Predicted Transmembrane Segment Research Articles

Inhibitory proteins block substrate access by occupying the active site cleft of Bacillus subtilis intramembrane protease SpoIVFB.

Intramembrane proteases (IPs) function in numerous signaling pathways that impact health, but elucidating the regulation of membrane-embedded proteases is challenging. We examined inhibition of intramembrane metalloprotease SpoIVFB by proteins BofA and SpoIVFA. We found that SpoIVFB inhibition requires BofA residues in and near a predicted transmembrane segment (TMS). This segment of BofA occupies the SpoIVFB active site cleft based on cross-linking experiments. SpoIVFB inhibition also requires SpoIVFA. The inhibitory proteins block access of the substrate N-terminal region to the membrane-embedded SpoIVFB active site, based on additional cross-linking experiments; however, the inhibitory proteins did not prevent interaction between the substrate C-terminal region and the SpoIVFB soluble domain. We built a structural model of SpoIVFB in complex with BofA and parts of SpoIVFA and substrate, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that conserved BofA residues interact to stabilize a TMS and a membrane-embedded C-terminal region. The model also predicts that SpoIVFA bridges the BofA C-terminal region and SpoIVFB, forming a membrane-embedded inhibition complex. Our results reveal a novel mechanism of IP inhibition with clear implications for relief from inhibition in vivo and design of inhibitors as potential therapeutics.

OCIAD1 is a host mitochondrial substrate of the hepatitis C virus NS3-4A protease.

The hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) protease is a key component of the viral replication complex and the target of protease inhibitors used in current clinical practice. By cleaving and thereby inactivating selected host factors it also plays a role in the persistence and pathogenesis of hepatitis C. Here, we describe ovarian cancer immunoreactive antigen domain containing protein 1 (OCIAD1) as a novel cellular substrate of the HCV NS3-4A protease. OCIAD1 was identified by quantitative proteomics involving stable isotopic labeling using amino acids in cell culture coupled with mass spectrometry. It is a poorly characterized membrane protein believed to be involved in cancer development. OCIAD1 is cleaved by the NS3-4A protease at Cys 38, close to a predicted transmembrane segment. Cleavage was observed in heterologous expression systems, the replicon and cell culture-derived HCV systems, as well as in liver biopsies from patients with chronic hepatitis C. NS3-4A proteases from diverse hepacivirus species efficiently cleaved OCIAD1. The subcellular localization of OCIAD1 on mitochondria was not altered by NS3-4A-mediated cleavage. Interestingly, OCIAD2, a homolog of OCIAD1 with a cysteine residue in a similar position and identical subcellular localization, was not cleaved by NS3-4A. Domain swapping experiments revealed that the sequence surrounding the cleavage site as well as the predicted transmembrane segment contribute to substrate selectivity. Overexpression as well as knock down and rescue experiments did not affect the HCV life cycle in vitro, raising the possibility that OCIAD1 may be involved in the pathogenesis of hepatitis C in vivo.

A study on the anomalies associated with the human sterna in south Indian population

Chondrocalcinosis (CC) is a common cause of joint pain and arthritis that is caused by the deposition of calcium-containing crystals within articular cartilage. Although most cases are sporadic, rare familial forms have been linked to human chromosomes 8 (CCAL1) or 5p (CCAL2) (Baldwin et al. 1995; Hughes et al. 1995; Andrew et al. 1999). Here, we show that two previously described families with CCAL2 have mutations in the human homolog of the mouse progressive ankylosis gene (ANKH). One of the human mutations results in the substitution of a highly conserved amino acid residue within a predicted transmembrane segment. The other creates a new ATG start site that adds four additional residues to the ANKH protein. Both mutations segregate completely with disease status and are not found in control subjects. In addition, 1 of 95 U.K. patients with sporadic CC showed a deletion of a single codon in the ANKH gene. The same change was found in a sister who had bilateral knee replacement for osteoarthritis. Each of the three human mutations was reconstructed in a full-length ANK expression construct previously shown to regulate pyrophosphate levels in cultured cells in vitro. All three of the human mutations showed significantly more activity than a previously described nonsense mutation that causes severe hydroxyapatite mineral deposition and widespread joint ankylosis in mice. These results suggest that small sequence changes in ANKH are one cause of CC and joint disease in humans. Increased ANK activity may explain the different types of crystals commonly deposited in human CCAL2 families and mutant mice and may provide a useful pharmacological target for treating some forms of human CC.

Scanning mutagenesis studies reveal a potential intramolecular interaction within the C-terminal half of dengue virus NS2A involved in viral RNA replication and virus assembly and secretion.

The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMSs; pTMS1 to pTMS8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. In the study described here, we explored the function of DENV NS2A by introducing a series of double or triple alanine substitutions into the C-terminal half (pTMS4 to pTMS8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (8 within pTMS8) of 35 NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by a replicon assay. Three NS2A mutants with mutations within pTMS7, the CM20, CM25, and CM27 mutants, displayed similar phenotypes, low virus yields (>100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transient trans-packaging experiments, suggesting a defect in virus assembly and secretion. The sequencing of revertant viruses derived from CM20, CM25, and CM27 mutant viruses revealed a consensus reversion mutation, leucine (L) to phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, CM25, and CM27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants with mutations within pTMS4, pTMS6, and pTMS8, i.e., the CM2, CM3, CM13, CM31, and CM32 mutants. In conclusion, the results revealed the essential roles of pTMS4 to pTMS8 of NS2A in RNA replication and/or virus assembly and secretion. The intramolecular interaction between pTMS7 and pTMS4, pTMS6, or pTMS8 of the NS2A protein was also implicated. The reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly and secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication-defective and virus assembly- and secretion-defective mutants with mutations within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein.

Bestrophin‐3 expression in mouse glomeruli (694.1)

The bestrophin isoform, bestrophin‐3 (Best3), acts as a calcium‐activated chloride channel in cardiomyocytes and in vascular smooth muscle. It may have cell‐protective functions in other cell types. Best3 may be regulated by cyclic GMP and exists in different splice variants.We have examined expression and localization of Best3 in mouse kidney on the mRNA level using RT‐PCR and on the protein level by immunohistochemistry and Western blotting.Staining for Best3 was obtained in mouse glomeruli, and was also detected in cultured mouse podocytes. Western blot confirmed the presence of Best3 protein in kidney cortex. Best3 did not co‐localize with markers for endothelial cells or podocyte foot processes. However, it co‐localized with the intermediate filament nestin, suggesting localization of Best3 in podocyte primary processes and cell bodies. In kidney cortex, in glomerulus‐enriched fractions and in cultured podocytes the two shortest splice variants, but no full‐length mRNA, were detected.In conclusion, two variants of Best3, lacking some of the predicted transmembrane segments, are expressed in podocytes in mouse kidney, and are likely co‐localized with the intermediate filaments.

FRI0389 RDEA3170, a novel, high affinity URAT1 inhibitor binds to a central domain within URAT1

BackgroundURAT1 inhibitors, used for the treatment of gout, lower serum urate levels by blocking uric acid reabsorption in the kidney proximal tubule through inhibition of the uric acid transporter URAT1....

Design, expression, and purification of de novo transmembrane “hairpin” peptides

While our understanding of the folding and structure of water-soluble proteins has progressed to the point where they can be artificially designed and produced from first principles, there has been only limited work toward the de novo design of membrane proteins. Such studies have been hindered in large part due to the practical challenges in the production and characterization of multispanning transmembrane (TM) proteins that arise from their highly hydrophobic character. In this work, we used molecular biology cloning techniques to produce a library of partially randomized Ala- and Ile-rich de novo helix-loop-helix (hairpin) TM constructs as models for tertiary TM-TM folding. From this plasmid DNA library, we selected sequences corresponding to hairpins with 0, 1, or 2 putative TM segments. While purification protocols could be adapted for application with a broad range of designed protein hairpins, bacterial expression of constructs with multiple predicted TM segments was limited as it is with native membrane proteins. Examples of the peptide hairpins obtained were characterized by circular dichroism spectroscopy, tryptophan fluorescence, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We found that hairpins composed of two TM segments display characteristic behavior on detergent solubilization, such as an increase in helical structure (vs. that in aqueous buffer), and sequence-dependent migration rates in SDS-PAGE analysis-features that may serve as structural hallmarks to verify dual TM topology in hairpin sequences.

Why a Membrane‐Anchored Chitinase (CHT7) in Tribolium?

Insect chitinases (CHTs) and chitinase‐like proteins, which belong to family 18 glycosylhydrolases (GH‐18), have been detected in molting fluid and gut tissues and are predicted to mediate the digestion of chitin present in the exoskeleton and peritrophic matrix (PM) in the gut to chitooligosaccharides. Based on amino acid sequence similarity and phylogenetic analysis, insect chitinase family proteins have been classified into eight groups (group I to VIII). The chitinases belonging to different groups have distinctly different developmental patterns of expression and tissue specificity, suggestive of distinct biological functions.CHT7s belong to Group III chitinase contain two catalytic domains and one CBD. The catalytic domain 1 of this group of chitinases exhibits greater sequence similarity to one another than to the catalytic domain 2 in the same protein(s), suggesting distinct functions and/or evolutionary origins for each of these two catalytic domains. This group of chitinases, unlike most insect CHTs, possesses a predicted transmembrane segment at the N‐terminal region. The recombinant T. castaneum CHT7 that was expressed in Hi‐5 insect cells was bound to the cell membrane. Apparently, the catalytic domains of this CHT face the extracellular space as revealed by its ability to hydrolyze an artificial chitin substrate added to the medium.DsRNA mediated post‐transcription gene silencing (RNAi) for several chitinases and chitinase‐like genes in Tribolium castaneum and demonstrated that chitinases belong to groups I (TcCHT5), II (TcCHT10) and V (TcIDGF4) play roles of insect molting. In other hand, RNAi for TcCHT7 did not affect any types of molting such as larval‐larval, larval‐pupal and pupal‐adult. The resulting pupae (and adults), however, failed to wing‐expansion and abdominal contraction suggesting that TcCHT7 may function in tissue differentiation rather than in molting.

Conservation of the Human Integrin-Type Beta-Propeller Domain in Bacteria

Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca2+-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca2+-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem copies are found.

Rpe65 Isomerase Associates with Membranes through an Electrostatic Interaction with Acidic Phospholipid Headgroups

Opsins are light-sensitive pigments in the vertebrate retina, comprising a G protein-coupled receptor and an 11-cis-retinaldehyde chromophore. Absorption of a photon by an opsin pigment induces isomerization of its chromophore to all-trans-retinaldehyde. After a brief period of activation, opsin releases all-trans-retinaldehyde and becomes insensitive to light. Restoration of light sensitivity to the apo-opsin involves the conversion of all-trans-retinaldehyde back to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle. The critical isomerization step in this pathway is catalyzed by Rpe65. Rpe65 is strongly associated with membranes but contains no membrane-spanning segments. It was previously suggested that the affinity of Rpe65 for membranes is due to palmitoylation of one or more Cys residues. In this study, we re-examined this hypothesis. By two independent strategies involving mass spectrometry, we show that Rpe65 is not palmitoylated nor does it appear to undergo other post-translational modifications at significant stoichiometry. Instead, we show that Rpe65 binds the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and cardiolipin, but not phosphatidic acid. No binding of Rpe65 to basic phospholipids or neutral lipids was observed. The affinity of Rpe65 to acidic phospholipids was strongly pH-dependent, suggesting an electrostatic interaction of basic residues in Rpe65 with negatively charged phospholipid headgroups. Binding of Rpe65 to liposomes containing phosphatidylserine or phosphatidylglycerol, but not the basic or neutral phospholipids, allowed the enzyme to extract its insoluble substrate, all-trans-retinyl palmitate, from the lipid bilayer for synthesis of 11-cis-retinol. The interaction of Rpe65 with acidic phospholipids is therefore biologically relevant.

Model of the Exofacial Substrate-Binding Site and Helical Folding of the Human Glut1 Glucose Transporter Based on Scanning Mutagenesis

Transmembrane helix 9 of the Glut1 glucose transporter was analyzed by cysteine-scanning mutagenesis and the substituted cysteine accessibility method (SCAM). A cysteine-less (C-less) template transporter containing amino acid substitutions for the six native cysteine residues present in human Glut1 was used to generate a series of 21 mutant transporters by substituting each successive residue in predicted transmembrane segment 9 with a cysteine residue. The mutant proteins were expressed in Xenopus oocytes, and their specific transport activities were directly compared to that of the parental C-less molecule whose function has been shown to be indistinguishable from that of native Glut1. Only a single mutant (G340C) had activity that was reduced (by 75%) relative to that of the C-less parent. These data suggest that none of the amino acid side chains in helix 9 is absolutely required for transport function and that this helix is not likely to be directly involved in substrate binding or translocation. Transport activity of the cysteine mutants was also tested after incubation of oocytes in the presence of the impermeant sulfhydryl-specific reagent, p-chloromercuribenzene sulfonate (pCMBS). Only a single mutant (T352C) exhibited transport inhibition in the presence of pCMBS, and the extent of inhibition was minimal (11%), indicating that only a very small portion of helix 9 is accessible to the external solvent. These results are consistent with the conclusion that helix 9 plays an outer stabilizing role for the inner helical bundle predicted to form the exofacial substrate-binding site. All 12 of the predicted transmembrane segments of Glut1 encompassing 252 amino acid residues and more than 50% of the complete polypeptide sequence have now been analyzed by scanning mutagenesis and SCAM. An updated model is presented for the outward-facing substrate-binding site and relative orientation of the 12 transmembrane helices of Glut1.

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV1000) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV100) were more appropriate for discriminating an all-or-none leakage (α=0) from the graded leakage with low values of α. While disrupting LUV1000, CyaA caused a highly cation-selective leakage (α~15) whereas its mutated form with decreased channel K+/Cl− selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K+E516K) exhibited much lower selectivity (α∼6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.

Quinone-reactive proteins devoid of haem b form widespread membrane-bound electron transport modules in bacterial respiration

Many quinone-reactive enzyme complexes that are part of membrane-integral eukaryotic or prokaryotic respiratory electron transport chains contain one or more haem b molecules embedded in the membrane. In recent years, various novel proteins have emerged that are devoid of haem b but are thought to fulfil a similar function in bacterial anaerobic respiratory systems. These proteins are encoded by genes organized in various genomic arrangements and are thought to form widespread membrane-bound quinone-reactive electron transport modules that exchange electrons with redox partner proteins located at the outer side of the cytoplasmic membrane. Prototypic representatives are the multihaem c-type cytochromes NapC, NrfH and TorC (NapC/NrfH family), the putative iron-sulfur protein NapH and representatives of the NrfD/PsrC family. Members of these protein families vary in the number of their predicted transmembrane segments and, consequently, diverse quinone-binding sites are expected. Only a few of these enzymes have been isolated and characterized biochemically and high-resolution structures are limited. This mini-review briefly summarizes predicted and experimentally demonstrated properties of the proteins in question and discusses their role in electron transport and bioenergetics of anaerobic respiration.

NADH as Donor.

The number of NADH dehydrogenases and their role in energy transduction in Escherchia coli have been under debate for a long time. Now it is evident that E. coli possesses two respiratory NADH dehydrogenases, or NADH:ubiquinone oxidoreductases, that have traditionally been called NDH-I and NDH-II. This review describes the properties of these two NADH dehydrogenases, focusing on the mechanism of the energy converting NADH dehydrogenase as derived from the high resolution structure of the soluble part of the enzyme. In E. coli, complex I operates in aerobic and anaerobic respiration, while NDH-II is repressed under anaerobic growth conditions. The insufficient recycling of NADH most likely resulted in excess NADH inhibiting tricarboxylic acid cycle enzymes and the glyoxylate shunt. Salmonella enterica serovar Typhimurium complex I mutants are unable to activate ATP-dependent proteolysis under starvation conditions. NDH-II is a single subunit enzyme with a molecular mass of 47 kDa facing the cytosol. Despite the absence of any predicted transmembrane segment it has to be purified in the presence of detergents, and the activity of the preparation is stimulated by an addition of lipids.

Two isoforms of PSAP/MTCH1 share two proapoptotic domains and multiple internal signals for import into the mitochondrial outer membrane

Presenilin 1-associated protein (PSAP) was first identified as a protein that interacts with presenilin 1. It was later reported that PSAP is a mitochondrial protein that induces apoptosis when overexpressed in cultured cells. PSAP is also known as mitochondrial carrier homolog 1 (Mtch1). In this study, we show that there are two proapoptotic PSAP isoforms generated by alternative splicing that differ in the length of a hydrophilic loop located between two predicted transmembrane domains. Using RT-PCR and Western blot assays, we determined that both isoforms are expressed in human and rat tissues as well as in culture cells. Our results indicate that PSAP is an integral mitochondrial outer membrane protein, although it contains a mitochondrial carrier domain conserved in several inner membrane carriers, which partially overlaps one of the predicted transmembrane segments. Deletion of this transmembrane segment impairs mitochondrial import of PSAP. Replacement of this segment with each of two transmembrane domains, with opposite membrane orientations, from an unrelated protein indicated that one of them allowed mitochondrial localization of the PSAP mutant, whereas the other one did not. Our interpretation of these results is that PSAP contains multiple mitochondrial targeting motifs dispersed along the protein but that a transmembrane domain in the correct position and orientation is necessary for membrane insertion. The amino acid sequence within this transmembrane domain may also be important. Furthermore, two independent regions in the amino terminal side of the protein are responsible for its proapoptotic activity. Possible implications of these findings in PSAP function are discussed.

Structural basis of natural ligand binding and activation of the Class II G-protein-coupled secretin receptor

The secretin receptor is prototypic of Class II GPCRs (G-protein-coupled receptors), based on its structural and functional characteristics and those of its natural agonist ligand. Secretin represents a linear 27-residue peptide with diffuse pharmacophoric domain. The secretin receptor includes the typical signature sequences for this receptor family within its predicted transmembrane segments and the highly conserved six cysteine residues contributing to three intradomain disulfide bonds within its long N-terminus. This domain is critical for secretin binding based on receptor mutagenesis and photoaffinity labelling studies. Full agonist analogues of secretin incorporating a photolabile moiety at various positions throughout the pharmacophore covalently label residues within this region, while only N-terminal probes have labelled the core helical bundle domain. Combining insights coming from receptor structural studies, peptide structure-activity relationship considerations, photoaffinity labelling, and application of fluorescence techniques has resulted in the development of a working model of the secretin-receptor complex. This supports the initial docking of the peptide agonist within a cleft in the receptor N-terminus, providing the opportunity for an endogenous sequence within that domain to interact with the core of the receptor. This interaction is believed to be key in the molecular basis of conformational change associated with activation of this receptor. The site of action of this endogenous agonist could also provide a possible target for small molecule agonists to act.

Role of LRAT on the Retinoid Isomerase Activity and Membrane Association of Rpe65

Absorption of a photon by a vertebrate opsin pigment induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical re-isomerization of the chromophore via an enzyme pathway called the visual cycle. The retinoid isomerase in this pathway is Rpe65, a membrane-associated protein in the retinal pigment epithelium (RPE) with no predicted membrane-spanning segments. It has been suggested that Rpe65 is S-palmitoylated by lecithin:retinol acyl transferase (LRAT) on Cys(231), Cys(329), and Cys(330), and that this palmitoylation is required for isomerase activity and the association of Rpe65 with membranes. Here we show that the affinity of Rpe65 for membranes is similar in wild-type and lrat(-/-) mice. The isomerase activity of Rpe65 is also similar in both strains when all-trans-retinyl palmitate is used as substrate. With all-trans-retinol substrate, isomerase activity is present in wild-type but undetectable in RPE homogenates from lrat(-/-) mice. Substitution of Cys(231), Cys(329), and Cys(330) with Ser or Ala did not affect the affinity of Rpe65 for membranes. Further, these Cys residues are not palmitoylated in Rpe65 by mass spectrometric analysis. Global inhibition of protein palmitoylation by 2-bromopalmitate did not affect the solubility or isomerase activity of Rpe65. Finally, we show that soluble and membrane-associated Rpe65 possesses similar isomerase specific activities. These results indicate that LRAT is not required for isomerase activity beyond synthesis of retinyl-ester substrate, and that the association of Rpe65 with membranes is neither dependent upon LRAT nor the result of S-palmitoylation. The affinity of Rpe65 for membranes is probably an intrinsic feature of this protein.

Segments Crucial for Membrane Translocation and Pore-forming Activity of Bordetella Adenylate Cyclase Toxin

Bordetella adenylate cyclase toxin-hemolysin (CyaA, AC-Hly, or ACT) permeabilizes cell membranes by forming small cation-selective (hemolytic) pores and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Both AC delivery and pore formation were previously shown to involve a predicted amphipathic alpha-helix(502-522) containing a pair of negatively charged Glu(509) and Glu(516) residues. Another predicted transmembrane alpha-helix(565-591) comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA. Substitutions of Glu(516) increased specific hemolytic activity of CyaA by two different molecular mechanisms. Replacement of Glu(516) by positively charged lysine residue (E516K) increased the propensity of CyaA to form pores, whereas proline (E516P) or glutamine (E516Q) substitutions extended the lifetime of open single pore units. All three substitutions also caused a drop of pore selectivity for cations. Substitutions of Glu(570) and Glu(581) by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA.

Membrane Topology of the STT3 Subunit of the Oligosaccharyl Transferase Complex

The highly conserved membrane protein STT3 is part of the oligosaccharyl transferase complex in the endoplasmic reticulum of eukaryotic cells. Various experimental observations strongly suggest that STT3 contains the active site of the complex. Here, we report a detailed topology study of STT3 from two different organisms, Saccharomyces cerevisiae and mouse, using in vivo and in vitro topology mapping assays. Our results suggest that STT3 has 11 transmembrane helices and an overall N(cyt)-C(lum) orientation.

Zonadhesin assembly into the hamster sperm acrosomal matrix occurs by distinct targeting strategies during spermiogenesis and maturation in the epididymis.

Zonadhesin is the only sperm protein known to bind in a species-specific manner to the zona pellucida. The zonadhesin precursor is a mosaic protein with a predicted transmembrane segment and large extracellular region composed of cell adhesion, mucin, and tandem von Willebrand D domains. Because the precursor possesses a predicted transmembrane segment and localizes to the anterior head, the mature protein was presumed to be a sperm surface zona pellucida-binding protein. In this study of hamster spermatozoa, we demonstrate that zonadhesin does not localize to the sperm surface but is instead a constituent of the acrosomal matrix. Immunoelectron microscopy revealed that distinct targeting pathways during spermiogenesis and sperm maturation in the epididymis result in trafficking of zonadhesin to the acrosomal matrix. In round spermatids, zonadhesin localized specifically to the acrosomal membrane, where it appeared to be evenly distributed between the outer and inner membrane domains. Subsequent redistribution of zonadhesin resulted in its elimination from the inner acrosomal membrane and restriction to the outer acrosomal membrane of the apical and principal segments and the contents of the posterior acrosome. During sperm maturation in the epididymis, zonadhesin dissociated from the outer acrosomal membrane and became incorporated into the forming acrosomal matrix. These data suggest an important structural role for zonadhesin in assembly of the acrosomal matrix and further support the view that the species specificity of zona pellucida adhesion is mediated by egg-binding proteins contained within the acrosome rather than on the periacrosomal plasma membrane.