Amino acid sequence requirements for the association of apocytochrome c with mitochondria.

Sprinkle, James R.; Hakvoort, Theodorus B. M.; Koshy, Thomas I.; Miller, David D.; Margoliash, E.

doi:10.1073/pnas.87.15.5729

Cited by 29 publications

(13 citation statements)

References 34 publications

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“…Recently, Margoliash and coworkers presented evidence that apocytochrome c can be reversibly imported into isolated mitochondria, suggesting that the early steps of translocation do not depend on heme attachment (15,41). These authors report that apocytochrome c can be efficiently concentrated inside mitochondria, even when the protein is altered by the substitution of serine residues for the cysteine residues that are involved in the thioether linkages to heme.…”

Section: Discussionmentioning

confidence: 93%

Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.

Dumont¹,

Cardillo²,

Hayes³

et al. 1991

Mol. Cell. Biol.

138

127

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Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase. To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae. Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm. Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase. However, detectable amounts of this altered apocytochrome c are also found inside mitochondria. The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase. Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes. These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation. Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.

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Section: Discussionmentioning

confidence: 93%

Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.

Dumont¹,

Cardillo²,

Hayes³

et al. 1991

Mol. Cell. Biol.

138

127

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“…The cofactors prevent the misfolding and aggregation of the positively charged N terminus of the preproteins in an ATP-dependent manner in the cytosol. Interestingly, the positively charged amino acids at the N terminus influence the uptake of prepeptides into the mitochondria (50). The preproteins bind to the receptors at the surface of the outer mitochondrial membrane.…”

Section: Discussionmentioning

confidence: 99%

Characterization of mammalian translocase of inner mitochondrial membrane (Tim44) isolated from diabetic newborn mouse kidney

Wada

Kanwar

1998

Proc. Natl. Acad. Sci. U.S.A.

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Mammalian translocase of mitochondrial inner membrane (mTim44) was isolated during representational difference analysis of cDNA from diabetic mouse kidney. Streptozotocin-induced diabetic mouse kidney cDNA was prepared and subtracted by normal mouse kidney cDNA. By using one of the isolated cDNA fragments as a screening probe, full-length cDNA of mTim44 was isolated from ZAP kidney cDNA library. At the nucleotide level, mTim44 did not exhibit significant homology with any known genes; however, at the amino acid level, it had 50% similarity and 29% identity with yeast Tim44. C-terminal FLAG epitope-tagged mTim44 fusion protein was transiently expressed in COS7 cells. By using anti-FLAG epitope M2 monoclonal antibody, mTim44 was found to have its subcellular localization associated with mitochondria. By immunoelectron microscopy, mTim44 was seen in the paracrystalline structures within the mitochondria, as well as in their cristae. Mitochondrial import assay of in vitro translated mTim44 indicated that its precursor product (Ϸ50 kDa) was imported and proteolytically processed to a mature Ϸ44-kDa protein, and its translocation was inner membrane potential (⌬⌿)-dependent. Imported mTim44 was protected from protease digestion in which outer membranes were selectively permeabilized with digitonin. The mature form of mTim44 could be recovered in the supernatant of sonicated mitochondrial membrane fraction treated with 0.1 M Na 2 CO 3 , pH 11.5. The data indicate that mTim44 is a mitochondrial inner membrane protein, one of the members of the mammalian TIM complex and up-regulated in hyperglycemic states.

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“…If export of C2Asig reflects the existence of an additional element that retards folding prior to export, then the mature (28) or apocyt c, which is the eucaryotic counterpart of cyt c2 (25,34,36). An N-terminal a-helical region of eucaryotic apocyt c has been implicated in the natural import of this protein into mitochondria without an organellar targeting sequence; however, additional elements must exist because mutant proteins lacking the N terminus are still imported (25,34,36).…”

Section: Discussionmentioning

confidence: 99%

“…An N-terminal a-helical region of eucaryotic apocyt c has been implicated in the natural import of this protein into mitochondria without an organellar targeting sequence; however, additional elements must exist because mutant proteins lacking the N terminus are still imported (25,34,36). Members of this c-type cytochrome family share considerable sequence similarity throughout the polypeptide chain (24).…”

Section: Discussionmentioning

confidence: 99%

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The Rhodobacter sphaeroides cytochrome c2 signal peptide is not necessary for export and heme attachment

Brandner

Donohue

1994

J Bacteriol

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Rhodobacter sphaeroides cytochrome c2 (cyt c2) is a member of the heme-containing cytochrome c protein family that is found in the periplasmic space of this gram-negative bacterium. This exported polypeptide is made as a higher-molecular-weight precursor with a typical procaryotic signal peptide. Therefore, cyt c2 maturation is normally expected to involve precursor translocation across the cytoplasmic membrane, cleavage of the signal peptide, and covalent heme attachment. Surprisingly, synthesis as a precursor polypeptide is not a prerequisite for cyt c2 maturation because deleting the entire signal peptide does not prevent export, heme attachment, or function. Although cytochrome levels were reduced about threefold in cells containing this mutant protein, steady-state cyt c2 levels were significantly higher than those of other exported bacterial polypeptides which contain analogous signal peptide deletions. Thus, this mutant protein has the unique ability to be translocated across the cytoplasmic membrane in the absence of a signal peptide. The covalent association of heme with this mutant protein also suggests that the signal peptide is not required for ligand attachment to the polypeptide chain. These results have uncovered some novel aspects of bacterial c-type cytochrome biosynthesis.

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Amino acid sequence requirements for the association of apocytochrome c with mitochondria.

Cited by 29 publications

References 34 publications

Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.

Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.

Characterization of mammalian translocase of inner mitochondrial membrane (Tim44) isolated from diabetic newborn mouse kidney

The Rhodobacter sphaeroides cytochrome c2 signal peptide is not necessary for export and heme attachment

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