A peptide corresponding to an export-defective mutant OmpA signal sequence with asparagine in the hydrophobic core is unable to insert into model membranes

We have examined the comparative membrane interaction properties of synthetic peptides corresponding to the wild-type and an export-defective, mutated signal sequence from the Escherichia coli outer membrane protein, OmpA. As part of a collaborative study of the effects of various alterations on the function of the OmpA signal sequence and the biophysical properties of the corresponding synthetic peptides, we incorporated the small, neutral polar residue, asparagine, into the hydrophobic core in place of Ile-8. This seemingly minor perturbation to the signal sequence caused a complete block of export in vivo (J. Goldstein, S. Lehnhardt, and M. Inouye, following paper). We now explore in detail the difference in the properties of the wild-type and the Ile-8----Asn OmpA signal peptides. The fluorescent residue Trp was substituted in both peptides in place of the wild-type Phe at position 15. This mutation is silent phenotypically and provides a superb probe of membrane interaction. We find that the Asn substitution leaves the conformational properties of the signal sequence essentially unchanged, but prevents any significant interaction of the peptide with a lipid bilayer. Asparagines are very underrepresented among known signal sequences. We believe this low frequency to be due to the lowering of mean residue hydrophobicity caused by incorporation of Asn and the consequent reduced ability to bind and insert into membranes.     

Signal peptidases recognize a structural feature at the cleavage site of secretory proteins

The cloning of the gene for staphylococcal nuclease A in the pIN-III-OmpA secretion vector results in a hybrid protein which is processed by signal peptidase I, yielding an active form of the nuclease that is secreted across the cytoplasmic membrane (Takahara, M., Hibler, D., Barr, P. J., Gerlt, J. A., and Inouye, M. (1985) J. Biol. Chem. 260, 2670-2674). Using oligonucleotide-directed site-specific mutagenesis, we have constructed a set of mutants at the cleavage site area of the precursor hybrid protein designed to alter progressively the predicted secondary structure of the cleavage site. Our results show that processing becomes increasingly defective as the turn probability decreases. These results are consistent with the structural requirement that we found for the processing of lipoprotein by signal peptidase II (Inouye, S., Duffaud, G., and Inouye, M. (1986) J. Biol. Chem. 261, 10970-10975). We conclude that secretory precursor proteins have a distinct secondary structural requirement at their cleavage site for processing by signal peptidase I, as well as by signal peptidase II.     

Pyridoxal 5'-phosphate-dependent histidine decarboxylase. Nucleotide sequence of the hdc gene and the corresponding amino acid sequence

The nucleotide sequence of a 1.3-kilobase NaeI fragment from Morganella morganii AM-15 that contains the gene for histidine decarboxylase has been determined. The gene was initially identified among total chromosomal digests using a mixed sequence oligonucleotide probe corresponding to amino acids 11-16 of histidine decarboxylase and then cloned on a 5.5-kilobase PstI fragment. The structural gene contains 1131 nucleotides and encodes 377 amino acids with the sequence: (sequence: in text). The independently determined NH2-terminal sequence of this enzyme (Tanase, S., Guirard, B. M., and Snell, E. E. (1985) J. Biol. Chem. 260, 6738-6746) and the amino acid sequences of two tryptic peptides reported in the accompanying paper (Hayashi, H., Tanase, S., and Snell, E. E. (1986) J. Biol. Chem. 261, 11003-11009) are localized in the sequence presented here; the lysine that binds pyridoxal phosphate is situated at residue 232, whereas the serine that binds the adduct formed between pyridoxal phosphate and the inhibitor alpha-fluoromethylhistidine is positioned at residue 322.     

Nucleoside diphosphate kinase from Myxococcus xanthus. I. Cloning and sequencing of the gene

By photoaffinity labeling with a photolysable analog of GTP, 8-N3GTP, we were able to find at least five distinct GTP-binding proteins in Myxococcus xanthus; two of them located in the membrane and the other three in the soluble fraction. The amino-terminal sequence of the 16-kDa GTP-binding protein from the soluble fraction was determined, and the gene that encodes this protein was isolated and cloned using degenerate oligonucleotides as a probe. The DNA sequence of the gene was determined, which did not show similarity with other known proteins. The gene product was overexpressed in Escherichia coli, by using the lacZ promoter, to a level of 13% of the soluble protein. Attempts to isolate deletion mutants were unsuccessful, although double crossing-over events leading to a deletion mutation of the gene were detected by Southern blot hybridization. This result indicates that this gene is essential for cell growth. In the following paper (Mu?oz-Dorado, J., Inouye, S., and Inouye, M. (1990) J. Biol. Chem. 265, 2707-2712), the gene product was biochemically characterized and identified to be a nucleoside diphosphate kinase.     

In vivo effect of asparagine in the hydrophobic region of the signal sequence

On the basis of the biophysical studies on the synthetic mutant (Ile-8----Asn) OmpA signal peptide in the preceding paper (Hoyt, D. C., and Gierasch, L.M. (1991) J. Biol. Chem. 266, 14406-14412), the in vivo effects of the same mutation were examined by fusing the mutant OmpA signal sequence to Staphylococcus aureus nuclease or TEM beta-lactamase. The mutation in which the isoleucine residue at position 8 of the OmpA signal sequence of Escherichia coli was replaced with a neutral polar residue, asparagine, resulted in a defective signal peptide. The mutant signal sequence was unable to be processed, and the precursor molecule accumulated in the cytoplasmic as well as in the membrane fractions, indicating that the Ile-8----Asn OmpA signal sequence is not competent for translocating nuclease A or beta-lactamase across the membrane. This result is consistent with the in vitro studies on the Ile-8----Asn OmpA signal peptide, which indicated that the mutant signal peptide was unable to penetrate into the hydrophobic core of the lipid bilayer. Other asparagine or glutamine substitution mutations in the hydrophobic region of the OmpA signal sequence were also examined. Interestingly, the OmpA signal sequence with either Ile-8----Gln, Val-10----Asn, or Leu-12----Asn mutation was completely defective as the Ile-8----Asn OmpA signal sequence, while the Ile-6----Asn and Ala-9----Asn OmpA nucleases were able to be processed to secrete nuclease, although the processing occurred at a much slower rate than the wild-type OmpA nuclease. These results indicate that the defects depend on the position of the lesion in the hydrophobic core of the OmpA signal sequence.     

A positive residue in the hydrophobic core of the Escherichia coli lipoprotein signal peptide suppresses the secretion defect caused by an acidic amino terminus.

The signal peptide of secretory proteins requires a basic amino terminus followed by a stretch of hydrophobic residues to effect efficient translocation of precursor proteins. Replacement of the positively charged amino-terminal residues of prolipoprotein by acidic amino acids decreased the rate of precursor translocation (Inouye, S., Soberon, X., Franceschini, T., Nakamura, K., Itakura, K., and Inouye, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3438-3441; Vlasuk, G. P., Inouye, S., Ito, H., Itakura, K., and Inouye, M. (1983) J. Biol. Chem. 258, 7141-7148). We demonstrate here that an arginine residue, but not an aspartate, when localized at position 9 of the hydrophobic region of the lipoprotein signal peptide, is able to suppress intramolecularly the processing defect caused by an acidic amino terminus. Furthermore, when present at position 14 of the signal peptide, this positive residue, but not aspartate, was able to support efficient translocation of unmodified prolipoprotein. This demonstrates that a positive residue can restore the function of a severely defective signal peptide and need not be localized at the amino terminus to do so. Both aspartate and arginine substitution at position 14 of the lipoprotein signal peptide stimulated prolipoprotein synthesis. This effect was position-specific, did not require precursor translocation, and was dominant to the inhibition of synthesis caused by an acidic amino terminus.     

Antigenic variation among group A streptococcal M proteins. Nucleotide sequence of the serotype 5 M protein gene and its relationship with genes encoding types 6 and 24 M proteins

The 1479-base pair (bp) nucleotide sequence of the serotype 5 M protein gene (smp5) from Streptococcus pyogenes contains three distinct types of tandemly repeated sequences, designated A, B, and C. Repeat A (21 bp x 6, in the 5'-half of smp5), shares no homology with the types 6 or 24 M protein genes (Hollingshead, S. K., Fischetti, V. A., and Scott, J. R. (1986) J. Biol. Chem. 261, 1677-1686; Mouw, A. R., Beachey, E. H., and Burdett, V. (1988) J. Bacteriol., in press). Repeat B (75 bp x 3.6, in the center of smp5) is also present in the M6, but not in the M24 gene. Repeat C (105 bp x 2.7, just distal to the B repeats) shares homology with repeats in both the M6 and M24 genes. All three genes share extensive homology in their 3'-halves and in 5' sequences encoding the N-terminal signal peptides, but between these two regions there are highly variable sequences that are responsible for antigenic diversity. These relationships suggest that both intergenic and intragenic recombination has occurred during the evolution of distinct M protein serotypes. All three M proteins contain conserved hydrophobic and proline-rich sequences at their C-terminal ends, suggestive of a membrane anchor and a peptidoglycan spanning region.     

Location of the ATP gamma-phosphate-binding sites on rat liver carbamoyl-phosphate synthetase I. Studies with the ATP analog 5'-p-fluorosulfonylbenzoyladenosine.

The gamma-phosphate subsites of the MgATP sites of rat liver carbamoyl-phosphate synthetase I have been defined by use of the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The synthetase utilizes two molecules of MgATP, apparently in mechanistically discrete steps and at separate MgATP sites. Sequence analysis has revealed internal duplication within the synthetase molecule (Nyunoya, H., Broglie, K.E., Widgren, E.E., and Lusty, C.J. (1985) J. Biol. Chem. 260, 9346-9356) and, based on sequence similarity with other nucleotide-binding proteins, potential ATP sites have been predicted for each of the duplicated sequences. The present FSBA studies have identified four peptides within carbamoyl-phosphate synthetase I that are involved in binding MgATP. Differential effects of N-acetylglutamate, a required allosteric activator, on the interaction of FSBA with the peptides were utilized to develop the following model for two distinct MgATP sites. Peptides 631-638 and 1327-1348 (with Cys1327 and/or Cys1337 modified by FSBA) apparently form part of the binding site for the MgATP involved in bicarbonate activation. Peptides 1310-1317 and 1445-1454 (with Tyr1450 modified by FSBA) apparently form part of the binding site for the MgATP involved in phosphorylation of enzyme-bound carbamate. Each of these MgATP sites contains a peptide from one of the internal duplicated regions of the enzyme molecule, which have previously been suggested as containing MgATP sites (Nyunoya, H., Broglie, K. E., Widgren, E. E., and Lusty, C. J. (1985) J. Biol. Chem. 260, 9346-9356; Powers-Lee, S. G., and Corina, K. (1987) J. Biol. Chem. 262, 9052-9056), as well as a peptide from the flexible C-terminal region.     

A functional prolipoprotein signal peptide with a deletion of four amino acid residues from the hydrophobic region

The deletion of several codons within the signal sequence coding region of the Escherichia coli lipoprotein gene has been accomplished by oligonucleotide-directed site-specific mutagenesis. The deletion of the Leu-13 residue in a mutant in which two glycine residues had previously been deleted from the hydrophobic region (Inouye, S., Vlasuk, G., Hsiung, H., and Inouye, M. (1984) J. Biol. Chem. 259, 3729-3733) was found to cause the accumulation of the unmodified form of the protein in the cytoplasm and cytoplasmic membrane. This mutation also caused a cessation in cell growth within 15 min after synthesis of the mutant protein was induced. A deletion of the Val-7 residue was capable of suppressing the effect of the Leu-13 deletion when both are present. However, by itself the Val-7 deletion appeared to have little effect on the glycine mutant. The ability of the signal sequence to mediate the secretion of the protein after the deletion of 4 residues from the hydrophobic region demonstrates a surprising degree of flexibility in the length of this region. The deletion mutations were also found to have an unusual effect on the rate of synthesis of lipoprotein.     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora crassa. Isolation and sequences of several cyanogen bromide peptides from the NH2-terminal portion of the peptide chain

Previous studies of the amino acid sequence of the NAD-specific glutamate dehydrogenase of Neurospora crassa (EC 1.4.1.2) resulted in the assignments of peptides to four fragments, the longest being the COOH-terminal 669 residues of the protein. A further study of peptides derived by cyanogen bromide cleavage by different separation methods has yielded additional peptides that have provided new information concerning the sequence and has given overlaps of previously known sequences. This has permitted establishment of 313 residues in one sequence (fragment II). This is in addition to a sequence of 43 residues (fragment I) at the NH2-terminal end and a sequence of 669 residues (fragment III) previously established at the COOH-terminal end of the molecule. The present status of our knowledge of the overall sequence is given in the accompanying papers, together with some views regarding the conformation of the protein (Haberland, M.E., Chen, C.-W., and Smith, E.L. (1980) J. Biol. Chem. 255, 7993-8000, and Austen, B.M., Haberland, M.E., and Smith, E.L. (1980) J. Biol. Chem. 255, 8001-8004).     

Novel multigene families encoding highly repetitive peptide sequences. Sequence analyses of rat and mouse proline-rich protein cDNAs

Multigene families encode the proline-rich proteins that are so prominent in human saliva and are dramatically induced in mouse and rat salivary glands by isoproterenol treatment and by feeding tannins. A cDNA encoding an acidic proline-rich protein of rat has been sequenced (Ziemer, M. A., Swain, W. F., Rutter, W. J., Clements, S., Ann, D. K., and Carlson D. M. (1984) J. Biol. Chem. 259, 10475-10480). This study presents the nucleotide sequences of five additional proline-rich protein cDNAs complementary to both mouse and rat parotid and submandibular gland mRNAs. Amino acid compositions deduced from the nucleotide sequences are typical for proline-rich proteins: 25-45% proline, 18-22% glycine, and 18-22% glutamine and generally an absence of sulfur-containing amino acids except for the initiator methionine. These proline-rich proteins display unusual repeating peptide sequences of 14-19 amino acids. The derived amino acid sequence of the cDNA insert of plasmid pMP1 from mouse has a 19-amino acid sequence which is repeated four times. The inserts of plasmids pUMP40 and pUMP4 also from mouse encode for 12 and 11 repeats of a 14-amino acid peptide, respectively. These repetitive sequences, and others from rat and mouse cDNAs and from human genomic clones, all show very high homologies and likely evolved from duplication of internal portions of an ancestral gene. Gene conversion could account for the high degree of conservation of nucleotide sequences of the repeat regions. Protein derived from the nucleotide sequences are all characterized by four general regions: a putative signal peptide, a transition region, the repetitive region, and a carboxyl-terminal region. The 5'-flanking sequences and sequences encoding the putative signal peptides are highly conserved (greater than 94%) in all six cDNAs. This sequence conservation may be important in the regulation of the biosynthesis of these unusual proteins.     

Internal deletions in the gene for an Escherichia coli outer membrane protein define an area possibly important for recognition of the outer membrane by this polypeptide

A series of overlapping deletions has been constructed in the ompA gene which encodes the 325-residue Escherichia coli outer membrane protein OmpA. Immunoelectron microscopy showed that the OmpA fragments were either located in the periplasmic space or were associated with the outer membrane. Apparently an area between residues 154 and 180 is required for this association; all proteins missing this area were found to be periplasmic. The nature of this association remained unknown; no membrane-protected tryptic fragments could be identified for any of these polypeptides. Hybrid genes were constructed encoding parts of the periplasmic maltose binding protein and an area of the ompA gene coding for residues 154-274. The corresponding proteins were not localized to the outer membrane but remained attached to the outer face of the plasma membrane, possibly because the normal mechanism of release from this membrane was impaired. In the OmpA protein the conspicuous sequence Ala180-Pro-Ala-Pro-Ala-Pro-Ala-Pro187 exists. Frameshift mutants were constructed to eliminate this sequence. There was no effect on the incorporation of the mutant proteins into the outer membrane. Thus, this "hinge" region is not involved in sorting. A proposal suggesting the existence of a sorting signal common to several outer membrane proteins (Benson, S. A., Bremer, E., and Silhavy, T. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3830-3834) was subsequently rejected (Bosch, D., Leunissen, J., Verbakel, J., de Jong, M., van Erp, H., and Tommassen, J. (1986) J. Mol. Biol. 189, 449-455; Freudl, R., Schwarz, H., Klose, M., Movva, N. R., and Henning, U. (1985) EMBO J. 4, 3593-3598). Although it is not known whether or not the outer membrane association observed represents a step in the normal sorting mechanism, it is concluded that it remains an open question whether or not a sorting signal, as proposed originally, exists in outer membrane proteins.     

Structural determinants in addition to the amino-terminal sorting sequence influence membrane localization of Escherichia coli lipoproteins.

The lipid-modified nine-residue amino-terminal sequence of the mature form of the major outer membrane lipoprotein of Escherichia coli contains information that is responsible for sorting to either the inner or outer membrane. Fusion of this sorting sequence to beta-lactamase is sufficient for localization of the resultant lipo-beta-lactamase to the outer membrane (J. Ghrayeb and M. Inouye, J. Biol. Chem. 259:463-467, 1984). Substitution of the serine adjacent to the amino-terminal lipid-modified cysteine residue of the sorting sequence with the negatively charged residue aspartate causes inner membrane localization (K. Yamaguchi, F. Yu, and M. Inouye, Cell 53:423-432, 1988). Fusion of the aspartate-containing nine-residue inner membrane localization signal to the normally outer membrane lipoprotein bacteriocin release protein does cause partial localization to the inner membrane. However, a single replacement of the glutamine adjacent to the amino-terminal lipid-modified cysteine residue of bacteriocin release protein with aspartate causes no inner membrane localization. Therefore, an aspartate residue itself lacks the information necessary for inner membrane sorting when removed from the structural context provided by the additional eight residues of the sorting sequence. Although the aspartate-containing inner membrane sorting sequence causes an almost quantitative localization to the inner membrane when fused to the otherwise soluble protein beta-lactamase, this sequence cannot prevent significant outer membrane localization when fused to proteins (bacteriocin release protein and OmpA) normally found in the outer membrane. Therefore, structural determinants in addition to the amino-terminal sorting sequence influence the membrane localization of lipoproteins.     

Structural determinants within residues 180-199 of the rodent alpha 5 nicotinic acetylcholine receptor subunit involved in alpha-bungarotoxin binding

Synthetic peptides corresponding to sequence segments of the nicotinic acetylcholine receptor (nAChR) alpha subunits have been used to identify regions that contribute to formation of the binding sites for cholinergic ligands. We have previously defined alpha-bungarotoxin (alpha-BTX) binding sequences between residues 180 and 199 of a putative rat neuronal nAChR alpha subunit, designated alpha 5 [McLane, K. E., Wu, X., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 9816-9824], and between residues 181 and 200 of the chick neuronal alpha 7 and alpha 8 subunits [McLane, K. E., Wu, X., Schoepfer, R., Lindstrom, J., & Conti-Tronconi, B. M. (1991) J. Biol. Chem. (in press)]. These sequences are relatively divergent compared with the Torpedo and muscle nAChR alpha 1 alpha-BTX binding sites, which indicates a serious limitation of predicting functional domains of proteins based on homology in general. Given the highly divergent nature of the alpha 5 sequence, we were interested in determining the critical amino acid residues for alpha-BTX binding. In the present study, the effects of single amino acid substitutions of Gly or Ala for each residue of the rat alpha 5(180-199) sequence were tested, using a competition assay, in which peptides compete for 125I-alpha-BTX binding with native Torpedo nAChR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the site of acetyl-S-enzyme formation on avian liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase

Avian liver mitochondrial hydroxymethylglutaryl-CoA synthase contains an active-site cysteine involved in forming the labile acetyl-S-enzyme intermediate. Identification of and assignment of function to this cysteine have been accomplished by use of an experimental strategy that relies upon generation and rapid purification of the S-acetylcysteine-containing active-site peptide under mildly acidic conditions that stabilize the thioester adduct. Automated Edman degradation techniques indicate the peptide's sequence to be Arg-Glu-Ser-Gly-Asn-Thr-Asp-Val-Glu-Gly-Ile-Asp-Thr-Thr-Asn-Ala-Cys-Tyr. The acetylated cysteine corresponds to position 129 in the sequence deduced from cDNA data for the hamster cytosolic enzyme [Gil, G., Goldstein, J.L., Slaughter, C.A., & Brown, M.S. (1986) J. Biol. Chem. 261, 3710-3716]. The acetyl-peptide sequence overlaps that reported for a tryptic peptide that contains a cysteine targeted by the affinity label 3-chloropropionyl-CoA [Miziorko, H. M., & Behnke, C. E. (1985) J. Biol. Chem. 260, 13513-13516]. Thus, availability of these structural data allows unambiguous assignment of the acetylation site on the protein as well as a refinement of the mechanism explaining the previously observed affinity labeling of the enzyme.     

Characterization of the carboxyl-terminal sequences responsible for protein retention in the endoplasmic reticulum.

The COOH-terminal sequence KDEL has been shown to be essential for the retention of several proteins in the lumen of the endoplasmic reticulum (Munro S., and Pelham, H. R. B. (1987) Cell 48, 899-907; Pelham, H. R. B. (1988) EMBO J. 7, 913-918; Mazzarella; R. A., Srinivasan, M., Haugejorden, S. M., and Green, M. (1990) J. Biol. Chem. 265, 1092-1101). We have previously demonstrated that variants to the KDEL retention signal, particularly at the initial two positions of the tetrapeptide, can be made without affecting its ability to direct intracellular retention when appended to the neuropeptide Y precursor (pro-NPY) (Andres, D. A., Dickerson, I. M., and Dixon, J. E. (1990) J. Biol. Chem. 265, 5952-5955). To further investigate the nature of the KDEL retention signal, oligonucleotide-directed mutagenesis and transfection was used to generate stable mouse anterior pituitary AtT-20 cell lines expressing pro-NPY mutants with variants of the KDEL sequence added to their direct carboxyl terminus. Analyses of dibasic processing and indirect immunofluorescent microscopy of AtT-20 subclones were consistent with the retention of the pro-NPY mutants bearing the COOH-terminal extensions QDEL, KEDL, or KDEI within the endoplasmic reticulum. A change in the final amino acid of the tetrapeptide from Leu to Val abolished retention completely, and the peptide hormone was processed and secreted. These results indicate that only a limited number of conservative changes can be made to the final two positions of the tetrapeptide without abolishing activity and suggest a highly specific interaction of the retention signal and the KDEL receptor.     

Structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. Sequences of the overlapping peptides, ordering the CNBr fragments, and the complete amino acid sequence

The amino acid sequence of the largest fragment, CNBr Ia (203 residues) has been reported (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749). The amino acid sequences of the second largest fragment, CNBr Ib (142 residues), and of the 12 smaller fragments are reported in accompanying papers (Moore, M. D., Behrens, P. Q., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10511-10519; Behrens, P. Q., Nakashima, H., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10520-10525). The complete amino acid sequence of hemocyanin component II has been established by isolation and analysis of 13 methionine-containing peptides from either a tryptic digest or a Staphylococcus aureus strain V8 protease digest of whole carboxamidomethylated hemocyanin II. Hemocyanin II is composed of 628 residues and has a molecular weight with two copper atoms of 72,946.     

Oligo-(R)-3-hydroxybutyrate modification of sorting signal enables pore formation by Escherichia coli OmpA

The outer membrane protein A (OmpA) of Escherichia coli is a well-known model for protein targeting and protein folding. Wild-type OmpA, isolated either from cytoplasmic inclusion bodies or from outer membranes, forms narrow pores of ∼ 80 pS in planar lipid bilayers at room temperature. The pores are well structured with narrow conductance range when OmpA is isolated using lithium dodecyl sulfate (LDS) or RapiGest surfactant but display irregular conductance when OmpA is isolated with urea or guanidine hydrochloride. Previous studies have shown that serine residues S163 and S167 of the sorting signal of OmpA (residues 163-169), i.e., the essential sequence for outer membrane incorporation, are covalently modified by oligomers of (R)-3-hydroxybutyrate (cOHB). Here we find that single-mutants S163 and S167 of OmpA, which still contain cOHB on one serine of the sorting signal, form narrow pores in planar lipid bilayers at room temperature with lower and more irregular conductance than wild-type OmpA, whereas double mutants S163:S167 and S163:V166 of OmpA, with no cOHB on the sorting signal, are unable to form stable pores in planar lipid bilayers. Our results indicate that modification of serines in the sorting signal of OmpA by cOHB in the cytoplasm enables OmpA to incorporate into lipid bilayers at room temperature as a narrow pore. They further suggest that cOHB modification may be an important factor in protein targeting and protein folding.     

Bilin attachment sites in the alpha, beta, and gamma subunits of R-phycoerythrin. Structural studies on singly and doubly linked phycourobilins

Three unique bilin peptides, a beta subunit peptide bearing a doubly linked phycourobilin (PUB), and two gamma subunit peptides with singly linked PUB groups, were obtained by enzymatic degradation of Gastroclonium coulteri R-phycoerythrin. These peptides were shown to have the sequences (Klotz, A. V., and Glazer, A. N. (1985) J. Biol. Chem. 260, 4856-4863): (Formula: see text) The sequence of peptide beta-3T was identical to that previously established for a doubly linked phycoerythrobilin (PEB) peptide derived from a B-phycoerythrin (Lundell, D. J., Glazer, A. N., DeLange, R. J., and Brown, D. M. (1984) J. Biol. Chem. 259, 5472-5480). Secondary ion mass spectrometry of beta-3T yielded a protonated molecular ion of 1629 mass units, the same as that given by the doubly linked PEB peptide (Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5481-5484), indicating that the doubly linked PUB and PEB tetrapyrroles were isomeric structures. High resolution 1H NMR analyses of peptides beta-3T, gamma-BV8, and gamma-DP provided unambiguous structural assignments for the singly and doubly linked PUB chromophores and indicated that the peptides in gamma-BV8 and gamma-DP were linked to ring A. The determination of which peptide fragment is linked to ring A and which to ring D in peptide beta-3T was not achieved in this study. 1H NMR analyses of three PEB-peptides from G. coulteri R-phycoerythrin--alpha-1 Cys(PEB)-Tyr-Arg, alpha-2 Leu-Cys(PEB)-Val-Pro-Arg, and beta-1 Met-Ala-Ala-Cys(PEB)-Leu-Arg--showed that they were identical to previously described corresponding chromopeptides from Porphyridium cruentum B-phycoerythrin, with the peptide linked to ring A of PEB in each instance (Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5485-5489). This is the first documented report on the structure of singly or doubly linked phycourobilins.     

Molecular cloning and sequence analysis of cDNAs encoding porcine kidney D-amino acid oxidase

Complementary DNAs encoding D-amino acid oxidase (EC 1.4.3.3, DAO), one of the principal and characteristic enzymes of the peroxisomes of porcine kidney, have been isolated from the porcine kidney cDNA library by hybridization with synthetic oligonucleotide probes corresponding to the partial amino acid sequences. Analysis of the nucleotide sequences of two clones revealed a complete 3211-nucleotide sequence with a 5'-terminal untranslated region of 198 nucleotides, 1041 nucleotides of an open reading frame that encoded 347 amino acids, and a 3'-terminal untranslated region of 1972 nucleotides. The deduced amino acid sequence was completely identical with the reported sequence of the mature enzyme [Ronchi, S., Minchiotti, L., Galliano, M., Curti, B., Swenson, R. P., Williams, C. H. J., & Massey, V. (1982) J. Biol. Chem. 257, 8824-8834]. These results indicate that the primary translation product does not contain a signal peptide at its amino-terminal region for its translocation into peroxisomes. RNA blot hybridization analysis suggests that porcine kidney D-amino acid oxidase is encoded by three mRNAs that differ in size: 3.3, 2.7, and 1.5 kilobases. Comparison of the sequences of the two cDNA clones revealed that multiple polyadenylation signal sequences (ATTAAA and AACAAA) are present in the 3'-untranslated region, making the different mRNA species. The efficiency of 3' processing of the RNA was quite different between the two signal sequences ATTAAA and AACAAA. Southern blot analysis showed the presence of a unique gene for D-amino acid oxidase in the porcine genome.