A cytosolic cytochrome b5-like protein in yeast cell accelerating the electron transfer from NADPH to cytochrome c catalyzed by Old Yellow Enzyme

A 410-nm absorbing species which enhanced the reduction rate of cytochrome c by Old Yellow Enzyme (OYE) with NADPH was found in Saccharomyces cerevisiae. It was solubilized together with OYE by the treatment of yeast cells with 10% ethyl acetate. The purified species showed visible absorption spectra in both oxidized and reduced forms, which were the same as those of the yeast microsomal cytochrome b5. At least 14 amino acid residues of the N-terminal region coincided with those of yeast microsomal b5, but the protein had a lower molecular weight determined to be 12,600 by SDS-PAGE and 9775 by mass spectrometry. The cytochrome b5-like protein enhanced the reduction rate of cytochrome c by OYE, and a plot of the reduction rates against its concentration showed a sigmoidal curve with an inflexion point at 6x10(-8) M of the protein.     

Presequence does not prevent folding of a purified mitochondrial precursor protein and is essential for association with a reticulocyte cytosolic factor(s)

Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) [EC 2.1.3.3] is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues, then is imported posttranslationally nto the mitochondrial matrix. We expressed rat pOTC in Escherichia coli, purified it in a denatured form, and showed that could be transported into isolated mitochondria in the presence of rabbit reticulocyte lysate [Murakami et al. (1988) J. Biol. Chem. 263, 18437-18442]. In order to compare the properties of the precursor and mature form of OTC, the rat mature OTC was synthesized in E. coli and purified. The recombinant OTC represented about 5% of the total bacterial protein and was present in both the supernatant and precipitate of the disrupted bacteria. The OTC, extracted from the precipitate with 8 M urea or 6 M guanidine.HCl, was essentially homogeneous, as judged by SDS-PAGE. When guanidine.HCl-denatured mature OTC was diluted and incubated at 0 degrees C for 40-60 h, it was reactivated to a specific activity of 170 mumol/min/mg protein at 37 degrees C (18% of that of the purified mature enzyme). Guanidine.HCl-denatured pOTC was activated to a specific activity of 125 mumol/min/mg protein under similar conditions. The native and reactivated OTC sedimented with an s20.w value of 6.2S, whereas the activated pOTC sedimented with an s20.w of 5.2S. The activated pOTC was more unstable than the reactivated OTC at 50 degrees C. These observations indicate that the presequence does not prevent pOTC from folding into an enzymatically active trimeric form, although the pOTC trimer appears to be less compact than the mature trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and characterization of a functional canine variant of cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.     

The membrane-interactive tail of cytochrome b(5) can function as a stop-transfer sequence in concert with a signal sequence to give inversion of protein topology in the endoplasmic reticulum

Sequence analyses of the C-terminal membrane intercalative region of the rat cytochrome b(5) indicated that this domain has, in addition to a signal sequence, a combined element of the classic stop-transfer sequence typically found in a variety of transmembrane proteins. Such bitopic protein arrangements arise by tandem but topogenically displaced activities of cleavable/noncleavable signal and stop-transfer sequences. A fusion precursor comprising an N-terminally linked prokaryotic signal sequence and the full-length of mammalian cytochrome b(5), including its C-terminal membrane insertion sequence, was engineered to investigate the outcome of this combination of signals on the targeting and topology of the cytochrome b(5) in the endoplasmic reticulum membrane. Precytochrome b(5) was cotranslationally translocated across the endoplasmic reticulum membrane. The signal-processed cytochrome b(5) was integrally anchored in the membrane with the globular domain facing the lumen. Thus, the topology of the signal sequence-directed cytochrome b(5) in the microsomal vesicle was reversed with respect to that of the native form. Posttranslational incubation of the precytochrome b(5) with microsomes resulted in a "loose" incorporation of the unprocessed form onto the surface of the vesicle. Our findings suggest that the membrane-insertion sequence of cytochrome b(5) has a functional stop-transfer sequence. We discuss the implications of these findings with respect to selective targeting of cytochrome b(5) to the endoplasmic reticulum membrane in the view that signal and stop-transfer sequences are often interchangeable or combined for topogenic functions.     

Molecular cloning and sequencing of genomic DNA encoding aminopeptidase I from Saccharomyces cerevisiae

Yeast aminopeptidase I is a vacuolar enzyme, which catalyzes the removal of amino acids from the NH2 terminus of peptides and proteins (Frey, J., and Rohm, K-H. (1978) Biochim. Biophys. Acta 527, 31-41). A yeast genomic DNA encoding aminopeptidase I was cloned from a yeast EMBL3A library and sequenced. The DNA sequence encodes a precursor protein containing 514 amino acid residues. The "mature" protein, whose NH2-terminal sequence was confirmed by automated Edman degradation, consists, based only on the DNA sequence, of 469 amino acids. A 45-residue presequence contains positively and negatively charged as well as hydrophobic residues, and its NH2-terminal residues could be arrayed in an amphiphilic alpha-helix. This presequence differs from the signal sequences which direct proteins across bacterial plasma membranes and endoplasmic reticulum or into mitochondria. It remains to be established how this unique presequence targets aminopeptidase I to yeast vacuoles and how this sorting utilizes classical protein secretory pathways. Further, the aminopeptidase I gene, localized previously by genetic mapping to yeast chromosome XI and called the LAP4 gene (Trumbly, R. J., and Bradley, G. (1983) J. Bacteriol. 156, 36-48), was determined by DNA blot analyses to be a single copy gene located on chromosome XI.     

Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).     

Erythrocyte Mr 28,000 transmembrane protein exists as a multisubunit oligomer similar to channel proteins

A novel Mr 28,000 erythrocyte transmembrane protein was recently purified and found to exist in two forms, "28kDa" and "gly28kDa," the latter containing N-linked carbohydrate (Denker, B. M., Smith, B. L., Kuhajda, F. P., and Agre, P. (1988) J. Biol. Chem. 263, 15634-15642). Although 28kDa protein resembles the Rh polypeptides biochemically, structural homologies were not identified by immunoblot or two-dimensional iodopeptide maps. The NH2-terminal amino acid sequence for the first 35 residues of purified 28kDa protein is 37% identical to the 26-kDa major intrinsic protein of lens (Gorin, M. B., Yancey, S. B., Cline, J., Revel, J.-P., and Horwitz, J. Cell 39, 49-59). Antisera to a synthetic peptide corresponding to the NH2-terminus of 28kDa protein gave a single reaction of molecular mass 28kDa on immunoblots of erythrocyte membranes. Selective digestions of intact erythrocytes and inside-out membrane vesicles with carboxypeptidase Y indicated the existence of a 5-kDa COOH-terminal cytoplasmic domain. Multiple studies indicated that 28kDa and gly28kDa proteins exist together as a multisubunit oligomer: 1) similar partial solubilizations in Triton X-100; 2) co-purification during ion exchange and lectin affinity chromatography; 3) cross-linking in low concentrations of glutaraldehyde; and 4) physical analyses of purified proteins and solubilized membranes in 1% (v/v) Triton X-100 showed 28kDa and gly28kDa proteins behave as a large single unit with Stokes radius of 61 A and sedimentation coefficient of 5.7 S. These studies indicate that the 28kDa and gly28kDa proteins are distinct from the Rh polypeptides and exist as a multisubunit oligomer. The 28kDa protein has NH2-terminal amino acid sequence homology and membrane organization similar to major intrinsic protein and other members of a newly recognized family of transmembrane channel proteins.     

Cumene hydroperoxide-supported demethylation reactions catalyzed by cytochrome P450 2B4 lacking the NH2-terminal sequence

Catalytic activities of cytochrome P450 2B4 lacking NH2-terminal amino acids 2-27 (wt Delta2B4) and that of truncated 2B4 containing a Pro to Ser mutation at position 221 were examined in a system supported by cumene hydroperoxide. Demethylation activities of either truncated 2B4 with N-methylaniline, N,N-dimethylaniline, and d-benzphetamine were lower than those of liver microsomal 2B4, whereas the rate of 1-phenylethanol oxidation to acetophenone catalyzed by liver microsomal and truncated 2B4 enzymes was nearly the same. The Km and Vmax values for cumene hydroperoxide in the demethylation of N-methylaniline by wt Delta2B4 were 20% and 28%, respectively, of those obtained for 2B4. The reaction with wt Delta2B4 displayed a lesser dependence on phospholipid than did that with 2B4, and a complex relationship between activity and substrate concentration. The results suggest that the NH2-terminal region contributes to interaction of oxidant, substrate, and phospholipid in cumene hydroperoxide-supported reactions catalyzed by cytochrome P450 2B4.     

The role of the Tim8p-Tim13p complex in a conserved import pathway for mitochondrial polytopic inner membrane proteins

Tim23p is imported via the TIM (translocase of inner membrane)22 pathway for mitochondrial inner membrane proteins. In contrast to precursors with an NH2-terminal targeting presequence that are imported in a linear NH2-terminal manner, we show that Tim23p crosses the outer membrane as a loop before inserting into the inner membrane. The Tim8p-Tim13p complex facilitates translocation across the intermembrane space by binding to the membrane spanning domains as shown by Tim23p peptide scans with the purified Tim8p-Tim13p complex and crosslinking studies with Tim23p fusion constructs. The interaction between Tim23p and the Tim8p-Tim13p complex is not dependent on zinc, and the purified Tim8p-Tim13p complex does not coordinate zinc in the conserved twin CX3C motif. Instead, the cysteine residues seemingly form intramolecular disulfide linkages. Given that proteins of the mitochondrial carrier family also pass through the TOM (translocase of outer membrane) complex as a loop, our study suggests that this translocation mechanism may be conserved. Thus, polytopic inner membrane proteins, which lack an NH2-terminal targeting sequence, pass through the TOM complex as a loop followed by binding of the small Tim proteins to the hydrophobic membrane spanning domains.     

Reconstitution of mitochondrial protein transport with purified ornithine carbamoyltransferase precursor expressed in Escherichia coli

Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues and imported posttranslationally into the mitochondrial matrix. The rat pOTC was synthesized in Escherichia coli using an expression vector containing a thermoinducible lambda pL promoter. The recombinant pOTC represented 5-10% of the total bacterial protein and was present in the precipitate of the disrupted bacteria. The precipitate was washed and pOTC was extracted with 8 M urea or 0.1% cetyltrimethylammonium bromide. The extracted pOTC was essentially homogeneous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified pOTC was cleaved to the intermediate-sized product of 37,000 Da by a processing protease partially purified from the matrix fraction of rat liver mitochondria. The purified recombinant pOTC, but not the mature form of OTC synthesized in E. coli and purified, competed with the in vitro-synthesized, radiolabeled pOTC for uptake and processing by the isolated rat liver mitochondria. The radiolabeled and purified recombinant pOTC could be imported into the isolated mitochondria and processed to the mature form in an energy- and rabbit reticulocyte lysate-dependent manner. When the purified pOTC was subjected to sucrose gradient centrifugation, it sedimented as a large aggregate of greater than 60 S in the absence of reticulocyte lysate, whereas it sedimented as a complex of about 5 S in the presence of the lysate. These observations together with our previous results indicate that a protein factor(s) present in the lysate interacts with pOTC and holds it in an import-competent form.     

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.     

Latent membrane perturbation activity of a mitochondrial precursor protein is exposed by unfolding.

We have purified milligram amounts of an importable mitochondrial precursor protein [the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase (DHFR)]. This has made it possible, for the first time, to perform detailed studies on the conformation of a precursor protein and its interaction with lipid membranes. The precursor protein closely resembled authentic mouse DHFR with respect to secondary structure (measured by CD spectra) and stability towards urea (measured by tryptophan fluorescence and enzyme activity). With this precursor protein, the presequence thus does not significantly alter the folding of the attached 'passenger protein'. In contrast to the corresponding presequence peptide, the native precursor exhibited only weak ability to disrupt vesicles with a low mol% of negatively charged lipids, suggesting that the passenger protein masks the amphiphilic properties of the presequence. The membrane-perturbing properties of the precursor were greatly enhanced by increasing the vesicles' content of negatively charged lipid or by denaturing the precursor in 5 M urea. Interaction with vesicles rich in acidic phospholipid was accompanied by partial unfolding of the precursor, suggesting that such a conformational change may also be involved in the interaction of the precursor with the mitochondrial membranes.     

The yeast F1-ATPase beta subunit precursor contains functionally redundant mitochondrial protein import information.

The NH2 terminus of the yeast F1-ATPase beta subunit precursor directs the import of this protein into mitochondria. To define the functionally important components of this import signal, oligonucleotide-directed mutagenesis was used to introduce a series of deletion and missense mutations into the gene encoding the F1-beta subunit precursor. Among these mutations were three nonoverlapping deletions, two within the 19-amino-acid presequence (delta 5-12 and delta 16-19) and one within the mature protein (delta 28-34). Characterization of the mitochondrial import properties of various mutant F1-beta subunit proteins containing different combinations of these deletions showed that import was blocked only when all three deletions were combined. Mutant proteins containing all possible single and pairwise combinations of these deletions were found to retain the ability to direct mitochondrial import of the F1-beta subunit. These data suggest that the F1-beta subunit contains redundant import information at its NH2 terminus. In fact, we found that deletion of the entire F1-beta subunit presequence did not prevent import, indicating that a functional mitochondrial import signal is present near the NH2 terminus of the mature protein. Furthermore, by analyzing mitochondrial import of the various mutant proteins in [rho-] yeast, we obtained evidence that different segments of the F1-beta subunit import signal may act in an additive or cooperative manner to optimize the import properties of this protein.     

The NH2-terminal 14-16 amino acids of mitochondrial and bacterial thiolases can direct mature ornithine carbamoyltransferase into mitochondria

Unlike most mitochondrial matrix proteins, the mitochondrial 3-oxoacyl-CoA thiolase [EC 2.3.1.16] is synthesized with no cleavable presequence and possesses information for mitochondrial targeting and import in the mature protein. This mitochondrial thiolase is homologous with the mature portion of peroxisomal 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase [EC 2.3.1.9] of Zoogloea ramigera along the entire sequence. A hybrid gene encoding the NH2-terminal 16 residues (MALLRGVFIVAAKRTP) of the mitochondrial thiolase fused to the mature portion of rat ornithine carbamoyltransferase [EC 2.1.3.3] (lacking its own presequence) was transfected into COS cells, and subcellular localization of the fusion protein was analyzed. Cell fractionation and immunocytochemical analyses showed that the fusion protein was localized in the mitochondria. These results indicate that the NH2-terminal 16 residues of the mitochondrial thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. The fusion protein containing the NH2-terminal 14 residues (MSTPSIVIASARTA) of the bacterial thiolase was also localized in the mitochondria. On the other hand, the fusion protein containing the corresponding portion (MQASASDVVVVHGQRTP) of the peroxisomal thiolase appeared not to be localized to the mitochondria. These results show that the import signal of mitochondrial 3-oxoacyl-CoA thiolase originated from the NH2-terminal portion of the ancestral thiolase. The ancestral enzyme might have already possessed a mitochondrial import activity when mitochondria appeared first, or that it might have acquired the import activity during evolution by accumulation of point mutations in the NH2-terminal portion of the enzyme.     

Presequence binding factor-dependent and -independent import of proteins into mitochondria.

A cytosolic protein factor(s) is involved in the import of precursor proteins into mitochondria. PBF (presequence binding factor) is a protein factor which binds to the precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) but not to the mature OTC, and is required for the mitochondrial import of pOTC. The precursors for aspartate aminotransferase and malate dehydrogenase as well as pOTC synthesized in a reticulocyte lysate were efficiently imported into the mitochondria. However, the precursors synthesized in the lysate depleted for PBF by treatment with pOTC-Sepharose were not imported. Readdition of the purified PBF to the depleted lysate fully restored the import. pOTC synthesized in the untreated lysate sedimented as a complex with a broad peak of around 9 S, whereas pOTC synthesized in the PBF-depleted lysate sedimented at an expected position of monomer (2.5 S). When the purified PBF was readded to the depleted lysate, pOTC sedimented as a complex of about 7 S. In contrast to most mitochondrial proteins, rat 3-oxoacyl-CoA thiolase is synthesized with no cleavable presequence and an NH2-terminal portion of the mature protein functions as a mitochondrial import signal. The thiolase synthesized in the PBF-depleted lysate could be efficiently imported into the mitochondria, and readdition of PBF had little effect on the import. The thiolase synthesized in the untreated, the PBF-depleted, or the PBF-readded lysate sedimented at an expected position of monomer (2.5 S). These observations provide support for the existence of PBF-dependent and -independent pathways of mitochondrial protein import.