Transport of Cytoplasmically Synthesized Proteins into the Mitochondria in a Cell Free System from Neurospora crassa

Synthesis and transport of mitochondrial proteins were followed in a cell-free homogenate of Neurospora crassa in which mitochondrial translation was inhibited. Proteins synthesized on cytoplasmic ribosomes are transferred into the mitochondrial fraction. The relative amounts of proteins which are transferred in vitro are comparable to those transferred in whole cells. Cycloheximide and puromycin inhibit the synthesis of mitochondrial proteins but not their transfer into mitochondria. The transfer of immunoprecipitable mitochondrial proteins was demonstrated for matrix proteins, carboxyatractyloside-binding protein and cytochrome c. Import of proteins into mitochondria exhibits a degree of specificity. The transport mechanism differentiates between newly synthesized proteins and preexistent mitochondrial proteins, at least in the case of matrix proteins. In the cell-free homogenate membrane-bound ribosomes are more active in the synthesis of mitochondrial proteins than are free ribosomes. The finished translation products appear to be released from the membrane-bound ribosomes into the cytosol rather than into the membrane vesicles. The results suggest that the transport of cytoplasmically synthesized mitochondrial proteins is essentially independent of cytoplasmic translation; that cytoplasmically synthesized mitochondrial proteins exist in an extramitochondrial pool prior to import; that the site of this pool is the cytosol for at least some of the mitochondrial proteins; and that the precursors in the extramitochondrial pool differ in structure or conformation from the functional proteins in the mitochondria

Evidence for the Molten Globule State of Human apo-Ceruloplasmin

The conformational features of copper-free ceruloplasmin (CP), as compared to the holo-protein, were evaluated utilizing far- and near-UV circular dichroism and fluorescence spectroscopy. The results obtained indicate that apo-CP maintains the secondary structure of the holo-protein, while the tertiary interactions are much weaker. In addition, the removal of copper from the holo-protein leads to the exposure of hydrophobic patches to solvent, as shown by the fact that apo-CP, at variance from the holo-protein, binds the hydrophobic probe ANS. It is proposed that the CP molecule, upon copper removal, acquires the conformational features typical of a molten globule, which might be the conformational state of CP during its biosynthesis before metal incorporation

Isolation and partial characterization of poly (A)-containing 7.5S messenger RNA from rat liver mitochondria.

Poly(A)-containing low molecular weight (7.5S) messenger RNA was isolated in a highly purified form from both polyribosomes and post-polysomal supernatant of rat liver mitochondria. Both mRNA's contain rather short poly(A) tracts (40-70 mononucleotides) according to a profile of their elution from poly(U)-Sepharose column with a gradient of formamide concentration. Both mRNA's when added to a preincubated mitochondrial lysate programmed the synthesis of a hydrophobic polypeptide of a molecular weight about 9000 daltons which was soluble in the neutral chloroform-methanol mixture

Enzymatic synthesis of DNA complementary to mitochondrial mRNA via reverse transcription.

The poly(A)-containing mitochondrial mRNAs of rat liver were tested for their ability to serve as templates for the DNA synthesis by means of reverse transcription in the presence of the oligo(dT) primer and the RNA-directed DNA-polymerase from avian myeloblastosis virus. The mT-mRNA does not support the DNA synthesis in the standard conditions sufficient for effective reverse transcription of rabbit globin mRNA and of poly(A) in the presence of oligo(dT) primers. After a mild alkaline treatment of the mRNA and subsequent polyadenylation of the 3'-termini of the generated fragments with ATP:RNA adenyltransferase from E.coli the poly(A) (+) polyribonucleotides are able to serve as templates for reverse transcription in the presence of oligo(dT) and the reverse transcriptase. A conclusion is made that a "structural stop" exists in mitochondrial mRNA non-translable regions adjacent to the poly(A) terminal sequence. The "structural stop" is suggested to be caused by post-transcriptional modification of mRNA (methylation, etc.) or by a particularly stable secondary structure in this region of the mRNA molecules

Interaction of Lactoferrin with Ceruloplasmin

When added to human blood serum, the iron-binding protein lactoferrin (LF) purified from breast milk interacts with ceruloplasmin (CP), a copper-containing oxidase. Selective binding of LF to CP is evidenced by the results of polyacrylamide gel electrophoresis, immunodiffusion, gel filtration, and affinity chromatography. The molar stoichiometry of CP:LF in the complex is 1:2. Near-uv circular dichroism spectra of the complex showed that neither of the two proteins undergoes major structural perturbations when interacting with its counterpart. K(d) for the CP/LF complex was estimated from Scatchard plot as 1.8 x 10(-6) M. The CP/LF complex is found in various fluids of the human body. Upon injection into rat of human LF, the latter is soon revealed within the CP/LF complex of the blood plasma, from where the human protein is substantially cleared within 5 h