Molecular cloning of matrin 3. A 125-kilodalton protein of the nuclear matrix contains an extensive acidic domain

We report here the cloning and sequencing of matrin 3, an acidic internal matrix protein, from a rat insuloma cDNA library. The nucleotide sequence has a single open reading frame encoding a polypeptide of 845 amino acids. The Genbank and National Biomedical Research Foundation databases did not contain any sequences similar to that of matrin 3. The primary structure consists of 33% charged residues and is generally hydrophilic. The amino-terminal region (residues 1-120) is positively charged and contains a large number of amino acids with free hydroxyl groups (26 of the first 100 residues) as in the lamins and several non-lamin intermediate filament proteins. A highly acidic domain (approximately 170 amino acids) near the carboxyl terminus, in which 32% of the amino acid residues are acidic (Glu or Asp), is a characteristic found in other nuclear proteins (Earnshaw, W. C. (1987) J. Cell Biol. 105, 1479-1482). A putative nuclear targeting signal sequence (Ser-Lys-Lys-Lys-Leu-Lys-Lys-Val-Glu) is located in the middle of the highly acidic domain. The corresponding human deduced partial amino acid sequence is 96% identical to the rat sequence, indicating that matrin 3 is a highly conserved protein.     

Import of proteins into mitochondria: nucleotide sequence of the gene for a 70-kd protein of the yeast mitochondrial outer membrane.

The nucleotide sequence of the yeast chromosomal gene coding for the 70-kd protein of the mitochondrial outer membrane was determined. The deduced amino acid sequence of the protein agrees with the experimentally determined size and amino acid composition of the purified protein and correctly predicts the fragments obtained by cleaving the protein at its single tryptophan residue. The deduced NH2-terminal sequence features an uninterrupted stretch of 28 uncharged amino acids flanked on both sides by basic amino acids. By sequencing a truncated version of the gene it was found that the corresponding polypeptide product lacks the 203 carboxy-terminal amino acids of the authentic 70-kd protein. As shown in the accompanying paper, this protein fragment still becomes attached to the mitochondrial outer membrane in vivo.     

A 70-kd protein of the yeast mitochondrial outer membrane is targeted and anchored via its extreme amino terminus

The major 70-kd protein of the yeast mitochondrial outer membrane is made on cytosolic ribosomes and imported into the outer membrane without proteolytic cleavage. We have attempted to identify the sequences which target the protein to the mitochondria and which permanently anchor it to the lipid bilayer of the outer membrane. By manipulating the cloned gene we have deleted 13 different regions throughout the polypeptide; in addition, we have fused amino-terminal regions of different length to beta-galactosidase. Each altered gene was introduced into yeast and the intracellular fate of the corresponding polypeptide product was determined by subcellular fractionation. All the information for targeting and anchoring the 70-kd protein (617 amino acids) was contained within the amino-terminal 41 amino acids. When this entire region was deleted, the protein was recovered with the cytosol fraction. However, several restricted deletions within this amino-terminal region appeared to affect targeting and anchoring differentially: most of the altered protein remained in the cytosol but a small fraction was misrouted into the mitochondrial matrix space. We suggest that targeting is mediated by a region which includes the 11 amino-terminal amino acids whereas the permanent membrane anchor is provided by a typical transmembrane sequence between residues 9 and 38.     

Primary structure of the chromosomal protein HMb from the archaebacteria Methanosarcina barkeri

The amino acid sequence of the protein HMb, a protein of 93 residues (Mr 10757) which represents the major acid-soluble component of the Methanosarcina barkeri nucleoprotein complex, has been established from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid, arginine and methionine residues. The protein HMb is mainly characterized by a high amount of charged residues (15% of acidic residues and 26.8% of basic residues) which are distributed all along the polypeptide chain. The amino acid sequence of the protein HMb is not homologous to any eubacterial, archaebacterial or eukaryotic chromosomal proteins known up to now.     

Amino acid sequence of subunit V of bovine heart cytochrome oxidase, the heme alpha-containing subunit.

The complete amino acid sequence of the heme alpha-containing subunit V of bovine heart cytochrome oxidase was determined to be: H2N-Ser-His-Gly-Ser-His-Glu-Thr-Asp-Glu-Glu-Phe-Asp-Ala-Arg-Trp-Val-Thr-Tyr-Phe-Asn-Lys-Pro-Asp-Ile-Asp-Ala-Trp-Glu-Leu-Arg-Lys-Gly-Met-Asn-Thr-Leu-Val-Gly-Tyr-Asp-Leu-Val-Pro-Glu-Pro-Lys-Ile-Ile-Asp-Ala-Ala-Leu-Arg-Ala-Cys-Arg-Arg-Leu-Asn-Asp-Phe-Ala-Ser-Ala-Val-Arg-Ile-Leu-Glu-Val-Val-Lys-Asp-Lys-Ala-Gly-Pro-His-Lys-Glu-Ile-Tyr-Pro-Tyr-Val-Ile-Gln-Glu-Leu-Arg-Pro-Thr-Leu-Asn-Glu-Leu-Gly-Ile-Ser-Thr-Pro-Glu-Glu-Leu-Gly-Leu-Asp-Lys-Val-COOH. The subunit V is a single polypeptide which consists of 109 amino acid residues. The protein contains 48.6% hydrophobic residues and 34.0% hydrophilic residues and it is an acidic protein having a net charge of -3 at neutral pH. The molecular weight of subunit V was calculated to be 12,436 and that for the heme alpha-containing polypeptide was 13,295.     

Abolition of anion-activation of mitochondrial F1-ATPase by the partial ADP-induced hysteretic inhibition

An M_r 21 000 polypeptide, designated APPG, has been purified by reverse-phase, high-performance liquid chromatography (RP-HPLC), from acid extracts of porcine anterior pituitary glands. This acidic protein possesses an isoelectric point of 4.9. Amino acid analysis shows that it is not a glycoprotein and estimates it to contain about 173 amino acids. NH₂-terminal sequence analysis allowed the determination of the first 50 residues unambiguously. A computer data bank search using a mutation data matrix and comparison with 269 012 protein segments indicated that this is a novel polypeptide sequence. However, this search revealed suggestive sequence homologies to a number of peptides of known sequence, including duck proinsulin (30%), Rous sarcoma virus transforming protein TVFV60 (24%) and pig secretin (26%).     

Structural studies on rat prostatic binding protein. The primary structure of component C2 from subunit S

The amino acid sequence of component C2, the polypeptide specific for subunit S of prostatic binding protein, the major secretory glycoprotein of the rat ventral prostate, has been determined. Its structure was established using the manual Edman degradation on the most relevant fragments obtained by enzymatic digestion of the S-carboxamidomethylated component C2 and the native subunit S and by chemical cleavage of the remaining undigestible 'cores' with cyanogen bromide. Component C2 contains 92 amino acids corresponding to a molecular weight of 10619. It is a slightly acidic polypeptide in which the acidic and basic residues are unevenly distributed. The N terminus is blocked and three cysteine residues are almost evenly distributed over the peptide chain. A highly polar region is found in position 23-34 and two hydrophobic segments are located in the C-terminal part of the molecule. Component C2 is compared with component C1 of subunit F and their high sequence homology reveals an evolutionary relationship.     

Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine

The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.     

Amino acid sequence of bovine cardiac troponin I

Troponin I (TnI) is the inhibitory subunit of troponin, the thin filament regulatory complex which confers calcium sensitivity to striated muscle actomyosin ATPase activity. We have determined the amino acid sequence of TnI from adult bovine cardiac muscle. This protein is a single polypeptide chain of 211 amino acids with an acetylated amino terminus, a calculated molecular weight of 23,975, and a net charge of +17 at neutral pH. There was no evidence for heterogeneity of the sequence. Comparison with other available TnI sequences shows an amino-terminal extension of 27-33 residues which is present in cardiac but not skeletal TnI. The remainder of the polypeptide is common to both cardiac and skeletal TnI. In the amino-terminal half of the common polypeptide, only 29% of the residues are invariant in all sequences. The carboxyl-terminal half (residues 124-210) is much more highly conserved, with 66% invariant residues. Bovine cardiac TnI and rabbit cardiac TnI are very similar in sequence: only 12 of 26 residues are identical in the amino-terminal segments, but the remaining residues of the proteins are 97% identical.     

S-layer protein gene of Acetogenium kivui: cloning and expression in Escherichia coli and determination of the nucleotide sequence.

Acetogenium kivui is anaerobically growing thermophilic bacterium with a gram-positive type of cell wall structure. The outer surface is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer polypeptide was cloned in Escherichia coli on two overlapping fragments by using the plasmid pUC18 as the vector. It was expressed under control of a cloned Acetogenium promoter or the lacZ gene. We determined the complete sequence of the structural gene. The mature polypeptide comprises 736 amino acids and is preceded by a typical procaryotic signal sequence of 26 amino acids. It i weakly acidic, weakly hydrophilic, and contains a relatively high proportion of hydroxyamino acids, including two clusters of serine and threonine residues. An N-terminal region of about 200 residues is homologous to the N-terminal part of the middle wall protein, one of the two S-layer proteins of Bacillus brevis, and there is also an internal homology within the N-terminal region of the A. kivui polypeptide.     

Isolation, molecular and functional properties of the C-terminal domain of colicin A

Partial proteolytic digestion of colicin A with bromelain allowed the isolation of a 20-kd fragment. This fragment has been purified to homogeneity and its molecular properties have been studied. The sequence of the 54 N-terminal amino acid residues has been determined by automated Edman degradation. This sequence is identical to that of the predicted amino acid sequence of the 20-kd C-terminal part of the colicin A polypeptide deduced from the nucleotide sequence of the caa gene. This polypeptide can produce channels in phospholipid planar bilayers of the same size as those formed by colicin A. However, the voltage-dependence for opening and closing was drastically altered in the peptide fragment channels. The latter, in contrast to colicin A channels, remained open over a wide range of voltage. Large negative potentials were required to close the peptide fragment channels although opening took place in the same voltage range as for colicin A ionic pores.     

The Complete Primary Structure of Molluscan Shell Protein 1 (MSP-1), an Acidic Glycoprotein in the Shell Matrix of the Scallop Patinopecten yessoensis

The complete primary structure of MSP-1, a major water-soluble glycoprotein in the foliated calcite shell layer of the scallop Patinopecten yessoensis, is reported. The full-length complementary DNA for MSP-1 isolated by polymerase chain reaction contained a sequence for a signal peptide of 20 amino acids followed by a polypeptide of 820 amino acids with calculated molecular mass of 74.5 kDa. The deduced amino acid sequence of MSP-1 includes a high proportion of Ser (32%), Gly (25%), and Asp (20%), and the predicted isoelectric point is 3.2; in these respects, MSP-1 is a typical acidic glycoprotein of mineralized tissues. A repeated modular structure characterizes MSP-1, with a sequence unit between 158 and 177 amino acids in length being repeated 4 times in tandem in the middle part of the protein. The repeated unit comprises 3 modules (SG, D, and K domains), each having a distinct amino acid composition and sequence. The SG domain is almost exclusively composed of Ser and Gly residues. The D domain is rich in Asp residues, potential N-glycosylation and phosphorylation sites. The K domain is rich in Gly residues and has a core of basic residues. The Asp residues are arranged more or less regularly in the D domains, exhibiting some repeated motifs such as Asp-Gly-Ser-Asp and Asp-Ser-Asp. Further, the 4 D domains indicate remarkable overall sequence similarities to each other. These observations suggest that the regular arrangements of COO⁻ groups in the D domain side chains may be important for specific control of crystal growth. Received September 19, 2000; accepted February 9, 2001     

Import of proteins into mitochondria: a 70 kilodalton outer membrane protein with a large carboxy-terminal deletion is still transported to the outer membrane.

The yeast mitochondrial outer membrane contains a major 70-kd protein which is coded by a nuclear gene. Two forms of this gene were isolated from a yeast genomic clone bank: the intact gene, and a truncated gene which had lost a large part of its 3' end during the cloning procedure. Upon transformation into yeast, both the intact and the truncated gene are expressed; the truncated gene generates a shortened protein missing 203 amino acids from the carboxy-terminus. This truncated polypeptide reacts with a monoclonal antibody against the authentic 70-kd protein and is transported to the mitochondrial outer membrane. By integrative transformation, we have constructed a yeast mutant which lacks the 70-kd protein and is unable to adapt to growth on a nonfermentable carbon source at 37 degrees C. This phenotypic lesion can be corrected by transforming the mutant with the intact, but not the truncated gene. The carboxy-terminal sequence of 203 amino acids is thus necessary for the function of the protein, but not for its targeting to the mitochondrion.     

Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies.

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate [HPI])-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1,036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and Mr estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%) of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.     

The first twelve amino acids of a yeast mitochondrial outer membrane protein can direct a nuclear-coded cytochrome oxidase subunit to the mitochondrial inner membrane.

We have used an in vivo complementation assay to test whether a given polypeptide sequence can direct an attached protein to the mitochondrial inner membrane. The host is a previously described yeast deletion mutant that lacks cytochrome oxidase subunit IV (an imported protein) and, thus neither assembles cytochrome oxidase in its mitochondrial inner membrane nor grows on the non-fermentable carbon source, glycerol. Growth on glycerol and cytochrome oxidase assembly are restored to the mutant if it is transformed with the gene encoding authentic subunit IV precursor, a protein carrying a 25-residue transient pre-sequence. No restoration is seen with a plasmid encoding a subunit IV precursor whose pre-sequence has been shortened to seven residues. Partial, but significant restoration is achieved by an artificial subunit IV precursor in which the authentic pre-sequence has been replaced by the first 12 amino acids of a 70-kd protein of the mitochondrial outer membrane. If this dodecapeptide is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein), the resulting fusion protein is imported into the matrix of yeast mitochondria in vitro and in vivo. Import in vitro requires an energized inner membrane. We conclude that the extreme amino terminus of the 70-kd outer membrane protein can direct an attached protein across the mitochondrial inner membrane.     

Characterization of the genes for the hexagonally arranged surface layer proteins in protein-producing Bacillus brevis 47: complete nucleotide sequence of the middle wall protein gene.

Bacillus brevis 47 contains two surface (S)-layer proteins, termed the outer wall protein (OWP) and the middle wall protein (MWP), which form a hexagonal array in the cell wall. The MWP and OWP genes are contained in the 9-kilobase-pair (kbp) BclI fragment and constitute an operon under coordinate control of their expression. The nucleotide sequence of a 3.8-kbp EcoRI-SacI fragment containing the entire MWP gene has been determined in this study. Together with the DNA sequence of the promoter region for the MWP-OWP gene operon (H. Yamagata, T. Adachi, A. Tsuboi, M. Takao, T. Sasaki, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 169:1239-1245, 1987) and that of the OWP gene (A. Tsuboi, R. Uchihi, R. Tabata, Y. Takahashi, H. Hashiba, T. Sasaki, H. Yamagata, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 168:365-373, 1986), the complete nucleotide sequence of the MWP-OWP gene operon has been determined. The MWP gene encodes a secretory precursor of the MWP, consisting of a total of 1,053 amino acid residues with a signal peptide of 23 amino acid residues at its amino-terminal end. Bacillus subtilis harboring the MWP gene synthesized an immunoreactive polypeptide with almost the same molecular weight as the authentic MWP, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid compositions deduced from the MWP and OWP genes were similar to the chemical amino acid compositions of other S-layer proteins in the predominance of acidic amino acids compared with basic amino acids and in the very low content of sulfur-containing amino acids. The acidic nature of the MWP and OWP was confirmed by isoelectric focusing on polyacrylamide gels. In addition, circular dichroism spectra indicated that the S-layer proteins in B. brevis 47 were composed of approximately 30% beta-sheet and 5% alpha-helical structures, with the remainder of the polypeptide backbone being aperiodic in nature.     

Amino acid sequences of cytochrome c-554(548) and cytochrome c'' from a halophilic denitrifying bacterium of the genus Paracoccus.

The amino acid sequences of the cytochromes c-554(548) and c' from the moderately halophilic bacterium Paracoccus sp., I.A.M. 203 (= A.T.C.C. 12084, N.C.I.B. 8669) have been determined. Cytochrome c-554(548) consists of a single polypeptide chain of 83 residues, and dimerizes strongly. The most similar protein of known sequence is the N-terminal half of the dihaem cytochrome c4, and other related proteins include the cytochrome c-554(547) of Thiobacillus neapolitanus and the cytochrome c-553 of Desulfovibrio vulgaris. Cytochrome c', which has a single polypeptide chain of 132 residues, is similar in sequence to cytochromes c' from phototrophic and denitrifying bacteria, but only shows about 36% sequence identity to the most similar protein of known sequence. Both of the Paracoccus proteins have a considerable excess of acidic amino acid side chains over basic ones, and a higher proportion of their basic amino acids is arginine than is usual in cytochromes c. Both these characteristics seem to be adaptations to increase the stability of the proteins in an environment of high ionic strength. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication 50140 (24 pp.) at the British Library (Lending Division), Boston Spa, Yorkshire LS23 7BQ, U.K. from which copies are available on prepayment.     

DNA cloning and amino acid sequence determination of a major constituent protein of mammalian nucleoli

Using a cDNA probe encoding the nucleolar protein N038 of Xenopus laevis, we have isolated clones that code for the corresponding mammalian protein from cDNA libraries of mouse embryonal carcinoma and fetal liver cells. The murine cDNA-derived amino acid sequence defines a polypeptide of 292 amino acids (including the initial methionine) of a total molecular weight of 32560 and identifies a single 1.5 kb mRNA on Northern blot hybridization. This polypeptide, which is highly homologous to the Xenopus protein N038, displays an organization in three major domains: (1) an aminoterminal portion of 119 amino acids, which shows a striking homology to nucleoplasmin of Xenopus; (2) a central portion of 68 amino acids that contains two extended acidic domains, a shorter of 13 residues and a longer of 29 residues, separated by an interval enriched in positively charged amino acids; (3) a carboxyterminal portion of 105 amino acids, which is almost identical to the reported partial amino acid sequence of human and rat nucleolar protein termed B23. The sequence comparisons show that the murine protein is the mammalian counterpart to the nucleolar protein N038 of Xenopus and is compatible with the idea that both proteins N038 represent the amphibian and murine equivalents to the human and rat nucleolar phosphoprotein B23. Special sequence features and predicted conformations of this protein are discussed in relation to the specific localization and the possible functions of this major nucleolar protein.     

The primary structure and functional characterization of the neutral histidine-rich polypeptide from human parotid secretion

The neutral histidine-rich polypeptide (HRP) from human parotid secretion was isolated by ion-exchange and gel-filtration chromatography. The complete amino acid sequence determined by automated Edman degradation of the protein, tryptic and Staphylococcus aureus V8 protease peptides, and digestion with carboxypeptidase A is: (Formula: see text) where Pse represents phosphoserine. The polypeptide contains 38 residues and has Mr 4929. The charged amino acids predominate with 7 histidine, 4 arginine, 3 lysine, 3 aspartic acid, 3 glutamic acid residues, and 1 phosphoserine. Assuming minimal charge contributions from histidine and one negative charge from phosphoserine at pH 7, the net charge of HRP is balanced by an equal contribution of basic and acidic residues. Furthermore, the distribution of hydrophilic and hydrophobic residues along the polypeptide chain indicates that there is no structural polarity. The polypeptide lacks threonine, alanine, valine, cysteine, methionine, and isoleucine. HRP did not display sequence similarity with any protein sequence in the National Biomedical Research Foundation Data Bank. HRP is an active inhibitor of hydroxyapatite crystal growth from solutions supersaturated with respect to calcium phosphate salts and therefore must play a role in the stabilization of mineral-solute interactions in oral fluid. In addition, HRP is a potent inhibitor of Candida albicans germination and therefore may be a significant component of the antimicrobial host defense system in the oral cavity.