Localization of the N-terminal methionine of rat liver cytochrome P-450 in the lumen of the endoplasmic reticulum

Recent cumulative evidence suggests that liver microsomal cytochrome P-450 (P-450) is exposed to the cytosol with the exception of the N-terminal peptide (amino acid residues 1 to 21), or two peptides (residues 1 to 60). We tested the localization of the N-terminal methionine residue of P-450IIB1 of rat liver microsomes in the natural membrane with the site-specific reagent fluorescein isothiocyanate. The N-terminus of isolated P-450 was stoichiometrically modified in solution with fluorescein isothiocyanate. In intact microsomes, the N-terminus was not modified but became accessible to the reagent when the membrane was dissolved with Triton X-100. Our results indicate that the N-terminus faces the lumen of the endoplasmic reticulum, and we propose that P-450 spans the membrane only once with amino acid residues 1 to 21.     

Chemical modification of rat liver microsomal cytochrome P-450: study of enzymic properties and membrane topology

Isolated rat liver cytochrome P-450IIB1 was alkylated and acetylated at primary amino groups, and the position of the modified amino acids in the protein was identified. Alkylation of up to nine amino groups did not disturb the interaction of reconstituted P-450 and NADPH-cytochrome-P-450 reductase in a way that hydroxylation of benzphetamine was altered, whereas deethylation of 7-ethoxycoumarin was gradually reduced in parallel with impaired 7-ethoxycoumarin binding. Acetylation of four lysine residues completely inhibited binding and metabolism of 7-ethoxycoumarin but not of benzphetamine. These results suggest the presence of different substrate binding sites on P-450. Exhaustive proteolysis of modified P-450 in proteoliposomes liberated all but the N-terminal modified peptide and 85 to 90% of the cytochrome's mass from intact proteoliposomes. These findings further support our previously proposed model of P-450 topology (Vergères, G., Winterhalter, K.H. and Richter, C. (1989) Biochemistry 28, 3650-3655), in which P-450 is anchored to the membrane with the N-terminal peptide only, the N-terminal methionine facing the lumenal interior.     

Membrane topology of guinea pig cytochrome P450 17 alpha revealed by a combination of chemical modifications and mass spectrometry

Cytochrome P450s in endoplasmic reticulum membranes function in the hydroxylation of exogenous and endogenous hydrophobic substrates concentrated in the membranes. The reactions require electron supplies from NADPH-cytochrome P450 reductase in the same membranes. The membranes play important roles in the reaction of cytochrome P450. The membrane topology of guinea pig P450 17alpha was investigated on the basis of the differences in reactivity to hydrophilic chemical modification reagents between those in the detergent-solubilized state and proteoliposomes. Recombinant guinea pig cytochrome P450 17alpha was purified from Escherichia coli and incorporated into liposome membranes. Lysine residues in the detergent-solubilized P450 17alpha and in the proteoliposomes were acetylated with acetic anhydride at pH 9.0, and the acidic amino acid residues were conjugated with glycinamide at pH 5.0 by the aid of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The modifications were performed under conditions where the denatured form, P420, was not induced. The modified P450 17alpha's were digested by trypsin, and the molecular weights of the peptide fragments were determined by MALDI-TOF mass spectrometry. From the increase in the molecular weights of the peptides, the positions of modifications could be deduced. In the detergent-solubilized state, 11 lysine residues and 7 acidic amino acid residues were modified, among which lysine residues at positions 29, 59, 490, and 492 and acidic residues at 211, 212, and/or 216 were not modified in the proteoliposomes. Both the N- and C-terminal domains and the putative F-G loop were concluded to be in or near the membrane-binding domains of P450 17alpha.     

Fluorescent energy transfer measurements on fluorescein isothiocyanate modified cytochrome P-450 LM2

The distance between the heme iron and the N-terminus of cytochrome P-450 LM2 was determined by fluorescence energy transfer measurements. Fluorescein isothiocyanate which was covalently bound to the N-terminal methionine was used as donor chromophor. The Ro value between fluorescein isothiocyanate and the heme was calculated to be 3.98 nm. The distance between the nitrogen of the N-terminal methionine and the heme was estimated with 2.84 +/- 0.23 nm excluding most likely the N-terminal amino acid of cytochrome P-450 LM2 to participate in the electron transfer to the heme iron. A cytochrome P-450 LM2 membrane model is proposed.     

Purification,reconstitution, and characterization of Na(+)/serine symporter,SstT, of Escherichia coli

A gene encoding Na(+)/serine symporter (SstT) of Escherichia coli has been cloned and sequenced in our laboratory [Ogawa et al. (1998) J. Bacteriol. 180, 6749-6752]. In an attempt to overproduce the protein and purify it, we first constructed a plasmid pTSTH in which the modified sstT gene (sstT gene with 8 successive codons for His at the 3'-terminus) is located downstream from the trc promoter. Upon induction by IPTG, the His-tagged SstT protein was overproduced (about 15% of total membrane proteins), and showed activity as high as the wild type SstT. The His-tagged SstT was solubilized with octylglucoside and purified to homogeneity using a nickel nitrilotriacetic acid (Ni(2+)-NTA) affinity resin. The N-terminal sequence (20 amino acid residues) of the purified protein showed that the sequence was identical to that deduced from the DNA sequence of the sstT gene and that the initiation methionine was excised. The purified His-tagged SstT was reconstituted into liposomes by the detergent dilution method. Reconstituted proteoliposomes mediated the transport of serine driven by an artificially imposed electrochemical Na(+) gradient. The K(m) and the V(max) values for serine transport with the proteoliposomes were 0.82 microM and 0.37 nmol/min/mg protein, respectively. Serine transport was inhibited by L-threonine, but not by other amino acids. The purified protein was stable for at least 6 months at -80 degrees C.     

Dynamic structures of adrenocortical cytochrome P-450 in proteoliposomes and microsomes: protein rotation study.

Purified adrenocortical microsomal cytochromes P-45017 alpha,lyase and P-450C21 were reconstituted with and without NADPH-cytochrome P-450 reductase in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles at a lipid to P-450 ratio of 35 (w/w) by cholate dialysis procedures. Trypsinolysis revealed that a considerable part of each P-450 molecule is deeply embedded in the lipid bilayer, on the basis of the observation of no detectable digestion for P-45017 alpha,lyase and the proteolysis-resistant membrane-bound heavy fragments for P-450C21. Rotational diffusion was measured in proteoliposomes and adrenocortical microsomes by observing the decay of absorption anisotropy, r(t), after photolysis of the heme-CO complex. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 1-2 ms to a time-independent value r3. Coexistence of a mobile population with an average rotational relaxation time phi of 138-577 microseconds and immobile (phi > or = 20 ms) populations of cytochrome P-450 was observed in both phospholipid vesicles and microsomes. Different tilt angles of the heme plane from the membrane plane were determined in proteoliposomes to be either 47 degrees or 63 degrees for P-45017 alpha,lyase from [r3/r(0)]min = 0.04 and either 38 degrees or 78 degrees for P-450C21 from [r3/r(0)]min = 0.19, when these P-450s were completely mobilized by incubation with 730 mM NaCl. Very different interactions with the reductase have been observed for the two P-450s in proteoliposomes. In the presence of the reductase, the mobile population of cytochrome P-450C21 was increased significantly from 79% to 96% due to dissociation of P-450 oligomers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-peptide antibodies and proteases as structural probes for the lactose/H+ transporter of Escherichia coli: a loop around amino acid residue 130 faces the cytoplasmic side of the membrane

From the amino acid sequence of the Escherichia coli lactose/H+ transporter, 7 hydrophilic segments were selected, 8-13 amino acids in length, and chemically synthesized, and anti-peptide antibodies were raised in rabbits. Apart from the antiserum to the synthetic COOH terminus (P408-417), which reacted strongly with the lactose/H+ transporter and has previously been used to localize the COOH terminus on the cytoplasmic face of the membrane, only those antibodies directed against the peptide corresponding to amino acid residues 125-135 (P125-135) exhibited a marked reaction with the transporter, while antibodies to the five other peptides reacted very weakly or not at all, suggesting that most of the hydrophilic segments are conformationally restricted or buried in the interior of the protein. Thermolysin treatment destroys the epitope on the transporter which is recognized by anti-P125-135 antibodies. Comparison of the kinetics and the extent of proteolysis of the transporter in right-side-out or inside-out cytoplasmic membrane vesicles or in reconstituted proteoliposomes suggests that the hydrophilic sequence from amino acid 125 to amino acid 135 is accessible to thermolysin only from one side, corresponding to the cytoplasmic face of the membrane. Furthermore, the experiments demonstrate that the transporter is inserted bimodally in a nonpreferential fashion into the proteoliposomes, confirming earlier results using antibodies to the synthetic COOH terminus of the transporter in conjunction with carboxypeptidase A treatment.     

Messenger RNA structure participating in the initiation of synthesis of cucumber mosaic virus coat protein

The sequence of the 5'-terminal 106 nucleotides of cucumber mosaic virus (strain Y) RNA 4, the mRNA coding for viral coat protein, has been determined. The first AUG was located at 77 nucleotides from the 5'-terminus and was confirmed to be an initiation codon by analysis of the N-terminal amino acid sequence of the protein. The nucleotide sequence (positions 77-106) beyond the AUG codon predicted the sequence of ten amino acids corresponding to the N-terminal region of the protein, which exactly matched the determined amino acid sequence containing an acetyl methionine as the N-terminal amino acid. The distance of the initiation codon AUG from the cap structure was 76 nucleotides and the longest among the mRNAs for coat protein of plant viruses so far reported (9-36 nucleotides). This noncoding region is rich in U residues (40%) and the number of G residues (21 nucleotides) is the largest among these mRNAs (usually 1 or 2 residues). A possible secondary structure is postulated for the region, which might be implicated in efficient translation of the RNA 4 in vivo.     

Molecular cloning of monkey liver cytochrome P-450 cDNAs: similarity of the primary sequences to human cytochromes P-450.

Three cDNAs coding for monkey cytochrome P-450 (P450) 2C, 2E and 3A (MKmp13, MKj1 and MKnf2, respectively) were isolated from a lambda gt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey, using cDNA fragments for human P450 2C, 2E and 3A as respective probes. MKmp13 and MKnf2 were 1901 and 2032 bp long, containing entire coding regions for polypeptides of 490 and 503 residues, respectively. The deduced N-terminal amino acid sequences of MKmp13 and MKnf2 were identical with those of P450-MK1 and P450-MK2, which had been purified from liver microsomes of untreated and polychlorinated biphenyl (PCB)-treated crab-eating monkeys, respectively. MKj1 was 1508 bp long, encoding a polypeptide of 449 residues, which is presumed to lack N-terminal 45 residues as compared with the sequence for human P450 2E1. Northern blot analysis indicated that monkey P450 2C, 2E and 3A mRNAs were expressed constitutively in monkey livers. P450 2E and 3A mRNAs were induced by both 3MC and PCB, while P450 2C mRNA was induced only by PCB. The deduced amino acid sequences of four monkey cytochrome P-450 cDNAs, including P450 1A1 (MKah1) which we isolated previously, were more than 92% identical with those of corresponding human cytochrome P-450 cDNAs.     

Structure of the murine Ia-associated invariant (Ii) chain as deduced from a cDNA clone

The invariant (Ii) chain is a membrane-spanning glycoprotein found intracellularly associated with class II major histocompatibility complex (MHC) molecules. Using hybrid-selected translation and the Ii-specific monoclonal antibody In-1, we have isolated a cDNA clone (pIi-5) coding for most of the Ii chain. Sequence analysis of this clone reveals an open reading frame encoding 169 amino acid residues. The protein is rich in methionine and contains two potential N-glycosylation sites. No stretch of uncharged amino acid residues, characteristic for a membrane-spanning segment, is found close to the COOH-terminal end. There is one, however, close to the NH2-terminal end. As it is know that approximately 20 amino acid residues of Ii chain are exposed on the cytoplasmic side, we conclude that the Ii chain spans the membrane exposing the NH2 terminus on the cytoplasmic side and the COOH terminus on the luminal side.     

Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11.5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.     

Toxic components of the venom of the Central Asian scorpion, Orthochirus scrobiculosus

Four polypeptide neurotoxins, possessing paralytic activity for mice, were isolated from the venom of the Central Asian black scorpion Orthochirus scrobiculosus. All these toxins, Os-1 - Os-4, were shown to be homogeneous by disc-electrophoresis and N-terminal group analyses. The amino acid composition of the toxins was determined, methionine residues being found in toxin Os-1. The neurotoxin Os-3 was subjected to tryptic and chymotryptic hydrolyses and its total amino acid sequence was established. It was shown that neurotoxin Os-3 consists of 67 amino acid residues with four intramolecular disulfide bonds.     

Membrane topology of ABC-type macrolide antibiotic exporter MacB in Escherichia coli

MacB is an ABC-type membrane protein that exports only macrolide compounds containing 14- and 15-membered lactones, cooperating with a membrane fusion protein, MacA, and a multifunctional outer membrane channel, TolC. We determined the membrane topology of MacB by means of site-specific competitive chemical modification of single cysteine mutants. As a result, it was revealed that MacB is composed of four transmembrane (TM) segments with a cytoplasmic N-terminal nucleotide binding domain of about 270 amino acid residues and a periplasmic large hydrophilic polypeptide between TM segments 1 and 2 of about 200 amino acid residues.     

Endoplasmic reticulum retention determinants in the transmembrane and linker domains of cytochrome P450 2C1

The cytochrome P450 2C1 N-terminal signal anchor sequence mediates direct retention of the protein in the endoplasmic reticulum and consists of a hydrophobic transmembrane domain, residues 3-20, followed by a hydrophilic linker, residues 21-28. Fusions of the N-terminal 21 or 28 amino acids of P450 2C1 to green fluorescent protein resulted in endoplasmic reticulum localization of the chimera in transfected cells. Disruption of microtubules by nocodazole treatment resulted in redistribution into a punctate pattern for the 1-21, but not for the 1-28, chimera indicating that the linker was preventing transport from the endoplasmic reticulum but was not required for retrieval to the endoplasmic reticulum from the pre-Golgi compartment. In the 1-28 chimera, mutations of residues 21-23 (KQS) in the linker resulted in redistribution of the chimera after nocodazole treatment. Mutations in the transmembrane domain affected both direct retention in the endoplasmic reticulum and retrieval from the pre-Golgi compartment, and although structural requirements for each process are distinct, in both cases the arrangement of amino acids and distribution of hydrophobicity are critical. In contrast, the linker region exhibits a sequence-specific requirement for direct retention in the endoplasmic reticulum.     

The single-copy gene psbS codes for a phylogenetically intriguing 22 kDa polypeptide of photosystem II.

Recombinant phages that encode the complete precursor polypeptide for the 22 kDa polypeptide associated with photosystem II have been serologically selected from two lambda gt11 expression libraries made from polyadenylated RNA of spinach seedlings. The cDNAs hybridize to a 1.3 kb RNA species. The precursor protein is comprised of 274 amino acid residues and carries an N-terminal transit peptide of probably 69 amino acid residues. The mature protein exhibits four predicted transmembrane segments and is shown to be an integral component of photosystem II originating in a single-copy gene. The unique characteristics of this protein are: (i) it is the result of a gene-internal duplication of an ancestor with two membrane spans, (ii) a striking resemblance to LHC I/II, CP24/CP29 apoproteins, and ELIPs, although it does not bind chlorophyll and is present in cyanobacteria, and, as these proteins, (iii) it integrates into the membrane with uncleaved routing signals that display remarkable resemblance to patterns found in bipartite transit peptides.     

Reconstitution of transport-active multidrug resistance protein 2 (MRP2; ABCC2) in proteoliposomes

The apical multidrug resistance protein MRP2 (symbol ABCC2) is an ATP-dependent export pump for anionic conjugates in polarized cells. MRP2 has only 48% amino acid identity with the paralog MRP1 (ABCC1). In this study we show that purified recombinant MRP2 reconstituted in proteoliposomes is functionally active in substrate transport. The Km values for ATP and LTC4 in the transport by MRP2 in proteoliposomes were 560 microM and 450 nM, respectively. This transport function of MRP2 in proteoliposomes was dependent on the amount of MRP2 protein present and was determined to 2.7 pmol x min(-1) x mg MRP2(-1) at 100 nM LTC4. Transport was competitively inhibited by the quinoline derivative MK571 with 50% inhibition at about 12 microM. Our data document the first reconstitution of transport-active purified recombinant MRP2. Binding and immunoprecipitation experiments indicated that MRP2 preferentially associates with the chaperone calnexin, but co-reconstitution studies using purified MRP2 and purified calnexin in proteoliposomes suggested that the LTC4 transport function of MRP2 is not dependent on calnexin. The purified, transport-active MRP2 may serve to identify additional interacting proteins in the apical membrane of polarized cells.     

Archaeal N-terminal protein maturation commonly involves N-terminal acetylation: a large-scale proteomics survey

We present the first large-scale survey of N-terminal protein maturation in archaea based on 873 proteomically identified N-terminal peptides from the two haloarchaea Halobacterium salinarum and Natronomonas pharaonis. The observed protein maturation pattern can be attributed to the combined action of methionine aminopeptidase and N-terminal acetyltransferase and applies to cytosolic proteins as well as to a large fraction of integral membrane proteins. Both N-terminal maturation processes primarily depend on the amino acid in penultimate position, in which serine and threonine residues are over represented. Removal of the initiator methionine occurs in two-thirds of the haloarchaeal proteins and requires a small penultimate residue, indicating that methionine aminopeptidase specificity is conserved across all domains of life. While N-terminal acetylation is rare in bacteria, our proteomic data show that acetylated N termini are common in archaea affecting about 15% of the proteins and revealing a distinct archaeal N-terminal acetylation pattern. Haloarchaeal N-terminal acetyltransferase reveals narrow substrate specificity, which is limited to cleaved N termini starting with serine or alanine residues. A comparative analysis of 140 ortholog pairs with identified N-terminal peptide showed that acetylatable N-terminal residues are predominantly conserved amongst the two haloarchaea. Only few exceptions from the general N-terminal acetylation pattern were observed, which probably represent protein-specific modifications as they were confirmed by ortholog comparison.     

The N-terminal region of Escherichia coli lactose permease mediates membrane contact of the nascent polypeptide chain

Plasmids encoding N-terminal segments of the Escherichia coli lactose permease (also referred to as lactose carrier) have been used to analyze the biosynthesis and membrane insertion of this complex integral protein of the cytoplasmic membrane. Such truncated polypeptides were found to be stably associated with the membrane and to resemble the full-length protein with respect to their solubilization characteristics. Membrane-bound and free cytoplasmic polysomes were prepared from plasmid-bearing cells and incubated in the presence of [35S]methionine to permit completion of polypeptides initiated in vivo. Under these conditions, lactose permease was found to be radiolabeled in the fraction of membrane-bound polysomes; beta-galactosidase, used as a control, was translated almost exclusively by free polysomes. From similar experiments with N-terminal segments of lactose permease, we estimate that at most a polypeptide of 120 amino acid residues emerging from the ribosome is needed to target the nascent chain to the lipid bilayer and to mediate attachment of the ribosome to the membrane during elongation. Additional data support the idea that even shorter N-terminal sequences of 50 and 71 amino acid residues contain sufficient 'information' to provide contact with the membrane.     

Cloning of cDNA for Vitellogenin of the Parasitoid Wasp, Pimpla nipponica (Hymenoptera: Apocrita: Ichneumonidae): Vitellogenin Primary Structure and Evolutionary Considerations

The cDNA for vitellogenin (Vg) of the parasitoid wasp Pimpla nipponica (Hymenoptera: Apocrita) was cloned and sequenced.¹ The deduced amino acid sequence with 1807 residues was obtained. The N-terminal 20 amino acids chemically determined for vitellin (Vn) agreed completely with the deduced 20 amino acids that follow the 16 amino acid residues for putative signal peptide. The cDNA clone for the Vg of the turnip sawfly Athalia rosae (Hymenoptera: Symphyta), previously obtained and partially sequenced, was also completely sequenced and the amino acid sequence deduced. Amino acid sequences were compared between these two species and also with known Vg sequences from other insects. Common to all these insects is the presence of two long regions with relatively well-conserved amino acid sequences, one near the N-terminal extending 267–282 residues (including two cysteines at conserved locations), and the other starting at position 450 to 655 and extending 279–283 residues, and of a region at the C-terminal extending some 200 residues (about 250 in Aedes aegypti due to the presence of a serine-rich stretch) with 10 cysteines at conserved locations. A molecular phylogenetic tree was constructed.     

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.