Showing papers on "Mitochondrial carrier published in 1983"
TL;DR: It is concluded that the binding observed represents an interaction with receptor sites and thus is an early step in the import pathway.
88 citations
TL;DR: Citrate synthase and other mitochondrial matrix proteins bind to the inner surface of the mitochondrial inner membrane and no binding was observed to the outer membrane or to the Outer membrane.
71 citations
TL;DR: This chapter describes the isolation and properties of the porin of the outer mitochondrial membrane from Neurospora crassa and gives pure and functionally active porin in high yield and can be easily scaled up.
Abstract: Publisher Summary The pore-forming protein, called mitochondrial porin, is a major component of outer mitochondrial membranes. This chapter describes the isolation and properties of the porin of the outer mitochondrial membrane from Neurospora crassa. Mitochondrial outer membranes are isolated by swelling-shrinking of the mitochondria and subsequent separation by a sucrose step centrifugation. Differential detergent extraction of the outer membrane followed by diethylaminoethyl (DEAE)-cellulose chromatography gives pure porin, which forms active pores after reconstitution into artificial bilayers. Porin isolation from whole mitochondria involves lysis of whole mitochondria with Genapol X-100 followed by chromatography on hydroxyapatite, Celite, and DEAE-cellulose. This procedure gives pure and functionally active porin in high yield and can be easily scaled up. Mitochondrial porin is synthesized on cytoplasmic free polysomes without an additional presequence. In contrast to the biogenesis of mitochondrial inner membrane or matrix proteins, assembly of the porin is not dependent on energization of the mitochondria.
30 citations
Journal Article•
TL;DR: It is concluded that the binding observed represents an interaction with receptor sites and thus is an early step in the import pathway.
16 citations
TL;DR: The conclusion is reached that the rate of proteolysis of mitochondrial translation products in the mitochondrial inner membrane depends on the physical state of the membrane, which in its turn is controlled by the relative content of unsaturated fatty acid chains in the mitochondria phospholipids.
Abstract: Our previous results [Kalnov, Novikova, Zubatov & Luzikov (1979) FEBS Lett. 101, 355-358; Biochem. J. 182, 195-202] suggested that in yeast the mitochondrial translation products localized in the mitochondrial inner membrane are rapidly broken down by a proteolytic system inherent in the membrane. In the present work, it is demonstrated that, on glucose repression in undividing cells of Saccharomyces cerevisiae, there is no proteolysis of the mitochondrial translation products. This effect is not likely to be associated with lower activity of the proteolytic system of the mitochondrial inner membrane. Nor is the cessation of proteolysis due to qualitative changes in the composition of mitochondrial translation products. What repression does cause is a considerable alteration in the physical state (i.e. structure of the lipid bilayer) of the mitochondrial inner membrane; this was established by experiments involving lipid-soluble spin probes. The conclusion is reached that the rate of proteolysis of mitochondrial translation products in the mitochondrial inner membrane depends on the physical state of the membrane, which in its turn is controlled by the relative content of unsaturated fatty acid chains in the mitochondrial phospholipids.
9 citations
TL;DR: The methods to analyze the kinetics of transfer of newly synthesized proteins into mitochondria and to identify precursors of mitochondrial proteins after labeling of intact cells are described.
Abstract: Publisher Summary The chapter describes the methods to analyze the kinetics of transfer of newly synthesized proteins into mitochondria and to identify precursors of mitochondrial proteins after labeling of intact cells. The methods with in vitro systems, which can be employed to investigate various problems, are described: (1) structure and properties of extramitochondrial precursor proteins; (2) identification of ribosomes involved in the synthesis of mitochondrial precursor proteins; (3) transfer of precursor proteins in reconstituted systems and dissection of the process into different steps; and (4) energy requirements of the transfer process. The methods described in this chapter focus on the study of integral proteins of the inner mitochondrial membrane of Neurospora crassa , the adenosine diphosphate–adenosine triphosphate (ADP–ATP) carrier, and subunit 9 of the oligomycin-sensitive ATPase. The transport of proteins into mitochondria is a multistep process and an understanding of the individual steps requires in vitro systems. The steps that can be resolved include (1) synthesis of precursor proteins on cytoplasmic ribosomes; (2) transfer through cytosol to the mitochondria; (3) binding to specific sites on the mitochondria; and (4) translocation across inner or outer membrane, or both, accompanied in most cases by proteolytic cleavage or by some other covalent modification.
9 citations
TL;DR: This chapter outlines a general approach for examining the import of a given mitochondrial protein and emphasizes some of the rationale and the methods available for doing so.
Abstract: Publisher Summary This chapter outlines a general approach for examining the import of a given mitochondrial protein and emphasizes some of the rationale and the methods available for doing so In examining the import of a particular mitochondrial protein, some questions must be answered as to whether the protein is initially synthesized as a larger precursor; the polypeptide can be taken up by mitochondria posttranslationally; import of the protein is mediated by binding to a mitochondrial outer membrane receptor; and import of the polypeptide require an energized inner membrane The import of polypeptides into the mitochondrial matrix, the inner membrane, or the intermembrane space requires an electrochemical gradient across the mitochondrial inner membrane If an imported mitochondrial polypeptide is made as a larger precursor, then the energy dependence of import is conveniently checked by demonstrating accumulation of this precursor in yeast cells pulse-labeled in the presence, but not in the absence, of 20μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) This assay is unsuitable for precursors that have the same molecular weight as the mature protein
4 citations
TL;DR: A yeast mitochondrial inner membrane hydrophobic protein 30K has been isolated and compared to subunit 32K of the yeast cytochrome bc 1 complex, showing that the two proteins are not identical.