics, yet were impaired in unfolding of preproteins. The other mutant Hsp70 (Ssc1-3p') bound both Tim44 and preproteins, but the mitochondria did not import folded polypeptides and were impaired in import of unfolded preproteins; Ssc1-3p' was defective in its ATPase domain and did not undergo a nucleotide-dependent conformational change, resulting in permanent binding to Tim44. The following conclusions are suggested. (i) The import of loosely folded polypeptides (translocase function of mtHsp70) does not depend on formation of a detectable Hsp70-Tim44 complex. Two explanations are possible: a trapping mechanism by soluble mtHsp70, or a weak/very transient interaction of Ssc1-2p with Tim44 that leads to a weak force generation sufficient for import of loosely folded, but not folded, polypeptides. (ii) Import of folded preproteins (unfoldase function of mtHsp70) involves a reversible nucleotide-dependent interaction of mtHsp70 with Tim44, including a conformational change in mtHsp70. This is consistent with a model that the dynamic interaction of mtHsp70 with Tim44 generates a pulling force on preproteins which supports unfolding during translocation.

Adenine Nucleotides↗
PubMed1996

Yeast mitochondria lacking the phosphate carrier/p32 are blocked in phosphate transport but can import preproteins after regeneration of a membrane potential.

Two different functions have been proposed for the phosphate carrier protein/p32 of Saccharomyces cerevisiae mitochondria: transport of phosphate and requirement for import of precursor proteins into mitochondria. We characterized a yeast mutant lacking the gene for the phosphate carrier/p32 and found both a block in the import of phosphate and a strong reduction in the import of preproteins transported to the mitochondrial inner membrane and matrix. Binding of preproteins to the surface of mutant mitochondria and import of outer membrane proteins were not inhibited, indicating that the inhibition of protein import occurred after the recognition step at the outer membrane. The membrane potential across the inner membrane of the mutant mitochondria was strongly reduced. Restoration of the membrane potential restored preprotein import but did not affect the block of phosphate transport of the mutant mitochondria. We conclude that the inhibition of protein import into mitochondria lacking the phosphate carrier/p32 is indirectly caused by a reduction of the mitochondrial membrane potential (delta(gamma)), and we propose a model that the reduction of delta(psi) is due to the defective phosphate import, suggesting that phosphate transport is the primary function of the phosphate carrier/p32.

Adenosine Triphosphate↗
PubMed1996

[Quality assurance in roentgen diagnosis. Comparison of constancy test methods according to DIN and IEC and practical consequences in Germany].

Since 1987 constancy tests for X-ray imaging equipment are required by the German Röntgenordinance and are performed according to the DIN 6868 standards. On an international level, standards for these tests have been developed by the IEC (International Electrotechnical Commission, IEC 1223 series); however, these standards have so far not been applied in nation-wide quality programmes in any country. Typical aspects of both, DIN and IEC standards, are demonstrated for direct radiography and indirect radioscopy and indirect radiography as examples. DIN standards optimise test procedures for minimum effort and cost efficiency designed for execution even in small hospitals and practices by the users. For each type of x-ray equipment tests are performed with only one universal test device, which is precisely specified and independent of local conditions. Oplimisation is misation is missing for IEC test procedures; they are designed for more performance characteristics and need for each x-ray equipment more individual test devices, which are mostly open for local choice and need a considerably higher effort in respect of time consumption and test films compared to DIN. Adoption of the IEC standards for the constancy tests in Germany would involve considerable financial and organisational effort which is probably not counterbalanced by an increased benefit.

Equipment Safety↗
PubMed1995

The mitochondrial ClpB homolog Hsp78 cooperates with matrix Hsp70 in maintenance of mitochondrial function.

The mitochondrial heat shock protein Hsp78 is a member of the Hsp104/Clp family with unknown function. Saccharomyces cerevisiae deletion mutants of HSP78 show wild-type like growth. We report that deletion of the HSP78 gene in yeast strains with point mutations in the SSC1 gene (encoding matrix Hsp70) led to loss of mitochondrial DNA, indicating that at least one of the heat shock proteins Hsp78 and mt-Hsp70 is needed to maintain a rho+ state of the mitochondrial genome. Mitochondria isolated from these double mutants had a strongly reduced membrane potential, explaining defects in the rate of preprotein import. The lack of Hsp78 led to aggregation of the mutant mt-Hsp70 while other matrix chaperones stayed soluble. We conclude that Hsp78 is required to keep mutant forms of mt-Hsp70 soluble and suggest a cooperation of Hsp78 and mt-Hsp70 in maintenance of essential mitochondrial functions.

Cell Division↗