Cell-free synthesis of a larger-molecular-weight precursor of cytochrome c oxidase subunit V from rat liver and the distribution of its mRNA between free and membrane-bound polysomes

Poly(A)-rich RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germs. The labeled translation products were incubated with an antiserum against cytochrome c oxidase subunit V. After immunoprecipitation and affinity chromatography with protein-A-Sepharose, the isolated antigen-immunoglobulin complexes were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. Only one protein with an apparent molecular weight of 15 500 was visualized. In immunocompetition experiments with unlabeled individual cytochrome c oxidase subunits IV, V, VI or VII only subunit V could compete with the 15 500-Mr protein synthesized in vitro. Two-dimensional fingerprints of cytochrome c oxidase subunit V and the polypeptide synthesized in vitro showed a high degree of similarity. It is concluded that the cytochrome c oxidase subunit V is synthesized as a precursor with an amino-terminal extension of about 25 amino acids. It was possible to convert the precursor of cytochrome c oxidase subunit V synthesized in vitro to its mature form by intact mitochondria as well as by submitochondrial particles. A chain length of 830 +/- 70 nucleotides was estimated for the poly(A)-rich mRNA of the higher-molecular-weight precursor of rat liver cytochrome c oxidase subunit V. Assuming a molecular weight of 15 500 for the precursor a non-coding region of about 300 nucleotides must exist. In experiments on the site of synthesis it is shown that the poly(A)-rich RNA for the higher-molecular-weight precursor of cytochrome c oxidase subunit V is found in free, loosely and tightly membrane-bound polyribosomes.     

Sequencing of the nuclear gene for the yeast cytochrome c1 precursor reveals an unusually complex amino-terminal presequence.

Cytochrome c1 is a component of the mitochondrial respiratory chain in most eukaryotes. The protein is coded by nuclear DNA, synthesized as a larger precursor outside the mitochondria and then cleaved to the mature form in two successive steps during its import into the mitochondria. We have cloned the structural gene for yeast cytochrome c1 by functional complementation of a cytochrome c1-deficient yeast mutant with a yeast genomic library in the yeast-Escherichia coli 'shuttle' vector YEp 13. The complete nucleotide sequence of the gene and of its 5'- and 3'-flanking regions was determined. The deduced amino acid sequence of the yeast cytochrome c1 precursor reveals an unusually long transient amino-terminal presequence of 61 amino acids. This presequence consists of a strongly basic amino-terminal region of 35 amino acids, a central region of 19 uncharged amino acids and an acidic carboxy-terminal region of seven amino acids. This tripartite structure of the presequence resembles that of the precursor of cytochrome c peroxidase and supports a previous suggestion on the import pathways of these two precursors.     

Bilayer-penetrating properties enable apocytochrome c to follow a special import pathway into mitochondria.

In this study, we have investigated the protein/lipid interactions of two mitochondrial precursor proteins, apocytochrome c and pCOX IV-DHFR, which exhibit mitochondrial import pathways with different characteristics. In-vitro-synthesized apocytochrome c was found to bind efficiently and specifically to liposomes composed of negatively charged phospholipids and showed a (at least partial) translocation across a lipid bilayer, as reported previously for the chemically prepared precursor protein [Rietveld, A. & de Kruijff, B. (1984) J. Biol. Chem. 259, 6704-6707; Dumont, M. E. & Richards, F. M. (1984) J. Biol. Chem. 259, 4147-4156]. Negatively charged liposomes were shown to efficiently compete with mitochondria for import of in-vitro-synthesized apocytochrome c into the organelle, suggesting an important role for negatively charged phospholipids in the initial binding of apocytochrome c to mitochondria. In contrast, the purified and in-vitro-synthesized precursor fusion protein pCOX IV-DHFR, consisting of the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase was unable to translocate across a pure lipid bilayer. The data indicate that the ability of apocytochrome c to spontaneously translocate across the bilayer is not shared by all mitochondrial precursor proteins. The implications of the special protein/lipid interaction of apocytochrome c for import into mitochondria will be discussed.     

A novel cryptic peptide derived from the rat neuropeptide FF precursor reverses antinociception and conditioned place preference induced by morphine

Neuropeptide FF (NPFF) precursors from different species contain at least three known neuropeptides, i.e. FF (FLFQPQRF-NH(2)), AF (AGEGLSSPFWSLAAPQR-NH(2)) and SF (SLAAPQRF-NH(2)). We demonstrate that the rat NPFF precursor contains another bioactive sequence, NAWGPWSKEQLSPQA, spanning between positions 85 and 99. Synthetic NPFF precursor (85-99) (10 and 20 nmol, i.c.v.) blocked the expression of conditioned place preference induced by morphine (5 mg/kg, s.c.). This peptide alone (10 and 20 nmol, i.c.v.) had no influence on the baseline latency of a nociceptive reaction but reversed the antinociceptive activity of morphine (5 mg/kg, s.c.) in the tail-immersion test in rats. These data suggest the existence of a novel bioactive cryptic peptide within an already known NPFF precursor.     

Biogenesis of cytochrome c in Neurospora crassa. Synthesis of apocytochrome c, transfer to mitochondria and conversion to Holocytochrome c.

1. Precipitating antibodies specific for apocytochrome c and holocytochrome c, respectively, were employed to study synthesis and intracellular transport of cytochrome c in Neurospora in vitro. 2. Apocytochrome c as well as holocytochrome c were found to be synthesized in a cell-free homogenate. A precursor product relationship between the two components is suggested by kinetic experiments. 3. Apocytochrome c synthesized in vitro was found in the post-ribosomal fraction and not in the mitochondrial fraction, whereas holocytochrome c synthesized in vitro was mainly detected in the mitochondrial fraction. A precursor product relationship between postribosomal apocytochrome c and mitochondrial holocytochrome c is indicated by the labelling data. In the microsomal fraction both apocytochrome c and holocytochrome c were found in low amounts. Their labeling kinetics do not subbest a precursor role of microsomal apocytochrome c or holocytochrome c. 4. Formation of holocytochrome c from apocytochrome c was observed when postribosomal supernatant containing apocytochrome c synthesized in vitro was incubated with isolated mitochondria, but not when incubated in the absence of mitochondria. The cytochrome c formed under these conditions was detected in the mitochondria. 5. Conversion of labelled apocytochrome c synthesized in vitro to holocytochrome c during incubation of a postribosomal supernatant with isolated mitochondria was inhibited when excess isolated apocytochrome c, but not when holocytochrome c was added. 6. The data presented are interpreted to show that apocytochrome c is synthesized on cytoplasmic ribosomes and released into the supernatant. It is suggested that apocytochrome c migrates to the inner mitochondrial membrane, where the heme group is covalently linked to the apoprotein. The hypothesis is put forward that the concomitant change in conformation leads to trapping of holocytochrome c in the membrane. The problems of permeability of the outer mitochondrial membrane to apocytochrome c and the site and nature of the reaction by which the heme group is linked to the apoprotein are discussed.     

The N-terminal propeptide and the C terminus of the precursor to 20-kilo-dalton potato tuber protein can function as different types of vacuolar sorting signals

Two types of vacuolar sorting signals (VSSs), an asparagine-proline-isoleucine-arginine-leucine (NPIRL)-related VSS in the N-terminal propeptides (NTPPs) and a C-terminal VSS in the C-terminal propeptides (CTPPs), function differently in plant cells. A precursor to a 20-kDa protein of potato tuber (PT20) contains two NPIRL-related sequences, NPINL in a short NTPP and NPLDV close to the C terminus of the precursor. We made mutant forms of sweet potato sporamin (SPO), nPT20-SPO, in which the N-terminal pre-pro part was exchanged with that of the precursor to PT20, and SPO-PT20c, in which the C-terminal 13 amino acids of the precursor to PT20 was attached to the C terminus of delta pro-SPO which lacked NTPP. Both nPT20-SPO and SPO-PT20c were efficiently transported to the vacuoles in tobacco cells. Unlike nPT20-SPO, the vacuolar transport of SPO-PT20c was inhibited by wortmannin and by the C-terminal addition of Gly or Gly-Gly suggesting its similarity to the vacuolar transport of sporamin mediated by CTPP of barley lectin. Further analysis of the C-terminal sequence of PT20 indicated that the most C-terminal SFKQVQ sequence functions as the C-terminal VSS. These results suggest that the precursor to PT20 contains both NPIRL-like VSS in its NTPP and C-terminal VSS at the C terminus.     

Site-specific mutagenesis of cDNA clones expressing a poliovirus proteinase

The cleavage of poliovirus precursor polypeptides occurs at specific amino acid pairs that are recognized by viral proteinases. Most of the polio-specific cleavages occur at glutamine-glycine (Q-G) pairs that are recognized by the viral-encoded proteinase 3C (formerly called P3-7c). In order to carry out a defined molecular genetic study of the enzymatic activity of protein 3C, we have made cDNA clones of the poliovirus genome. The cDNA region corresponding to protein 3C was inserted into an inducible bacterial expression vector. This recombinant plasmid (called pIN-III-C3-7c) utilizes the bacterial lipoprotein promoter to direct the synthesis of a precursor polypeptide that contains the amino acid sequence of protein 3C as well as the amino- and carboxy-terminal Q-G cleavage signals. These signals have been previously shown to allow autocatalytic production of protein 3C in bacteria transformed with plasmid pIN-III-C3-7c. We have taken advantage of the autocatalytic cleavage of 3C in a bacterial expression system to study the effects of site-specific mutagenesis on its proteolytic activity. One mutation that we have introduced into the cDNA region encoding 3C is a single amino acid insertion near the carboxy-terminal Q-G cleavage site. The mutant recombinant plasmid (designated pIN-III-C3-mu 10) directs the synthesis of a bacterial-polio precursor polypeptide that is like the wild-type construct (pIN-III-C3-7c). However, unlike the wild-type precursor, the mutant precursor cannot undergo autocatalytic cleavage to generate the mature proteinase 3C. Rather, the precursor is able to carry out cleavage at the amino-terminal Q-G site but not at the carboxy-terminal site. Thus, we have generated an altered poliovirus proteinase that is still able to carry out at least part of its cleavage activities but is unable to be a suitable substrate for self-cleavage at its carboxy-terminal Q-G pair.     

The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain.

The major mitochondrial processing activity removing presequences from nuclear encoded precursor proteins is present in the soluble fraction of fungal and mammalian mitochondria. We found that in potato, this activity resides in the inner mitochondrial membrane. Surprisingly, the proteolytic activity co-purifies with cytochrome c reductase, a protein complex of the respiratory chain. The purified complex is bifunctional, as it has the ability to transfer electrons from ubiquinol to cytochrome c and to cleave off the presequences of mitochondrial precursor proteins. In contrast to the nine subunit fungal complex, cytochrome c reductase from potato comprises 10 polypeptides. Protein sequencing of peptides from individual subunits and analysis of corresponding cDNA clones reveals that subunit III of cytochrome c reductase (51 kDa) represents the general mitochondrial processing peptidase.     

The nature of the reaction of an essential tyrosine residue of bovine heart mitochondrial ATPase with 4-chloro-7-nitrobenzofurazan and related compounds

The import and processing of cytochrome c1 and the iron sulfur protein of the cytochrome b-c1 complex were studied in Zajdela hepatoma ascites cells. Both peptides were synthesized as larger percursor molecules which were approximately 2-3 kDa and 5-6 kDa larger than the mature forms of apocytochrome c1 and apo-iron sulfur protein, respectively. Comparison of these precursors to those reported for functionally homologous peptides in yeast and Neurospora indicate significant size changes have occurred in mammals. Rhodamine 6G, a specific vital stain for mitochondria, is a potent inhibitor of precursor processing in isolated hepatoma cells. Both precursor to cytochrome c1 and precursor to FeS accumulate in the soluble and particulate fractions obtained by digitonin treatment of tumor cells treated with Rhodamine 6G. Appearance of the mature peptides was abolished. The precursors are unstable, however, and disappear from the cytosolic and membrane fractions during a 10 min chase. Comparison of the effects of Rhodamine 6G and carbonylcyanide m-chlorophenylhydrazone on precursor processing shows that: (a) Rhodamine 6G is a more effective inhibitor of processing, (b) it has less of an inhibitory effect on cellular protein synthesis, and (c) it inhibits processing under conditions in which it appears to have little influence on coupled respiration in whole cells. The data suggest that the most likely mode of action of Rhodamine 6G is on the matrix processing step.     

Phenethyl alcohol disorders phospholipid acyl chains and promotes translocation of the mitochondrial precursor protein apocytochrome c across a lipid bilayer.

The interaction of phenethyl alcohol with model membranes and its effect on translocation of the chemically prepared mitochondrial precursor protein apocytochrome c across a lipid bilayer was studied. Phenethyl alcohol efficiently penetrates into monolayers and causes acyl chain disordering judged from deuterium nuclear magnetic resonance measurements with specific acyl chain-deuterated phospholipids. Translocation of apocytochrome c across a phospholipid bilayer was stimulated on addition of phenethyl alcohol indicating that the efficiency of translocation of this precursor protein is enhanced due to a disorder of the acyl chain region of the bilayer.     

Interaction with mitochondrial membranes of a synthetic peptide with a sequence common to extra peptides of mitochondrial precursor proteins

A hepta-peptide, Arg-Leu-Leu-Pro-Ser-Leu-Gly, which has a sequence involved in the extra peptides of mitochondrial proteins, was synthesized chemically. The peptide was found to bind specifically to mitochondria, but not to microsomes. The binding was blocked by pretreatment of mitochondria with trypsin but was not affected by the presence of apocytochrome c. The synthetic peptide inhibited the binding to mitochondria of the precursor protein of ATPase inhibitor, which was synthesized in vitro, but did not inhibit that of the precursor of the 9 K stabilizing factor, which has an entirely different extra-peptide sequence. The peptide also did not inhibit the binding of apocytochrome c. These results suggest the existence of a common protein receptor on mitochondrial membranes that facilitates entrance of a group of mitochondrial precursor proteins, including pre-ATPase inhibitor.     

Biogenesis of cytochrome c1. Role of cytochrome c1 heme lyase and of the two proteolytic processing steps during import into mitochondria

The biogenesis of cytochrome c1 involves a number of steps including: synthesis as a precursor with a bipartite signal sequence, transfer across the outer and inner mitochondrial membranes, removal of the first part of the presequence in the matrix, reexport to the outer surface of the inner membrane, covalent addition of heme, and removal of the remainder of the presequence. In this report we have focused on the steps of heme addition, catalyzed by cytochrome c1 heme lyase, and of proteolytic processing during cytochrome c1 import into mitochondria. Following translocation from the matrix side to the intermembrane-space side of the inner membrane, apocytochrome c1 forms a complex with cytochrome c1 heme lyase, and then holocytochrome c1 formation occurs. Holocytochrome c1 formation can also be observed in detergent-solubilized preparations of mitochondria, but only after apocytochrome c1 has first interacted with cytochrome c1 heme lyase to produce this complex. Heme linkage takes place on the intermembrane-space side of the inner mitochondrial membrane and is dependent on NADH plus a cytosolic cofactor that can be replaced by flavin nucleotides. NADH and FMN appear to be necessary for reduction of heme prior to its linkage to apocytochrome c1. The second proteolytic processing of cytochrome c1 does not take place unless the covalent linkage of heme to apocytochrome c1 precedes it. On the other hand, the cytochrome c1 heme lyase reaction itself does not require that processing of the cytochrome c1 precursor to intermediate size cytochrome c1 takes place first. In conclusion, cytochrome c1 heme lyase catalyzes an essential step in the import pathway of cytochrome c1, but it is not involved in the transmembrane movement of the precursor polypeptide. This is in contrast to the case for cytochrome c in which heme addition is coupled to its transport directly across the outer membrane into the intermembrane space.     

Different transport pathways of individual precursor proteins in mitochondria

Transport of mitochondrial precursor proteins into mitochondria of Neurospora crassa was studied in a cell-free reconstituted system. Precursors were synthesized in a reticulocyte lysate programmed with Neurospora mRNA and transported into isolated mitochondria in the absence of protein synthesis. Uptake of the following precursors was investigated: apocytochrome c, ADP/ATP carrier and subunit 9 of the oligomycin-sensitive ATPase. Addition of high concentrations of unlabelled chemically prepared apocytochrome c (1-10 microM) inhibited the appearance in the mitochondrial of labelled cytochrome c synthesized in vitro because the unlabelled protein dilutes the labelled one and because the translocation system has a limited capacity [apparent V is 1-3 pmol X min-1 X (mg mitochondrial protein)-1]. Concentrations of added apocytochrome c exceeding the concentrations of precursor proteins synthesized in vitro by a factor of about 10(4) did not inhibit the transfer of ADP/ATP carrier or ATPase subunit 9 into mitochondria. Carbonylcyanide m-chlorophenylhydrazone, an uncoupler of oxidate phosphorylation, inhibited transfer in vitro of ADP/ATP carrier and of ATPase subunit 9, but not of cytochrome c. These findings suggest that cytochrome c and the other two proteins have different import pathways into mitochondria. It can be inferred from the data presented that different 'receptors' on the mitochondria. It can be inferred from the data presented that different 'receptors' on the mitochondrial surface mediate the specific recognition of precursor proteins by mitochondria by mitochondria as a first step in the transport process.     

Phospholipids as a possible instrument for translocation of nascent proteins across biological membranes

The interaction of phospholipids with precursor proteins, particularly with the mitochondrial precursor protein apocytochromec is reviewed and integrated with other aspects of protein insertion and translocation, leading to a model for (apo)cytochrome c import into mitochondria, in which phospholipids play a dominant role.     

A water-lipid interface induces a highly dynamic folded state in apocytochrome c and cytochrome c, which may represent a common folding intermediate.

In this study, we have used CD and NMR techniques to investigate the secondary structure of (apo-) cytochrome c both in solution and when associated with micelles. In aqueous solution, the holoprotein cytochrome c is tightly folded at secondary and tertiary levels and differs strongly from its random-coiled precursor. However, in the presence of 12-PN/12-Pglycol (9:1) micelles, we observed a remarkable resemblance between the CD spectra of these partially helical proteins. The water-lipid interface induces a secondary folding of apocytochrome c, whereas cytochrome c is suggested to partially lose its tertiary structure. The exchange of all amide protons and, using deuterium-labeled proteins, of all amide deuterons with the solvent was monitored by NMR. A rapid exchange rate was observed, indicating that these folding states are highly dynamic. Saturation-transfer NMR of micelle-associated apocytochrome c showed that the exchange takes place at the (sub-) second time scale. The holoprotein in the presence of micelles was found to have two distinct exchange rates: (1) a fast rate, comparable to that found for the micelle-associated precursor and 4.5 times slower than that of the random-coiled apocytochrome c, and (2) a slow rate which is 75 times slower than the precursor in solution. Urea denaturation studies showed the micelle-bound proteins to have a low helix stability, which explains the inability of the lipid-induced secondary structure to prevent its labile protons from rapid exchange. The uniqueness of this lipid-induced highly dynamic folding state of (apo-) cytochrome c is demonstrated by comparison with amphiphilic polypeptides like melittin, and its implications for membrane translocation and functioning are discussed.     

Transport of proteins into mitochondria: translocational intermediates spanning contact sites between outer and inner membranes

Translocational intermediates of precursor proteins of ATPase F1 beta subunit and cytochrome c1 across mitochondrial membranes were analyzed using two different approaches, transport at low temperature and transport after binding of precursor proteins to antibodies. Under both conditions precursors were partially transported into mitochondria in an energy-dependent manner. They were processed by the metalloprotease in the matrix but a major proportion of the polypeptide chains was still present at the outer face of the outer mitochondrial membrane. We conclude that transfer of precursors into the inner membrane or matrix space occurs through "translocation contact sites"; precursor polypeptides to F1 beta and cytochrome c1 enter the matrix space with the amino terminus first; and a membrane potential is required for the transmembrane movement on an amino-terminal "domain-like" structure but not for completing translocation of the major part of the polypeptides.     

Gene knockout of amyloid precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in primary mouse cortical neurons and embryonic fibroblasts

Alzheimer's disease is characterised by the accumulation of amyloid-beta peptide, which is cleaved from the copper-binding amyloid-beta precursor protein. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for amyloid-beta precursor protein and amyloid-beta in copper homeostasis. Amyloid-beta precursor protein is a member of a multigene family, including amyloid precursor-like proteins-1 and -2. The copper-binding domain is similar among amyloid-beta precursor protein family members, suggesting an overall conservation in its function or activity. Here, we demonstrate that double knockout of amyloid-beta precursor protein and amyloid precursor-like protein-2 expression results in significant increases in copper accumulation in mouse primary cortical neurons and embryonic fibroblasts. In contrast, over-expression of amyloid-beta precursor protein in transgenic mice results in significantly reduced copper levels in primary cortical neurons. These findings provide cellular neuronal evidence for the role of amyloid-beta precursor protein in copper homeostasis and support the existing hypothesis that amyloid-beta precursor protein and amyloid precursor-like protein-2 are copper-binding proteins with functionally interchangeable roles in copper homeostasis.     

Diversity of SREBP-1 gene expression in rat adipose tissue depots in response to refeeding after food restriction

The SREBP-1c mRNA level and precursor (microsomal) form of SREBP-1 abundance were significantly higher in epididymal and perirenal than in subcutaneous white adipose tissue of control rats. Moreover, the SREBP-1c mRNA level and an amount of precursor form of SREBP-1 were significantly higher in the epididymal and perirenal white adipose tissue of rats maintained on restricted diet and refed ad libitum for 48 h as compared to the control animals. No significant effects of food restriction/refeeding on SREBP-1c mRNA level and an amount of precursor form of SREBP-1 were found in subcutaneous white adipose tissue. The mature (nuclear) form of SREBP-1 was significantly increased in the epididymal, perirenal and subcutaneous white adipose tissue of the food restricted/refed animals. The activity, protein level and the mRNA abundance of malic enzyme (one of the target genes for SREBP-1) increased significantly in the epididymal, perirenal and subcutaneous white adipose tissue of the food restricted/refed rats as compared to the control animals, however the increase in perirenal and epididymal was higher than in the subcutaneous white adipose tissue. The results presented suggest that SREBP-1c is differently expressed in various rat white adipose tissue depots both under basal (control) and dieting conditions.     

Import of proteins into mitochondria. Import and maturation of the mitochondrial intermembrane space enzymes cytochrome b2 and cytochrome c peroxidase in intact yeast cells

The import of cytochrome b2 and cytochrome c peroxidase into mitochondria was investigated by pulse-chase experiments with intact yeast cells combined with subcellular fractionation. Import and processing of the precursors of these intermembrane space proteins is blocked by uncouplers of oxidative phosphorylation, indicating that an "energized" inner membrane is required. Cytochrome b2 is processed in two steps. The first step involves energy-dependent transport across both mitochondrial membranes and cleavage by a matrix-located protease to yield an intermediate which is smaller than the precursor, but larger than the mature protein. The second step involves conversion of the intermediate to the mature form. Whereas the precursor and the mature form are soluble, the intermediate is membrane-bound and exposed to the intermembrane space. The maturation of cytochrome c peroxidase is much slower than that of cytochrome b2. Proteolytic processing rather than import is rate-limiting since cytochrome c peroxidase precursor labeled during a 3-min pulse is already found attached to the outer face of the mitochondrial inner membrane. Import of cytochrome b2 and probably also of cytochrome c peroxidase thus involves energy-dependent transport to the matrix and cleavage by a matrix-localized protease. Maturation of cytochrome b2 proceeds in the sequence: soluble precursor leads to membrane-bound intermediate form leads to soluble mature form.