Transport of ADP/ATP carrier into mitochondria

Precursor to ADP/ATP carrier synthesized in vitro is transferred into isolated mitochondria to a protease-resistant location. This process requires an electrical potential across the inner membrane. We show now that precursor imported in a cell-free system exhibits the same resistance to protease as the mature endogenous carrier. Furthermore, transferred protein, but not receptor-associated precursor, binds carboxy-atractyloside, a specific inhibitor of the mature carrier and can be isolated by the purification procedure for the mature carrier. At least 70% of the precursor associated with mitochondria in the presence of a membrane potential acquires this functional characteristic. Finally, the binding of carboxyatractyloside can be modulated by treatment of the imported protein with sulfhydryl reagents in a manner indistinguishable from the authentic carrier protein. We conclude that import in vitro leads to correct assembly and orientation of the ADP/ATP carrier in the mitochondria

Distinct steps in the import of ADP/ATP carrier into mitochondria

Transport of the precursor to the ADP/ATP carrier from the cytosol into the mitochondrial inner membrane was resolved into several consecutive steps. The precursor protein was trapped at distinct stages of the import pathway and subsequently chased to the mature form. In a first reaction, the precursor interacts with a protease-sensitive component on the mitochondrial surface. It then reaches intermediate sites in the outer membrane which are saturable and where it is protected against proteases. This translocation intermediate can be extracted at alkaline pH. We suggest that it is anchored to the membrane by a so far unknown proteinaceous component. The membrane potential delta psi-dependent entrance of the ADP/ATP carrier into the inner membrane takes place at contact sites between outer and inner membranes. Completion of translocation into the inner membrane can occur in the absence of delta psi. A cytosolic component which is present in reticulocyte lysate and which interacts with isolated mitochondria is required for the specific binding of the precursor to mitochondria

Proteinaceous receptors for the import of mitochondrial precursor proteins

Mild trypsin treatment of isolated Neurospora mitochondria strongly inhibits their ability to bind and import the precursors of several mitochondrial proteins. Evidence is presented for two proteins, the ADP/ATP carrier and the mitochondrial porin, that specific binding of the precursors to the outer surface of the mitochondria is affected by the protease treatment. We suggest that the receptors that mediate the import of these two precursors are proteinaceous. Treatment of mitochondria with elastase also inhibits the binding and import of the ADP/ATP carrier and the porin. In contrast the import of the precursors of subunits 2 and 9 of the mitochondrial proton-translocating ATPase was unaffected by elastase treatment at the concentrations used. We suggest that the import pathways of the latter two proteins are distinct from those of the ADP/ATP carrier and the porin

Isolation and characterization of a laminin-binding protein from rat and chick muscle.

A major laminin-binding protein (LBP), distinct from previously described LBPs, has been isolated from chick and rat skeletal muscle (Mr 56,000 and 66,000, respectively). The purified LBPs from the two species were shown to be related antigenically and to have similar NH2-terminal amino acid sequences and total amino acid compositions. Protein blots using laminin and laminin fragments provided evidence that this LBP interacts with the major heparin-binding domain, E3, of laminin. Studies on the association of this LBP with muscle membrane fractions and reconstituted lipid vesicles indicate that this protein can interact with lipid bilayers and has properties of a peripheral, not an integral membrane protein. These properties are consistent with its amino acid sequence, determined from cDNAs (Clegg et al., 1988). Examination by light and electron microscopy of the LBP antigen distribution in skeletal muscle indicated that the protein is localized primarily extracellularly, near the extracellular matrix and myotube plasmalemma. While a form of this LBP has been identified in heart muscle, it is present at low or undetectable levels in other tissues examined by immunocytochemistry indicating that it is probably a muscle-specific protein. As this protein is localized extracellularly and can bind to both membranes and laminin, it may mediate myotube interactions with the extracellular matrix

Mitochondrial Protein Import

The role of nucleoside triphosphates (NTPs) in mitochondrial protein import was investigated with the precursors of N. crassa ADP/ATP carrier, F1-ATPase subunit β, F0-ATPase subunit 9, and fusion proteins between subunit 9 and mouse dihydrofolate reductase. NTPs were necessary for the initial interaction of precursors with the mitochondria and for the completion of translocation of precursors from the mitochondrial surface into the mitochondria. Higher levels of NTPs were required for the latter reactions as compared with the early stages of import. Import of precursors having identical presequences but different mature protein parts required different levels of NTPs. The sensitivity of precursors in reticulocyte lysate to proteases was decreased by removal of NTPs and increased by their readdition. We suggest that the hydrolysis of NTPs is involved in modulating the folding state of precursors in the cytosol, thereby conferring import competence

Influence of cell surface characteristics on adhesion of Saccharomyces cerevisiae to the biomaterial hydroxylapatite

The influence of the physicochemical properties of biomaterials on microbial cell adhesion is well known, with the extent of adhesion depending on hydrophobicity, surface charge, specific functional groups and acid–base properties. Regarding yeasts, the effect of cell surfaces is often overlooked, despite the fact that generalisations may not be made between closely related strains. The current investigation compared adhesion of three industrially relevant strains of Saccharomyces cerevisiae (M-type, NCYC 1681 and ALY, strains used in production of Scotch whisky, ale and lager, respectively) to the biomaterial hydroxylapatite (HAP). Adhesion of the whisky yeast was greatest, followed by the ale strain, while adhesion of the lager strain was approximately 10-times less. According to microbial adhesion to solvents (MATS) analysis, the ale strain was hydrophobic while the whisky and lager strains were moderately hydrophilic. This contrasted with analyses of water contact angles where all strains were characterised as hydrophilic. All yeast strains were electron donating, with low electron accepting potential, as indicated by both surface energy and MATS analysis. Overall, there was a linear correlation between adhesion to HAP and the overall surface free energy of the yeasts. This is the first time that the relationship between yeast cell surface energy and adherence to a biomaterial has been described

PROCESSING OF HYDROXYLAPATITE COATINGS ON TITANIUM ALLOY BONE PROSTHESES

Processing of hydroxylapatite Sol-gel films on titanium alloy bone prostheses. A method utilizing non-line-of-Sight ion beam implantation and/or rapid thermal processing to provide improved bonding of layers of hydroxylapatite to titanium alloy Substrates while encouraging bone ingrowth into the hydroxylapatite layers located away from the substrate, is described for the fabrication of prostheses. The first layer of hydroxylapatite is mixed into the Substrate by the ions or rapidly thermally annealed, while Subsequent layers are heat treated or densified using ion implantation to form layers of decreasing density and larger crystallization, with the outermost layers being Suitable for bone ingrowth

NMR and Raman spectroscopic study of as-sprayed coatings and coatings incubated in simulated body fluid

Hydroxylapatite (HA) is a frequently used bioceramic material for replacement of bone matter subjected to low loading conditions, for osseoconductive coatings on implants and for utilisation as a drug carrier. Plasma spraying is widely used to coat hydroxylapatite onto titanium alloys in hip endoprostheses. However, the high temperature of the plasma jet changes the crystallinity and decomposes hydroxylapatite. This affects in turn its bioconductivity. In this study, HA was coated onto Ti6Al4V substrates by atmospheric plasma spraying (APS). Also, a bionert TiO2 bond coat was applied between the HA coating and the titanium alloy. By means of some sensitive analytical techniques, notably nuclear magnetic resonance (NMR) and Raman spectroscopy, the structural decomposition of HA during plasma-spraying and the in vitro reconstruction mechanisms of distorted (oxy)hydroxylapatite to well-ordered hydroxylapatite were investigated. The advantages of such a bond coat were also shown.thesi

NF-KB protein purification from bovine spleen: Nucleotide stimulation and binding site specificity

The activity of the enhancer for the κ immunoglobulin light chain gene critically depends on the presence in the nucleus of the NF-κB protein. We purified NF-κB over 50,000-fold and identified two protein species, 42 and 44 kDa, that could be eluted and renatured from a sodium dodecyl sulfate/polyacrylamide gel to give specific DNA-binding activity. Binding of the purified bovine NF-κB as well as that from human and murine B- or T-lymphoid cell extracts was dramatically stimulated by nucleoside triphosphates. This effect distinguished NF-κB from a related factor, H2-TF1. Purified NF-κB interacted efficiently with regulatory sequences that function during either B- or T-lymphocyte activation, including the human immunodeficiency virus enhancer and a NF-κB binding site we detected in the interleukin 2 enhancer