A sensitive in vitro protein synthesizing system from Ehrlich ascites mitochondria

The S-25 fraction prepared from digitonin washed mitochondria is highly active in poly(U) directed phenylanine incorporation when supplemented with t-RNA. Ribosomes prepared from the S-25 fraction contain 58S monomeric ribosomes and 40S and 29S subunits. Further, these ribosomes contain 21S and 13S rRNA. No detectable cytoplasmic specific ribosomal particles and also rRNA was detected in the mitochondrial S-25 preparation. Ribosomes from mitochondrial S-25 have specific requirement for mitochondrial specific supernatant factors for complete activity.     

The biogenesis of rat mitochondrial ATPase. Two subunits are synthesized inside the mitochondria

Isolated mitochondria from regenerating rat liver synthesize at least five different polypeptides with molecular weights ranging from 19 000 to 43 000. Among these, two polypeptides with molecular weights of 22 000 and 25 ooo could be identified as ATPase subunits. It has previously been shown that these subunits, designated 6 and 7, are lacking in the ATPase complex that is formed in vivo when mitochondrial protein synthesis is blocked by thiamphenicol treatment. The 22 000 Mr protein is enriched in the fraction containing the fully assembled ATPase complex, whereas the 25 000 Mr protein is not.     

Nuclear and mitochondrial genes in the biogenesis of Saccharomyces cerevisiae mitochondria

The mechanisms of interaction of nuclear and mitochondrial genes in biogenesis of mitochondria are discussed in this review. Brief characterization of yeast mitochondrial genes and their products is presented. The mechanism of nuclear and mitochondrial control of expression of the mosaic genes in mitochondria is described. The data on the processing of imported mitochondrial proteins synthesized on cytoplasmic ribosomes are presented. The possibility of existence of common proteins encoded for by common genes and possessing similar functions in the cytoplasm and mitochondria is discussed. A hypothesis is put forward considering the role of common proteins in coordination of nuclear and mitochondrial genes' expression in biogenesis of mitochondria.     

The biogenesis of rat-liver mitochondrial ATPase. Evidence that the N, N'-dicyclohexyl carbodiimide-binding protein is synthesized outside the mitochondria

1. Radioactive N,N'-dicyclohexyl carbodiimide (DCCD) is bound as effectively to the N, N'-dicyclohexyl carbodiimide- and oligomycin-sensitive ATPase complex in submitochondrial particles of normal rat liver as to the similar but partially N,N'-dicyclohexyl carbodiimide- and oligomycin-insensitive complex of thiamphenicol-treated rats. The latter complex is deficient in 3 subunits (subunit 6, 7 and 10). 2. Radioactive N,N'-dicyclohexyl carbodiimide is exclusively bound to the subunits present in the bands 8 and 11 of SDS-PAA gels of the purified ATPase complex. These subunits, most likely the dimer and monomer of the N,N'-dicyclohexyl carbodiimide-binding protein, are products of the cytoplasmic protein synthesis. 3. The results together indicate that the N,N'-dicyclohexyl carbodiimide-insensitivity of the ATPase complex formed during in vitro inhibition of mitochondrial protein synthesis, is not caused by a lack of inhibitor binding protein. The same holds for the oligomycin-insensitivity.     

Stimulation of in vitro mitochondrial protein synthesis by yeast cytoplasmic extracts is caused by guanyl nucleotides

Micromolar concentrations of GDP or GTP stimulate protein synthesis by isolated yeast mitochondria 3- to 10-fold even if alpha-ketoglutarate and an ATP-regenerating system are present. No stimulation is observed with GMP, UTP, CTP, TTP, and the nonhydrolyzable GTP analogues guanyl(beta, gamma-methylene) diphosphate and guanyl imidodiphosphate. This stimulatory effect of exogenously added guanyl nucleotides may answer the long standing question why protein synthesis by isolated mitochondria is so slow. It can also explain previous reports by two other laboratories that a high speed supernatant from yeast cells stimulates protein synthesis by isolated mitochondria. The supernatant contains nondialyzable GMP which is converted to GDP under the conditions used for assaying mitochondrial protein synthesis. The stimulatory effect of high speed supernatants is abolished by 5'-nucleotidase (which degrades GMP) or by trypsin (which destroys supernatant protein(s) necessary for converting GMP to GDP). No evidence was obtained that the stimulatory effect of high speed supernatants was caused by precursors to cytoplasmically made cytochrome c oxidase subunits.     

Biogenesis of mitochondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions

The ability in vitro of yeast mitochondrial and microsomal fractions to synthesize lipid de novo was measured. The major phospholipids synthesized from sn-[2-(3)H]glycerol 3-phosphate by the two microsomal fractions were phosphatidylserine, phosphatidylinositol and phosphatidic acid. The mitochondrial fraction, which had a higher specific activity for total glycerolipid synthesis, synthesized phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid, together with smaller amounts of neutral lipids and diphosphatidylglycerol. Phosphatidylcholine synthesis from both S-adenosyl[Me-(14)C]methionine and CDP-[Me-(14)C]choline appeared to be localized in the microsomal fraction.     

A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria

The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to decreased levels of MOM22 and reduced protein import through MOM22. Furthermore, MOM72 does not function as a general backup receptor for MOM19 suggesting that these two proteins have distinct precursor specificities. These findings demonstrate that the import receptor MOM19 fulfills an important role in the biogenesis of mitochondria and that it is essential for the formation of mitochondria competent in respiration and phosphorylation.