Import of mitochondrial carriers mediated by essential proteins of the intermembrane space.

In order to reach the inner membrane of the mitochondrion, multispanning carrier proteins must cross the aqueous intermembrane space. Two essential proteins of that space, Tim10p and Tim12p, were shown to mediate import of multispanning carriers into the inner membrane. Both proteins formed a complex with the inner membrane protein Tim22p. Tim10p readily dissociated from the complex and was required to transport carrier precursors across the outer membrane; Tim12p was firmly bound to Tim22p and mediated the insertion of carriers into the inner membrane. Neither protein was required for protein import into the other mitochondrial compartments. Both proteins may function as intermembrane space chaperones for the highly insoluble carrier proteins.

Analysis of the sorting signals directing NADH-cytochrome b5 reductase to two locations within yeast mitochondria.

Mitochondrial NADH-cytochrome b5 reductase (Mcr1p) is encoded by a single nuclear gene and imported into two different submitochondrial compartments: the outer membrane and the intermembrane space. We now show that the amino-terminal 47 amino acids suffice to target the Mcr1 protein to both destinations. The first 12 residues of this sequence function as a weak matrix-targeting signal; the remaining residues are mostly hydrophobic and serve as an intramitochondrial sorting signal for the outer membrane and the intermembrane space. A double point mutation within the hydrophobic region of the targeting sequence virtually abolishes the ability of the precursor to be inserted into the outer membrane but increases the efficiency of transport into the intermembrane space. Import of Mcr1p into the intermembrane space requires an electrochemical potential across the inner membrane, as well as ATP in the matrix, and is strongly impaired in mitochondria lacking Tom7p or Tim11p, two components of the translocation machineries in the outer and inner mitochondrial membranes, respectively. These results indicate that intramitochondrial sorting of the Mcr1 protein is mediated by specific interactions between the bipartite targeting sequence and components of both mitochondrial translocation systems.

The mitochondrial processing peptidase behaves as a zinc-metallopeptidase.

The yeast mitochondrial processing peptidase (MPP) and its subunits were purified in Escherichia coli under conditions for which the enzyme retains most of its processing activity in the absence of externally added divalent cation. The holoenzyme exhibited a Km value of 1.35 microM and a Vmax value of 0.25 microM/min and was inhibited by metal chelators in a time-dependent manner. Measurement of the metal content showed that both, MPP and beta-MPP, contained 0.86 and 1.05 atoms of Zn2+ per molecule, respectively. An enzymatically inactive MPP mutant carrying a mutation of the first histidine of the putative metal-ion binding HXXEH motif in beta-MPP retained less than 0.2 atom of Zn2+ per molecule. A metal-free enzyme (apoenzyme) was prepared from the holoenzyme and shown to be devoid of any processing activity. Incubation of the apoenzyme with 50 nM and 500 nM Zn2+ restored 50% and 80% of the processing activity, respectively. However, no reactivation occurred at concentrations of Zn2+ higher than 1 microM. Addition of 500 nM Mn2+ or higher concentrations (up to 50 microM) reactivated only 50% of the processing activity. The holoenzyme was competitively inhibited by molar excess of Zn2+ (Ki of 3.1 microM) but not by molar excess of Mn2+. Taken together, our data suggest that the authentic MPP is a Zn2+ rather than a Mn2+ metallopeptidase.

Interaction of the duplicated segment carried by Clostridium thermocellum cellulases with cellulosome components.

The function of the non-catalytic, duplicated segment found in C. thermocellum cellulases was investigated. Rabbit antibodies reacting with the duplicated segment of endoglucanase CelD cross-reacted with a variety of cellulosome components ranging between 50 and 100 kDa. 125I-labeled forms of CelD and of xylanase XynZ carrying the duplicated segment bound to a set of cellulosome proteins ranging between 66 and 250 kDa, particularly to the 250 kDa SL (or S1) subunit. 125I-labeled forms of CelD and XynZ devoid of the duplicated segment failed to bind to any cellulosome protein. The duplicated segment appears thus to serve to anchor the various cellulosome subunits to the complex by binding to SL, which may be a scaffolding element of the cellulosome.

High activity of inclusion bodies formed in Escherichia coli overproducing Clostridium thermocellum endoglucanase D.

The formation of cytoplasmic inclusion bodies by Escherichia coli overproducing Clostridium thermocellum endoglucanase D (EGD) was investigated. EGD was found in inclusion bodies as a 68 kDa form, whereas the size of the cytoplasmic form was 65 kDa. Upon solubilization with urea followed by dialysis, the 68 kDa form was converted to the 65 kDa species. Proteolysis occurred within the COOH-terminal, reiterated region of the 68 kDa form, which is conserved among most C. thermocellum endoglucanases, but is not required for catalytic activity. The specific activity of the enzyme embedded in inclusion bodies was close to that of the purified protein. Thus, inclusion body formation does not involve denaturation of the catalytic domain of EGD, but, more likely, the participation of the reiterated, conserved region in intermolecular interactions.

Involvement of separate domains of the cellulosomal protein S1 of Clostridium thermocellum in binding to cellulose and in anchoring of catalytic subunits to the cellulosome.

Fragments of the 250 kDa S1 subunit of the Clostridium thermocellum cellulosome were obtained by protease-induced or spontaneous degradation. All detectable fragments, down to a mass of about 30 kDa, retained the ability to bind to 125I-labelled endoglucanase CelD, one of the catalytic subunits of the cellulosome. Several fragments were able to bind both to cellulose and to CelD. However, some fragments that could still bind to CelD did not have the ability to bind to cellulose. Therefore, S1, a putative scaffolding protein of the cellulosome, is likely to carry two separate types of domains, one of which binds to cellulose, while the other type binds to the various catalytic subunits of the complex.

Complementing structural information of modular proteins with small angle neutron scattering and contrast variation.

Many macromolecules in the cell function by forming multi-component assemblies. We have applied the technique of small angle neutron scattering to study a nucleic acid-protein complex and a multi-protein complex. The results illustrate the versatility and applicability of the method to study macromolecular assemblies. The neutron scattering experiments, complementing X-ray solution scattering data, reveal that the conserved catalytic domain of RNase E, an essential ribonuclease in Escherichia coli (E. coli), undergoes a marked conformational change upon binding a 5'monophosphate-RNA substrate analogue. This provides the first evidence in support of an allosteric mechanism that brings about RNA substrate cleavage. Neutron contrast variation of the multi-protein TIM10 complex, a mitochondrial chaperone assembly comprising the subunits Tim9 and Tim10, has been used to determine a low-resolution shape reconstruction of the complex, highlighting the integral subunit organization. It shows characteristic features involving protrusions that could be assigned to the six subunits forming the complex.

Properties conferred on Clostridium thermocellum endoglucanase CelC by grafting the duplicated segment of endoglucanase CelD.

The DNA sequence encoding the duplicated 22 amino acid segment of Clostridium thermocellum endoglucanase CelD was fused to the 3'-terminus of the celC gene encoding C.thermocellum endoglucanase CelC. The presence of the duplicated segment endowed CelC with the capacity to form cytoplasmic inclusion bodies containing active enzyme when the hybrid gene was expressed in Escherichia coli. Inclusion body formation prevented proteolytic cleavage of the duplicated segment. The intact hybrid protein CelC-Cel'D was purified from inclusion bodies and characterized. In contrast to CelC, CelC-Cel'D was able to bind to CipA, a protein acting as a scaffolding component of the C.thermocellum cellulase complex (cellulosome). However, the catalytic properties of CelC-Cel'D were similar to those of CelC. These results suggest that foreign proteins tagged with the duplicated segment could be incorporated into the cellulosome in order to modify the enzymatic properties of the complex. The formation of inclusion bodies by proteins carrying the duplicated segment may also prove a convenient means of purifying cloned gene products that are sensitive to proteolytic degradation.

Membrane protein import in yeast mitochondria.

The protein import pathway that targets proteins to the mitochondrial matrix has been extensively characterized in the past 15 years. Variations of this import pathway account for the sorting of proteins to other compartments as well, but the insertion of integral inner membrane proteins lacking a presequence is mediated by distinct translocation machinery. This consists of a complex of Tim9 and Tim10, two homologous, Zn(2+)-binding proteins that chaperone the passage of the hydrophobic precursor across the aqueous intermembrane space. The precursor is then targeted to another, inner-membrane-bound, complex of at least five subunits that facilitates insertion. Biochemical and genetic experiments have identified the key components of this process; we are now starting to understand the molecular mechanism. This review highlights recent advances in this new membrane protein insertion pathway.

Translocation arrest of an intramitochondrial sorting signal next to Tim11 at the inner-membrane import site.

The import of proteins from the cytosol into the mitochondrial matrix involves the concerted action of two separate import systems: the TOM system in the outer membrane, and the TIM system in the inner membrane. Here we report that the inner-membrane system also sorts proteins to the intermembrane space. Some intermembrane-space proteins, such as cytochromes b2 and c1, are synthesized with a complex pre-sequence consisting of a positively charged matrix targeting signal followed by an uncharged sequence that acts as sorting signal for the intermembrane space. We show that this sorting signal can be efficiently crosslinked to an inner-membrane protein of relative molecular mass 11K after the mature part of the precursor has been sorted to the intermembrane space. The 11K protein, which we term Tim11, is a component of the protein import system in the inner membrane.

Functional reconstitution of the import of the yeast ADP/ATP carrier mediated by the TIM10 complex.

Import of the ADP/ATP carrier (AAC) into mitochondria requires the soluble TIM10 complex to cross the intermembrane space. We report here that Tim9 and Tim10 purified from Escherichia coli can form a complex of the same size as the endogenous complex from yeast mitochondria. This shows that no other mitochondrial protein is required for the formation of the TIM10 complex. Co-expression of both proteins rendered Tim9 more soluble and allowed purification of the reconstituted complex in a single step. Urea/EDTA treatment of recombinant Tim10 allowed its import into tim10-ts mitochondria that lack endogenous Tim10 and cannot import AAC. In this way, we were able to (i) reconstitute the TIM10 complex in the intermembrane space and (ii) restore import of AAC to almost wild-type levels. The reconstituted TIM10 complex not only facilitated passage of AAC across the outer membrane but also ensured its accurate membrane insertion. We conclude that the TIM10 complex can be formed exclusively from Tim9 and Tim10 and that the reconstituted complex efficiently restores AAC import in a strain lacking the TIM10 complex.

Nascent chains: folding and chaperone interaction during elongation on ribosomes.

Monoclonal antibodies that detect folding intermediates in vitro were used to monitor the appearance of folded polypeptide chains during their synthesis on the ribosomes. Nascent immunoreactive chains of the bacteriophage P22 tail-spike protein and of the Escherichia coli beta 2 subunit of tryptophan-synthase were thus identified, suggesting that they can fold on the ribosomes. Moreover, the immunoreactivity of ribosome-bound tryptophan-synthase beta-chains of intermediate lengths was shown to appear with no detectable delay compared to their synthesis. This suggested that beta-chains start folding during their elongation on the ribosomes. However, newly synthesized incomplete beta-chains were shown to interact with chaperones while still bound to the ribosome. Because of the peculiar properties of the epitope recognized by the anti-tryptophan-synthase monoclonal antibody used, it could not be concluded whether the immunoreactivity of the nascent beta-chains resulted from their ability to fold cotranslationally or from their association with chaperones which might maintain them in an unfolded, immunoreactive state.

Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins.

Tim10p, a protein of the yeast mitochondrial intermembrane space, was shown previously to be essential for the import of multispanning carrier proteins from the cytoplasm into the inner membrane. We now identify Tim9p, another essential component of this import pathway. Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. Tim9p and Tim10p co-purify in successive chromatographic fractionations and co-immunoprecipitated with each other. Tim9p can be cross-linked to a partly translocated carrier protein. A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p. The sequence of Tim9p is 25% identical to that of Tim10p and Tim12p. A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect of tim10-1 and tim12-1 mutants. Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.