Cell-Free Synthesis of the Mitochondrial ADP/ATP Carrier Protein of Neurospora crassa

ADP/ATP carrier protein was synthesized in heterologous cell-free systems programmed with Neurospora poly(A)-containing RNA and homologous cell-free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl-tRNA (fMet-tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant. The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence

The moderating effect of brand orientation on inter-firm market orientation and performance

While prior research has shown that market and brand orientation are key contributors to successful business performance, research to date has not fully explored how inter firm collaboration for these two key orientations can enhance business performance. The purpose of the paper is to investigate the relationship between inter-firm market and performance; to test for the moderating role of brand orientation in that relationship. A total of 169 completed pairs of surveys were collected of small and medium enterprises operating internationally in a variety of industries in Switzerland. The results show that inter-firm market and brand orientation are two antecedents of marketing and financial performance. The impact of inter-firm market on marketing and financial performance is significant when the brand orientation is favorable. This study extends previous research by examining the moderating role of brand orientation on inter firm market orientation, which is important, especially for firms wanting to increase their brand reputation by entering into partnerships with other firms. Further research is indicated, to identify the key moderators of the driving force of inter-firm market in relation to business performance and the reason why maintaining a strong brand presence is important in the international marketplace

Import of cytochrome c into mitochondria

The import of cytochrome c into mitochondria can be resolved into a number of discrete steps. Here we report on the covalent attachment of heme to apocytochrome c by the enzyme cytochrome c heme lyase in mitochondria from Neurospora crassa. A new method was developed to measure directly the linkage of heme to apocytochrome c. This method is independent of conformational changes in the protein accompanying heme attachment. Tryptic peptides of [35S]cysteine-labelled apocytochrome c, and of enzymatically formed holocytochrome c, were resolved by reverse-phase HPLC. The cysteine-containing peptide to which heme was attached eluted later than the corresponding peptide from apocytochrome c and could be quantified by counting 35S radioactivity as a measure of holocytochrome c formation. Using this procedure, the covalent attachment of heme to apocytochrome c, which is dependent on the enzyme cytochrome c heme lyase, could be measured. Activity required heme (as hemin) and could be reversibly inhibited by the analogue deuterohemin. Holocytochrome c formation was stimulated 5–10-fold by NADH > NADPH > glutathione and was independent of a potential across the inner mitochondrial membrane. NADH was not required for the binding of apocytochrome c to mitochondria and was not involved in the reduction of the cysteine thiols prior to heme attachment. Holocytochrome c formation was also dependent on a cytosolic factor that was necessary for the heme attaching step of cytochrome c import. The factor was a heat-stable, protease-insensitive, low-molecular-mass component of unknown function. Cytochrome c heme lyase appeared to be a soluble protein located in the mitochondrial intermembrane space and was distinct from the previously identified apocytochrome c binding protein having a similar location. A model is presented in which the covalent attachment of heme by cytochrome c heme lyase also plays an essential role in the import pathway of cytochrome c

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

Processing of Thionin Precursors in Barley Leaves by a Vacuolar Proteinase

Thionins are synthesized as precursors with a signal peptide and a long C-terminal acidic peptide that is post-translationally processed. A fusion protein including the maltose-binding protein from Eschrrichia coli (MalE), thionin DG3 froin barley leaves, and its acidic C-terminal peptide has been used to obtain antibodies that recognize both domains of the precursor. In barley leaf sections. mature thionins accuinulated in the vacuolar content, while the acidic peptide was not detected in any cell fraction. Brefeldin A and inonensin inhibited processing of the precursor but its export from the microsomal fraction was not inhibited. Both purified vacuoles aiid an acid (pH 5.5) extract from leaves processed the fusion protein into a MalE-thionin and an acidic peptide fragment. A 70-kDa proteinase that effected this cleavage was purified froin the acid extract. Processing of the fusion protein by both lysed vacuoles and the purified proteinase was inhibited by Zn2+ and by Cu2+, but not by inhibitors of the previously described vacuolar processing thiol or aspartic proteinases. In vivo processing of the thionin precursor in leaf sections was also inhibited by Zn+, and Cu2+, Variants of the fusion protein with altered processing sites that represented thme of thionin precursors from different taxa were readily processed by the proteinase, whereas changing the polarity of either the C-terminal or N-terminal residues of the processing site prevented cleavage by the proteinase

Mitochondrial precursor proteins are imported through a hydrophilic membrane environment

We have analyzed how translocation intermediates of imported mitochondrial precursor proteins, which span contact sites, interact with the mitochondrial membranes. F1-ATPase subunit β(F1β) was trapped at contact sites by importing it into Neurospora mitochondria in the presence of low levels of nucleoside triphosphates. This F1β translocation intermediate could be extracted from the membranes by treatment with protein denaturants such as alkaline pH or urea. By performing import at low temperatures, the ADP/ATP carrier was accumulated in contact sites of Neurospora mitochondria and cytochrome b2 in contact sites of yeast mitochondria. These translocation intermediates were also extractable from the membranes at alkaline pH. Thus, translocation of precursor proteins across mitochondrial membranes seems to occur through an environment which is accessible to aqueous perturbants. We propose that proteinaceous structures are essential components of a translocation apparatus present in contact sites

Elastic interactions of active cells with soft materials

Anchorage-dependent cells collect information on the mechanical properties of the environment through their contractile machineries and use this information to position and orient themselves. Since the probing process is anisotropic, cellular force patterns during active mechanosensing can be modelled as anisotropic force contraction dipoles. Their build-up depends on the mechanical properties of the environment, including elastic rigidity and prestrain. In a finite sized sample, it also depends on sample geometry and boundary conditions through image strain fields. We discuss the interactions of active cells with an elastic environment and compare it to the case of physical force dipoles. Despite marked differences, both cases can be described in the same theoretical framework. We exactly solve the elastic equations for anisotropic force contraction dipoles in different geometries (full space, halfspace and sphere) and with different boundary conditions. These results are then used to predict optimal position and orientation of mechanosensing cells in soft material.Comment: Revtex, 38 pages, 8 Postscript files included; revised version, accepted for publication in Phys. Rev.