PubMed Health⌕ Search

Biomedical subjects

R Lill

Publications and source records attributed to R Lill.

At least 55 records · Page 3Linked to original sources
PubMed1997

The ABC transporter Atm1p is required for mitochondrial iron homeostasis.

The function of the ABC transporter Atm1p located in the mitochondrial inner membrane is not yet known. To study its cellular role, we analyzed a mutant in which ATM1 was disrupted. Delta atm1 cells are deficient in the holoforms, but not the apoforms of heme-carrying proteins both within and outside mitochondria, yet both synthesis and transport of heme are functional. Delta atm1 cells are hypersensitive for growth in the presence of oxidative reagents, and they contain increased levels of the antioxidant glutathione, in particular of its oxidized form. Mitochondria deficient in Atm1p accumulate 30-fold higher levels of free iron as compared to wild-type organelles, i.e. three-fold more than mitochondria deficient in frataxin, the protein mutated in Friedreich's ataxia. The increased mitochondrial iron content may be causative of the oxidative damage of heme-containing proteins in delta atm1 cells. Our data assign an important function to Atm1p in mitochondrial iron homeostasis.

ATP-Binding Cassette Transporters↗
PubMed1997

Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence.

During preprotein transport across the mitochondrial outer membrane, the N-terminal presequence initially binds to a surface-exposed site, termed cis site, of the protein translocation complex of this membrane (the TOM complex). The presequence then moves into the translocation pore and becomes exposed at the intermembrane space side. Membrane passage is driven by specific interaction of the presequence with the trans site. We have used chemical cross-linking to identify components in the vicinity of the translocating presequence. Preproteins bound to the surface-exposed cis site can be cross-linked via their N-terminal presequence to Tom20 and Tom22, demonstrating their direct association with this part of the preprotein. In addition, the presequence establishes an early contact to Tom40, a membrane-embedded protein of the TOM complex. Upon further entry of the preprotein into the translocation pore, the presequence loses its contact with Tom20/Tom22, but remains in firm association with Tom40. Our study suggests that Tom40 plays an important function in guiding the presequence of a preprotein across the mitochondrial outer membrane. We propose that Tom40 forms a major part of the trans presequence binding site.

Animals↗
PubMed1997

Phospholipid composition of highly purified mitochondrial outer membranes of rat liver and Neurospora crassa. Is cardiolipin present in the mitochondrial outer membrane?

Isolated mitochondrial outer membrane vesicles (OMV) are a suitable system for studying various functions of the mitochondrial outer membrane. For studies on mitochondrial lipid import as well as for studies on the role of lipids in processes occurring in the outer membrane, knowledge of the phospholipid composition of the outer membrane is indispensable. Recently, a mild subfractionation procedure was described for the isolation of highly purified OMV from mitochondria of Neurospora crassa (Mayer, A., Lill, R. and Neupert, W. (1993) J. Cell Biol. 121, 1233-1243). This procedure, which consists of swelling and mechanical disruption of mitochondria followed by two steps of sucrose density gradient centrifugation, was adapted for the isolation of OMV from rat liver mitochondria. Using the appropriate enzyme markers it is shown that the resulting OMV are obtained in a yield of 25%, and that their purity is superior to that of previous OMV preparations. Analysis of the phospholipid composition of the OMV showed that phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol are the major phospholipid constituents, and that cardiolipin is only present in trace amounts. The phospholipid composition is very similar to that of the highly purified OMV from mitochondria of Neurospora crassa, although the latter still contain a small amount of cardiolipin.

Animals↗
PubMed1996

Heme binding to a conserved Cys-Pro-Val motif is crucial for the catalytic function of mitochondrial heme lyases.

Covalent attachment of heme to the apoforms of mitochondrial cytochromes c and c1 requires the activity of cytochrome c heme lyase (CCHL) and cytochrome c1 heme lyase (CC1HL), respectively. The two enzymes differ in their cytochrome specificity, but they are related in sequence, and both contain conserved Cys-Pro-Val (CPV) motifs. By using various in vitro assays we investigated whether heme can bind directly to heme lyases and whether the CPV motif may be involved in heme binding. Heme stabilized CC1HL, as a model protein, in a folded, protease-resistant conformation, stimulated the refolding of CC1HL after urea denaturation, and inhibited the import of the CC1HL precursor into mitochondria. These effects were not observed with a point mutant, CC1HLSPV, in which cysteine was replaced by serine, and with CC1HLDeltaCPV, in which the motif was deleted. These results show that heme lyases can bind heme directly, and they identify the CPV sequence as a structural element important for this interaction. The phenotype of a yeast mutant expressing CC1HLSPV is in good agreement with such a role of the CPV motif. The mutant cells accumulate the heme-free intermediate form of cytochrome c1 and display a severe deficiency in the holo form. We suggest that the CPV motif forms a crucial part of the substrate binding site for heme.

Amino Acid Sequence↗
PubMed1996

Mitochondrial and cytosolic branched-chain amino acid transaminases from yeast, homologs of the myc oncogene-regulated Eca39 protein.

We have isolated a high copy suppressor of a temperature-sensitive mutation in ATM1, which codes for an ABC transporter of Saccharomyces cerevisiae mitochondria. The suppressor, termed BAT1, encodes a protein of 393 amino acid residues with an NH2-terminal extension that directs Bat1p to the mitochondrial matrix. A highly homologous protein, Bat2p, of 376 amino acid residues was found in the cytosol. Both Bat proteins show striking similarity to the mammalian protein Eca39, which is one of the few known targets of the myc oncogene. Deletion of a single BAT gene did not impair growth of yeast cells. In contrast, deletion of both genes resulted in an auxotrophy for branched-chain amino acids (Ile, Leu, and Val) and in a severe growth reduction on glucose-containing media, even after supply of these amino acids. Mitochondria and cytosol isolated from bat1 and bat2 deletion mutants, respectively, contained largely reduced activities for the conversion of branched-chain 2-ketoacids to their corresponding amino acids. Thus, the Bat proteins represent the first known isoforms of yeast branched-chain amino acid transaminases. The severe growth defect of the double deletion mutant observed even in the presence of branched-chain amino acids suggests that the Bat proteins, in addition to the supply of these amino acids, perform another important function in the cell.

Amino Acid Sequence↗
PubMed1996

Tom71, a novel homologue of the mitochondrial preprotein receptor Tom70.

The protein Tom71 is encoded by the open reading frame YHR117w (yeast chromosome VIII) and shares 53% amino acid sequence identity with Tom70, a protein import receptor of the mitochondrial outer membrane. We investigated the cellular function of Tom71 and addressed the question of whether Tom71 and Tom70 fulfill similar functions. Like Tom70, Tom71 is anchored to the mitochondrial outer membrane via its N terminus, thereby exposing a large C-terminal domain to the cytosol. Tom71 is associated with the protein import complex of this membrane and can be cross-linked to a protein with a molecular mass of 30-35 kDa. Disruption of the TOM71 gene does not reduce cell growth, except on nonfermentable carbon sources at elevated temperatures. Deletion of both the TOM71 and TOM70 genes does not acerbate this growth defect. In vitro import studies demonstrated no functional requirement for Tom71 in the import of several preproteins destined for each of the mitochondrial subcompartments. In particular, the import of Tom70-dependent preproteins is minimally affected by the deletion of Tom71, irrespective of the presence or absence of the Tom70 receptor. Thus, despite their strikingly similar biochemical properties, Tom71 and Tom70 do not perform identical functions.

Amino Acid Sequence↗
PubMed1996

Role of the N- and C-termini of porin in import into the outer membrane of Neurospora mitochondria.

The signals for targeting and assembly of porin, a protein of the mitochondrial outer membrane, have not been clearly defined. Targeting information has been hypothesized to be contained in the N-terminus, which may form an amphipathic alpha-helix, and in the C-terminal portion of the protein. Here, the role of the extreme N- and C-termini of porin from Neurospora crassa in its import into the mitochondrial outer membrane was investigated. Deletion mutants were constructed which lacked the N-terminal 12 or 20 residues or the C-terminal 15 residues. The porins truncated at their N-termini were imported in a receptor-dependent manner into the outer membrane of isolated mitochondria. When integrated into the outer membrane, these preproteins displayed an increased sensitivity to protease as compared to wild-type porin. In contrast, mutant porin truncated at its C-terminus did not acquire protease resistance upon incubation with mitochondria. Thus, unlike most other mitochondrial preproteins, porin appears to contain important targeting and/or assembly information at its C-terminus, rather than at the N-terminus.

Amino Acid Sequence↗
PubMed1996

The role of the N and C termini of recombinant Neurospora mitochondrial porin in channel formation and voltage-dependent gating.

To investigate the role of the N and C termini in channel function and voltage-dependent gating of mitochondrial porin, we expressed wild-type and mutant porins from Neurospora crassa as His-tag fusion products in Escherichia coli. Large quantities of the proteins were purified by chromatography across a nickle-nitrilotriacetic acid-agarose column under denaturing conditions. The purified His-tagged wild-type protein could be functionally reconstituted in the presence of detergent and sterol and behaved in black lipid bilayer membranes indistinguishably from native porin isolated from Neurospora crassa mitochondria. Mutants of porin lacking part of the N terminus (DeltaN2-12porin, DeltaN3-20porin), part of the C terminus (DeltaC269-283porin), or both (DeltaN2-12/DeltaC269-283porin) also showed channel forming activity. The mutant porin lacking the C terminus had a smaller single channel conductance than the wild-type protein, but its other biophysical properties were identical. DeltaN2-12porin and DeltaN3-20porin formed noisy channels with decreased channel stability. These channels were still voltage-dependent. DeltaN2-12/DeltaC269-283porin lost channel stability and had altered gating characteristics. These results are discussed with respect to different models that have been proposed in the literature for the structure of mitochondrial porin channels.

Amino Acid Sequence↗
PubMed1996

Mechanisms of protein import across the mitochondrial outer membrane.

Mitochondria import the majority of their proteins from the cytosol. At the mitochondrial outer membrane, import is initiated through a series of reactions, which include preprotein recognition, unfolding, insertion and translocation. These processes are facilitated by a multisubunit complex, the TOM complex. Specific roles can now be assigned to several components of this complex. Although the import machinery of the outer membrane can insert and translocate a few proteins on its own, completion of translocation o f most preproteins is dependent upon coupling to both the membrane potential and mt-Hsp70/ATP-driven transport across the inner membrane, mediated by the TIM complex.

Journal Article↗
PubMed1996

Biogenesis of mitochondrial proteins.

Numerous components have been identified that participate at various stages in the biogenesis of mitochondria. For many of these components, their specific functions have recently been defined through detailed investigations of the molecular mechanisms underlying protein targeting, translocation across the mitochondrial outer and inner membranes, membrane insertion, suborganellar sorting, and protein folding.

Biological Transport↗
PubMed1996

Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria.

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix and inner membrane were imported less efficiently. The reduced import was not due to impaired interaction of presequences with their specific binding site on the trans side of the outer membrane. Rather, the IMS domain of Tom22 appears to slightly enhance the efficiency of the transfer of these preproteins to the import machinery of the inner membrane.

Base Sequence↗
PubMed1995

Biogenesis of mitochondrial heme lyases in yeast. Import and folding in the intermembrane space.

Heme lyases are components of the mitochondrial intermembrane space facilitating the covalent attachment of heme to the apoforms of c-type cytochromes. The precursors of heme lyases are synthesized in the cytosol without the typical N-terminal mitochondrial targeting signal. Here, we have analyzed the mode of import and folding of the two heme lyases of the yeast Saccharomyces cerevisiae, namely of cytochrome c heme lyase and of cytochrome c1 heme lyase. For transport into mitochondria, both proteins use the general protein import machinery of the outer membrane. Import occurred independently of a membrane potential, delta psi, across the inner membrane and ATP in the matrix space, suggesting that the inner membrane is not required for transport along this direct sorting pathway. The presence of a large folded domain in heme lyases was utilized to study their folding in the intermembrane space. Formation of this domain occurred at the same rate as import, indicating that heme lyases fold either during or immediately after their transfer across the membrane. Folding was not affected by depletion of ATP and delta psi or by inhibitors of peptidylprolyl cis-trans isomerases, i.e. it does not involve homologs of known folding factors (like Hsp60 and Hsp70). The energy derived from folding cannot be regarded as a major driving force for import, since the folded domain could be imported into mitochondria with the same efficiency as the intact protein. We conclude that protein folding in the intermembrane space obeys principles different from those established for other subcellular compartments.

Adenosine Triphosphate↗
PubMed1995

MOM22 is a receptor for mitochondrial targeting sequences and cooperates with MOM19.

Recognition of targeting signals is a crucial step in protein sorting within the cell. So far, only a few components capable of deciphering targeting signals have been identified, and insights into the chemical nature of the interaction between the signals and their receptors are scarce. Using highly purified mitochondrial outer membrane vesicles, we demonstrate that MOM22 and MOM19, components of the protein import complex of the outer membrane, bind preproteins at the mitochondrial surface in a reversible fashion. Interaction specifically and directly occurs with the N-terminal presequence and is abolished after inactivation of either MOM22 or MOM19. Binding is salt sensitive, suggesting that recognition involves electrostatic forces between the positive charges of the presequence and the acidic cytosolic domain of MOM22. MOM19 and MOM22 can be cross-linked with high efficiency. We propose that the two proteins form a complex which functions as the presequence receptor at the mitochondrial surface and facilitates the movement of preproteins into the translocation pore.

Animals↗
PubMed1995

Translocation of apocytochrome c across the outer membrane of mitochondria.

Apocytochrome c follows a unique pathway into mitochondria. Import does not require the general protein translocation machinery, protease-sensitive components of the outer membrane, or a membrane potential across the inner membrane. We investigated the membrane binding and translocation steps of the import reaction using purified outer membrane vesicles (OMV) from Neurospora crassa mitochondria. OMV specifically bound, but did not import apocytochrome c. If, however, specific antibodies were enclosed inside OMV, apocytochrome c was accumulated in soluble form in the lumen. Import was reversible, since apocytochrome c became accessible to external protease after release from the antibodies. Thus, OMV are competent of translocating apocytochrome c into their lumen, but lack a binding partner which traps the apoprotein. In intact mitochondria, cytochrome c heme lyase (CCHL), a peripheral protein of the inner membrane, serves such a function by stably associating with apocytochrome c in a complex which is detectable by co-immunoprecipitation. We suggest a model for the import mechanism of apocytochrome c in which the apoprotein specifically associates with and reversibly passes across the outer membrane. Translocation is rendered unidirectional by stable association with CCHL which serves as a "trans side receptor." Finally, heme is attached by CCHL and the holoprotein folds into its native structure.

Apoproteins↗
PubMed1995

'Sheltered disruption' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex.

MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa. Using the newly developed procedure of 'sheltered disruption', we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom-22 gene and the other with a wild-type allele. Homokaryons bearing the mom-22 disruption could not be isolated, suggesting that mom-22 is an essential gene. The mutant nucleus can be forced to predominate in the heterokaryon through the use of specific nutritional and inhibitor resistance markers. Cultivation of the heterokaryon under conditions favoring the mutant nucleus resulted in selective depletion of MOM22. MOM22-depleted cells did not grow and contained mitochondria with an altered morphology and protein composition. Protein import into isolated, MOM22-depleted mitochondria was abolished for most precursor proteins destined for all subcompartments. In contrast, precursors of MOM19, MOM22 and MOM72 became inserted normally into the outer membrane, defining a novel MOM22-independent import pathway which remained intact in mutant mitochondria. Furthermore, the specific binding of the ADP/ATP carrier to the outer membrane was unaffected, but subsequent transport across the outer membrane did not occur. Our data show that MOM22 is an essential component of Neurospora cells specifically required for the biogenesis of mitochondria.

Biological Transport↗
PubMed1995

Mitochondrial protein import: reversible binding of the presequence at the trans side of the outer membrane drives partial translocation and unfolding.

The mechanism of translocation of matrix-targeted, cleavable preproteins across the mitochondrial outer membrane was studied using purified outer membrane vesicles. The N-terminal presequence interacts in a sequential and reversible fashion with two specific binding sites. The first one is provided by protease-sensitive receptors on the surface of the membrane (cis site); the second one is located at the inner face of the outer membrane (trans site). Binding to the trans site drives translocation of the N-terminal portion of the preprotein and, at the same time, unfolding of its mature part. We suggest that the outer membrane protein import machinery forms a translocation channel that permits reversible sliding of preproteins and prevents their lateral aggregation in the membrane. Although translocation can be initiated by the outer membrane, its completion requires coupling to the energetic system of the inner membrane. Our data assign an essential role to the presequence, not only for efficient targeting, but also for the translocation step.

Binding Sites↗
PubMed1995

Identification and initial characterization of the cytosolic protein Ycr77p.

The nucleotide sequence of yeast chromosome III encompassing the previously the previously described open reading frames (ORFs) YCR80w, YCR77c and YCR78c (Oliver et al., 1992) has been updated. In the corrected sequence, these ORFs are replaced by two new ORFs, YCR80w (453 bp) and YCR77c (2391 bp). In addition, the orientation of Ycr79c is reversed to give ORF Ycr79w, which has an unaltered nt sequence. The predicted translation products do not exhibit significant homology to known proteins. ORF Ycr77p encodes an 88 kDa, cytosolic protein. A fraction of the protein is associated with small membranous structures in a salt-sensitive fashion. Initial characterization revealed that the protein is not essential for yeast viability, growth on non-fermentable carbon sources, mating and sporulation. The chromosome III DNA sequence that was used for the analysis has the Accession Number X59720 in the GenBank/EMBL database.

Amino Acid Sequence↗