Identification and characterization of human SLP-2, a novel homologue of stomatin (band 7.2b) present in erythrocytes and other tissues

Human stomatin (band 7.2b) is a 31-kDa erythrocyte membrane protein of unknown function but implicated in the control of ion channel permeability, mechanoreception, and lipid domain organization. Although absent in erythrocytes from patients with hereditary stomatocytosis, stomatin is not linked to this disorder. A second stomatin homologue, termed SLP-1, has been identified in nonerythroid tissues, and other stomatin related proteins are found in Drosophila, Caenorhabditis elegans, and plants. We now report the cloning and characterization of a new and unusual stomatin homologue, human SLP-2 (stomatin-like protein 2). SLP-2 is encoded by an approximately 1.5-kilobase mRNA (GenBank(TM) accession no. AF190167). The gene for human SLP-2, HUSLP2, is present on chromosome 9p13. Its derived amino acid sequence predicts a 38,537-kDa protein that is overall approximately 20% similar to human stomatin. Northern and Western blots for SLP-1 and SLP-2 reveal a wide but incompletely overlapping tissue distribution. Unlike SLP-1, SLP-2 is also present in mature human erythrocytes ( approximately 4,000 +/- 5,600 (+/- 2 S.D.) copies/cell). SLP-2 lacks a characteristic NH(2)-terminal hydrophobic domain found in other stomatin homologues and (unlike stomatin) is fully extractable from erythrocyte membranes by NaOH, pH 11. SLP-2 partitions into both Triton X-100-soluble and -insoluble pools in erythrocyte ghost membranes or when expressed in cultured COS cells and migrates anomalously on SDS-polyacrylamide gel electrophoresis analysis with apparent mobilities of approximately 45,500, 44,600, and 34,300 M(r). The smallest of these protein bands is believed to represent the product of alternative translation initiated at AUGs beginning with nt 217 or 391, although this point has not been rigorously proven. Collectively, these findings identify a novel and unusual member of the stomatin gene superfamily that interacts with the peripheral erythrocyte cytoskeleton and presumably other integral membrane proteins but not directly with the membrane bilayer. We hypothesize that SLP-2 may link stomatin or other integral membrane proteins to the peripheral cytoskeleton and thereby play a role in regulating ion channel conductances or the organization of sphingolipid and cholesterol-rich lipid rafts.     

Fructose 2,6-bisphosphate, carbohydrate partitioning, and crassulacean Acid metabolism

To initiate structural studies of the ADPglucose pyrophosphorylase from spinach an improved purification procedure was devised. The modified purification scheme allowed the isolation of 20 to 30 milligrams pure enzyme from 10 kilogram of spinach leaves. Electrophoresis of the purified enzyme confirmed an earlier study which showed that the enzyme was putatively composed of two subunits (Copeland L, J Preiss 1981 Plant Physiol 68: 996-1001). The two subunits migrate as 51 and 54 kilodalton proteins upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both proteins can be detected on Western blots of leaf homogenates prepared under denaturing conditions suggesting that both subunits exist in vivo. Anion-exchange chromatography in the presence of urea allowed resolution of the 51 and 54 kilodalton proteins. They possess different N-terminal amino acid sequences and tryptic peptide maps. Western blot analysis reveals that the 51 and 54 kilodalton proteins are antigenically dissimilar. The 51 but not the 54 kilodalton protein is immunologically related to the ADPglucose pyrophosphorylase from maize endosperm and potato tuber.     

A conserved N-terminal motif in Rad54 is important for chromatin remodeling and homologous strand pairing

The Swi2/Snf2-related protein Rad54 is a chromatin remodeling enzyme that is important for homologous strand pairing catalyzed by the eukaryotic recombinase Rad51. The chromatin remodeling and DNA-stimulated ATPase activities of Rad54 are significantly enhanced by Rad51. To investigate the functions of Rad54, we generated and analyzed a series of mutant Rad54 proteins. Notably, the deletion of an N-terminal motif (amino acid residues 2-9), which is identical in Rad54 in Drosophila, mice, and humans, results in a complete loss of chromatin remodeling and strand pairing activities, and partial inhibition of the ATPase activity. In contrast, this conserved N-terminal motif has no apparent effect on the ability of DNA to stimulate the ATPase activity or of Rad51 to enhance the DNA-stimulated ATPase activity. Unexpectedly, as the N terminus of Rad54 is progressively truncated, the mutant proteins regain partial chromatin remodeling activity as well as essentially complete DNA-stimulated ATPase activity, both of which are no longer responsive to Rad51. These findings suggest that the N-terminal region of Rad54 contains an autoinhibitory activity that is relieved by Rad51.     

Inhibition of cathepsins and related proteases by amino acid, peptide, and protein hydroperoxides

Reaction of radicals in the presence of O2, and singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, and that this can result in inactivation of thiol-dependent enzymes. The major route for the cellular removal of damaged proteins is via catabolism mediated by proteosomal and lysosomal pathways; cysteine proteases (cathepsins) play a key role in the latter system. We hypothesized that inactivation of cysteine proteases by hydroperoxide-containing oxidised proteins may contribute to the accumulation of modified proteins within cells. We show here that thiol-dependent cathepsins, either isolated or in cell lysates, are rapidly and efficiently inactivated by amino acid, peptide, and protein hydroperoxides in a time- and concentration-dependent manner; this occurs with similar efficacy to equimolar H2O2. Inactivation involves reaction of the hydroperoxide with Cys residues as evidenced by thiol loss and formation of sulfenic acid intermediates. Structurally related, non-thiol-dependent cathepsins are less readily inactivated by these hydroperoxides. This inhibition, by oxidized proteins, of the system designed to remove modified proteins, may contribute to the accumulation of damaged proteins in cells subject to oxidative stress.     

Association of stomatin with lipid-protein complexes in the plasma membrane and the endocytic compartment

Membrane protein - microvilli - lipid raft - GPI-anchored protein - epithelial cell The 31 kDa integral membrane protein stomatin (protein 7.2b) has a monotopic structure and a cytofacial orientation. We have shown previously that stomatin is located in plasma membrane protruding structures and forms high-order homo-oligomers in the human epithelial cell line UAC, suggesting that this protein has a structural function in the cortical morphogenesis of the cells. It is also present in a pool of juxtanuclear vesicles. In this study, we show that stomatin colocalizes with the GPI-anchored proteins placental alkaline phosphatase (PLAP) and membrane folate receptor alpha (MFRalpha) endogenously expressed in UAC cells. This observation enabled us to demonstrate two different aspects of stomatin. First, using anti-PLAP antibody internalization, we show that the peri-centrosomal vesicles containing stomatin correspond to a subset of endosomes, which can also be labeled with the late endosomal/lysosomal marker LAMP-2. Secondly, we found that stomatin is partially present in detergent-insoluble membrane domains and co-patches with PLAP on the plasma membrane, after cross-linking of PLAP by antibodies. These data indicate that stomatin and GPI-anchored proteins are linked through lipid rafts and undergo the same sorting events. We propose that stomatin, through its affinity for lipid rafts, functions in concentrating GPI-anchored proteins in membrane microvillar structures. Consistent with this hypothesis, we found that stomatin is expressed exclusively in microvilli of the apical membrane in polarized Madin-Darby canine kidney (MDCK) cells.     

A novel gene encoding a sulfur-regulated outer membrane protein in Thiobacillus ferrooxidans.

Thiobacillus ferrooxidans is a Gram-negative chemolithotrophic bacterium able to oxidize ferrous iron, elemental sulfur and inorganic sulfur compounds. The oxidation of sulfur by T. ferrooxidans resulted in an expression of some outer membrane proteins (OMPs) at a level higher than that observed during ferrous iron oxidation. Among these OMPs, a protein with a molecular mass of 54 kDa was purified and 18 amino acids of the N-terminal sequence determined. Using a 54 bp PCR generated DNA product as a probe for the protein, we isolated a 4.5 kb Pst I DNA chromosomal fragment containing the corresponding gene. Sequencing 2169 bp of this fragment revealed the open reading frame codifying for the protein, consisting of 467 amino acids and a molecular mass of 49,674 Da. The mature protein was produced by the removal of a 32 amino acid signal peptide-like sequence from the N-terminus of a 499 amino acid peptide. Although no significant homology with any known protein has been found and its physiological role remains unclear, its high expression on sulfur substrates suggests a role in sulfide mineral oxidation.     

Cloning of complementary DNA encoding the pB1 component of the 54-kilodalton glycoprotein of boar seminal plasma

Complementary DNA (cDNA) encoding a protein component pB1 (also pAIF-1 and DQH) of the 54-kilodalton glycoprotein of boar seminal plasma was cloned and its nucleotide sequence was determined (Gene Bank accession no. AF047026). The pB1 precursor protein is a 130-amino-acid-long polypeptide containing a 25-amino-acid-long signal peptide. The amino acid sequence of the pB1 is homologous to that of SFP1_BOVIN (named also BSP-A1/A2, PDC-109/ major protein and SVSp109), SFP3_BOVIN (BSP-A3), SFP4 BOVIN (BSP-30 KD), and SP1_HORSE (HSP-1) seminal plasma proteins. The homology extends also for the signal peptide of SFP1_BOVIN protein. All these seminal plasma proteins contain two fibronectin type-II domains that differ from those found in other proteins such as colagenases, fibronectins, and mannose receptors. The first domain located in the N-terminal region of pB1 is four amino acids shorter than those present in other proteins. High homology is also observed between 3' noncoding regions of the nucleotide sequences of cDNAs of pB1_PIG and SFP1_BOVIN (Gene Bank accession nos. AF047026 and P02784, respectively).     

Euplotes crassus has genes encoding telomere-binding proteins and telomere-binding protein homologs.

We have identified two 1.6 kb macronuclear DNA molecules from Euplotes crassus that hybridize to the alpha subunit of the Oxytricha telomere protein. We have shown that one of these molecules encodes the 51 kDa Euplotes telomere protein while the other appears to encode a homolog of the telomere protein. Although this homolog clearly differs in sequence from the Euplotes telomere protein, the two proteins share extensive amino acid sequence identity with each other and with the alpha subunit of the Oxytricha telomere protein. In all three proteins 35-36% of the amino acids are identical, while 54-56% are similar. The most extended regions of sequence conservation map within the N-terminal section; this section has been shown to comprise the DNA-binding domain in the Euplotes telomere protein. Our findings suggest that some of the conserved amino acids may be involved in DNA recognition and binding. The gene encoding the telomere protein homolog contains two introns; one of these introns is only 24 bp in length. This is the smallest mRNA intron reported to date.     

Use of a novel strategy for the preparation and characterization of an antipeptide antibody capable of recognizing members of the annexin family

Annexins are structurally-related proteins which bind phospholipids in a Ca2+-dependent manner. We have used a novel coupling strategy to prepare an antiserum directed against a 17-amino acid synthetic peptide that resembles the sequence of a highly-conserved portion of these proteins. This antipeptide serum specifically recognizes 5 of 6 human annexins on Western blots, despite differences between the protein and peptide sequences of 3 or 4 amino acids. The antiserum does not recognize endonexin II, whose sequence differs from that of the peptide by 6 amino acids. The availability of multiple proteins with known amino acid sequence has allowed analysis of structural requirements for recognition by this antibody. In some situations, use of such an antibody may allow the identification of a protein as a member of a family.     

High-mobility group chromosomal proteins of wheat

Four proteins have been extracted from purified chromatin of wheat embryos with 0.35 M NaCl. These proteins are soluble in 2% (w/v) trichloroacetic acid and thus meet the original operational requirements to be classified as "high-mobility group" (HMG) chromosomal proteins. The proteins have been characterized by one- and two-dimensional electrophoresis, amino acid analysis, and peptide mapping. Three of the proteins (HMGb, c, and d) share the mammalian HMG characteristic of being rich in both acidic and basic amino acid residues. Unlike their putative mammalian counterparts, these plant HMG proteins contain less than 7 mol % proline. The fourth wheat protein (HMGa) is rich in both proline and in basic amino acid residues. This wheat protein, however, contains only about half the proportion of acidic residues found in mammalian HMG proteins--a characteristic also found in the trout testis HMG protein, H6. Comparative peptide maps show that none of the wheat HMG proteins are degradation products of other HMG proteins or the H1 histones. The peptide maps have not, however, been useful in establishing homologies with mammalian HMG proteins. Wheat HMG proteins are released from DNase I-treated nuclei and co-isolate with micrococcal nuclease-sensitive chromatin fractions. Similar observations concerning the HMG proteins of vertebrate animals have been considered consistent with a role for these proteins as structural components of actively transcribed chromatin.     

Cloning and nucleotide sequence of braC, the structural gene for the leucine-, isoleucine-, and valine-binding protein of Pseudomonas aeruginosa PAO.

The gene for the leucine-, isoleucine-, and valine-binding protein (LIVAT-BP) in Pseudomonas aeruginosa PAO was isolated, and its nucleotide sequence was determined. The gene consisted of 1,119 nucleotides specifying a protein of 373 amino acid residues. Determination of the N-terminal amino acid sequence of the LIVAT-BP purified from P. aeruginosa shock fluid suggested that the N-terminal 26 residues of the gene product are cleaved off posttranslationally, showing the characteristic features of procaryotic signal peptides. The amino acid composition of the mature product predicted from the nucleotide sequence was in good agreement with that of the purified LIVAT-BP. The plasmid carrying the LIVAT-BP gene restored the activity of the high-affinity branched-chain amino acid transport system (the leucine, isoleucine, valine [LIV-I] transport system) in the braC310 mutant of P. aeruginosa, confirming that braC is the structural gene for LIVAT-BP. The mutant LIVAT-BP lacking a 16-amino-acid peptide in the middle was found to be functional in the LIV-I transport system. LIVAT-BP showed extensive homology (51% identical) to the LIV- and leucine-specific-binding proteins of Escherichia coli K-12, which are coded for by the livJ and livK genes, respectively, suggesting that the role of the proteins in the LIV-I transport systems is analogous in both organisms.     

Both a short hydrophobic domain and a carboxyl-terminal hydrophilic region are important for signal function in the Escherichia coli leader peptidase

Leader peptidase, typical of inner membrane proteins of Escherichia coli, does not have an amino-terminal leader sequence. This protein contains an internal signal peptide, residues 51-83, which is essential for assembly and remains as a membrane anchor domain. We have employed site-directed mutagenesis techniques to either delete residues within this domain or substitute a charged amino acid for one of these residues to determine the important properties of the internal signal. The deletion analysis showed that a very small apolar domain, residues 70-76, is essential for assembly, whereas residues that flank it are dispensable for its function. However, point mutations with charged amino acid residues within the polar sequence (residues 77-82) slow or abolish leader peptidase membrane assembly. Thus, a polar region, Arg-Ser-Phe-Ile-Tyr-Glu, is important for the signal peptide function of leader peptidase, unlike other signals identified thus far.     

1H, 13C, and 15N resonance assignment of the SPFH domain of human stomatin

Stomatin, a 288-residue protein, is a component of the membrane skeleton of red blood cells (RBCs), which helps to physically support the membrane and maintains its function. In RBCs, stomatin binds to the glucose transporter GLUT-1 and may regulate its function. Stomatin has a stomatin/prohibitin/flotillin/HflK (SPFH) domain at the center of its polypeptide chain. There are 12 SPFH domain-containing proteins, most of which are localized at the cellular or subcellular membranes. Although the molecular function of the SPFH domain has not yet been established, the domain may be involved in protein oligomerization. The SPFH domain of the archaeal stomatin homolog has been shown to form unique oligomers. Here we report the ¹⁵N, ¹³C, and ¹H chemical shift assignments of the SPFH domain of human stomatin [hSTOM(SPFH)]. These may help in determining the structure of hSTOM(SPFH) in solution as well as in clarifying its involvement in protein oligomerization.     

Structural requirements for transport of preprocecropinA and related presecretory proteins into mammalian microsomes.

The presecretory protein preprocecropinA (which comprises 64 amino acid residues) as well as a synthetic hybrid between preprocecropinA and dihydrofolate reductase (which comprises 252 amino acid residues) are processed by and transported into mammalian microsomes. Transport of both precursor proteins can take place cotranslationally, i.e. with the aid of ribosome and signal recognition particle, or posttranslationally, i.e. independently of these ribonucleoparticles (RNPs). We investigated the role of the precursor structure with respect to competence for RNP-independent transport by constructing deletion mutants and hybrid proteins. The results demonstrate that the signal peptide is essential for RNP-independent transport. Furthermore, the signal peptide is sufficient for translocation of preprocecropinA derivatives up to 85 amino acid residues in size. However, the conformation of the precursor protein is decisive in the case of larger hybrid proteins.     

Correspondence of the functional epitopes of poxvirus and human interleukin-18-binding proteins

Molluscum contagiosum virus, a human poxvirus that causes persistent small benign skin tumors, encodes a variety of putative immune defense proteins. Three such proteins, MC51L, MC53L, and MC54L, have 20 to 35% amino acid sequence identities with human interleukin-18 (hIL-18)-binding protein (hIL-18BP), a naturally occurring antagonist of the proinflammatory cytokine IL-18. We previously demonstrated that seven amino acids within the immunoglobulin-like domain of hIL-18BP were important for high-affinity binding to hIL-18. Model building indicated that MC54L, which has been shown to bind hIL-18, contains five of the seven amino acids at corresponding positions in its immunoglobulin-like domain, the exceptions being the conservative substitution of isoleucine for a leucine and the nonconservative substitution of valine for a phenylalanine. We found that individual alanine substitutions for these six identical or highly conserved amino acids of MC54L caused changes in affinity and binding free energy for hIL-18 that were quantitatively similar to those produced by mutagenesis of hIL-18BP. Furthermore, when the nonconserved valine of MC54L was mutated to phenylalanine, making it more like hIL-18BP, its affinity for hIL-18 increased more than 10-fold. In addition, the carboxyl-terminal half of MC54L, which has no similarity with hIL-18BP, was dispensable for hIL-18 binding. Thus, despite their relatively low overall sequence identity, MC54L and hIL-18BP have similar hIL-18 binding sites and functional epitopes. On the other hand, MC51L and MC53L have nonconservative substitutions of three to six of the seven critical amino acids of hIL-18BP and neither protein bound hIL-18, suggesting that they may interact with unidentified ligands.