Membrane Polypeptides Research Articles

Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate.

Uptake of organic solutes and xenobiotics by mammalian cells is mediated by ATP-independent transporters, and four families of transporters have now been identified. To search for novel organic solute transporters, a liver cDNA library from an evolutionarily primitive marine vertebrate, the little skate Raja erinacea, was screened for taurocholate transport activity by using Xenopus laevis oocytes. In contrast to the organic anion transporters identified to date, a transport activity was identified in this library that required the coexpression of two distinct gene products, termed organic solute transporter alpha and beta (Ostalpha, Ostbeta). Ostalpha cDNA encodes for a protein of 352 aa and seven putative transmembrane (TM) domains. Ostbeta contains 182 aa and has at least one and perhaps two TM domains. There is no significant sequence identity between Ostalpha and Ostbeta, and only low identity with sequences in the databases; however, Ostalpha bears a resemblance to some G protein-coupled receptors, and Ostbeta exhibits 22% amino acid identity with the C-terminal TM and intracellular domains of protocadherin-gamma, a cell surface glycoprotein. Xenopus oocytes injected with the cRNA for both Ostalpha and Ostbeta, but not each separately, were able to take up taurocholate, estrone sulfate, digoxin, and prostaglandin E(2), but not p-aminohippurate or S-dinitrophenyl glutathione. Transport was sodium-independent, saturable, and inhibited by organic anions and steroids, including the major skate bile salt, scymnol sulfate. These results identify an organic anion transporter composed of a putative seven-helix TM protein and an ancillary membrane polypeptide.

Complete cross-validation and R-factor calculation of a solid-state NMR derived structure.

Cross-validation of a solid-state NMR-derived membrane polypeptide structure is demonstrated. An initial structure has been achieved directly from solid-state NMR derived orientational restraints based on a variety of anisotropic nuclear spin interactions. Refining the molecular structure involves setting up a penalty function that incorporates all available solid-state NMR experimental data and an energy function. A validation method is required to choose the optimal weighting factor for the total penalty function to balance the contribution from the experimental restraints and the energy function. Complete cross-validation has been used to avoid over-fitting the orientational restraints. Such cross-validation involves partitioning of the experimental data into a test set and a working set followed by checking the free R-value during the refinement process. This approach is similar to the method used in crystallography and solution NMR. Optimizing the weighting factor on the penalty function by cross-validation will increase the quality of the refined structure from solid-state NMR data. The complete cross-validation and R-factor calculation is demonstrated using experimental solid-state NMR data from gramicidin A, a monovalent cation channel in lipid bilayers.

Actin and annexins I and II are among the main endothelial plasmalemma-associated proteins forming early glucose adducts in experimental diabetes.

An immunochemical and biochemical study was performed to reveal which of the endothelial plasma membrane proteins become glycated during the early phases of diabetes. The blood front of the lung microvascular endothelial plasmalemma was purified by the cationic colloidal silica method from normal and diabetic (streptozotocin-induced) rats and comparatively analyzed by two-dimensional electrophoresis. No major qualitative differences in the general spectrum of endothelial plasmalemmal proteins were recorded between normoglycemic and hyperglycemic animals. By probing with anti-glucitollysine antibodies, we found that at 1 month after the onset of diabetes, several endothelial membrane polypeptides contained glucose covalently linked to their lysyl residues. Ten days of insulin treatment restored euglycemia in the diabetic animals and completely abolished the membrane nonenzymatic glycosylation. All the glycated polypeptides of the endothelial plasma membrane belong to the peripheral type and are associated with its cytoplasmic face (cell cortex). They were solubilized by buffers of high pH and were not detected in the lung cytosolic fraction (100,000 g). By microsequencing, the major proteins labeled by the anti-glucitollysine have been identified as being actin, annexin I, annexin II, the p34 subunit of the Arp2/3 complex, and the Ras suppressor protein-1. Conversely, the intrinsic endothelial membrane proteins do not seem to be affected by hyperglycemia. This defines the internal face of the endothelial plasma membrane, particularly the cortical cytoskeleton, as a preferential target for nonenzymatic glycosylation in diabetes, with possible consequences on the fluidity of the endothelial plasmalemma and impairment of the endothelial mechanotransducing ability.

Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling

To identify membrane-associated polypeptides present in Torpedo nicotinic acetylcholine receptor (AChR)-rich membranes, we used hydrophobic photolabeling with [ 3H]diazofluorene ([ 3H]DAF) and 1-azidopyrene (1-AP) to tag the membrane proteins which were then identified by amino-terminal sequence analysis of labeled fragments isolated from proteolytic digests by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reverse-phase high-performance liquid chromatography. In addition to AChR subunits, identified polypeptides include the 95 kDa α-subunit of the (Na ++K +)-ATPase, the 89 kDa voltage-gated chloride channel (CLC-0), the 105 kDa SITS-binding protein, and 32 and 34 kDa polypeptides identified as Torpedo homologues of the mitochondrial membrane ATP/ADP carrier protein and the voltage-dependent anion channel (VDAC), respectively. Further, individual amino acids that reacted with [ 3H]DAF and therefore likely to be in contact with lipid were identified in the transmembrane segment M3 of the α-subunit of the (Na ++K +)-ATPase and in a putative transmembrane β-strand in VDAC. Collectively these results demonstrate that [ 3H]DAF/1-AP photolabeling provides an effective method for tagging the membrane-associated segments of polypeptides in a way that makes it easy to isolate the labeled polypeptide or polypeptide fragments by fluorescence and then to identify amino acids at the lipid-protein interface by 3H release.

Characterization of plasma membrane domains enriched in lipid metabolites.

A subpopulation of plasma membrane vesicles enriched in membrane lipid metabolites has been isolated from petals of carnation flowers and leaves of canola seedlings. This was achieved by immunopurification from a microsomal membrane preparation using region-specific antibodies raised against a recombinant polypeptide of the plasma membrane H(+)-ATPase. The properties of this subpopulation of vesicles were compared with those of purified plasma membrane isolated by partitioning in an aqueous dextran-polyethylene glycol two-phase system. The lipid composition of the immunopurified vesicles proved to be clearly distinguishable from that of phase-purified plasma membrane, indicating that they represent a unique subpopulation of plasma membrane vesicles. Specifically, the immunopurified vesicles are highly enriched in lipid metabolites, including free fatty acids, diacylglycerol, triacylglycerol and steryl and wax esters, by comparison with the phase-purified plasma membrane. These findings can be interpreted as indicating that lipid metabolites generated within the plasma membrane effectively phase-separate by moving laterally through the plane of the membrane to form discrete domains within the bilayer. It is also apparent that these domains, once formed, are released as vesicles into the cytosol, presumably by microvesiculation from the surface of the plasmalemma. Such removal may be part of normal membrane turnover.

Sec, drugs and rock’n’roll: antibiotic targeting of bacterial protein translocation

A large number of bacterial proteins are active in extracytoplasmic locations. Targeting and membrane translocation of the vast majority of these secretory and membrane polypeptides is mediated by the Sec pathway. Protein secretion requires the co-ordinated and sequential action of targeting factors on the cis-side of the membrane, a complex membrane-embedded protein translocase and maturation enzymes on the trans-side. Recently, significant advances in the molecular genetics and biochemistry of the Sec pathway have revealed that several of the Sec pathway components are essential for bacterial viability and/or pathogenicity. Moreover, several biochemical assays and structural insights have become available. Importantly, some of the Sec components are unique to bacteria. These developments raise the possibility that the bacterial protein translocase and other Sec pathway components could become formidable targets for antibacterial drug discovery.

Evidence for two active branches for electron transfer in photosystem I.

All photosynthetic reaction centers share a common structural theme. Two related, integral membrane polypeptides sequester electron transfer cofactors into two quasi-symmetrical branches, each of which incorporates a quinone. In type II reaction centers [photosystem (PS) II and proteobacterial reaction centers], electron transfer proceeds down only one of the branches, and the mobile quinone on the other branch is used as a terminal acceptor. PS I uses iron-sulfur clusters as terminal acceptors, and the quinone serves only as an intermediary in electron transfer. Much effort has been devoted to understanding the unidirectionality of electron transport in type II reaction centers, and it was widely thought that PS I would share this feature. We have tested this idea by examining in vivo kinetics of electron transfer from the quinone in mutant PS I reaction centers. This transfer is associated with two kinetic components, and we show that mutation of a residue near the quinone in one branch specifically affects the faster component, while the corresponding mutation in the other branch specifically affects the slower component. We conclude that both electron transfer branches in PS I are active.

Membrane-bound structure and alignment of the antimicrobial beta-sheet peptide gramicidin S derived from angular and distance constraints by solid state 19F-NMR.

The antimicrobial properties of the cyclic beta-sheet peptide gramicidin S are attributed to its destabilizing effect on lipid membranes. Here we present the membrane-bound structure and alignment of a derivative of this peptide, based on angular and distance constraints. Solid-state 19F-NMR was used to study a 19F-labelled gramicidin S analogue in dimyristoylphosphatidylcholine bilayers at a lipid:peptide ratio of 80:1 and above. Two equivalent leucine side chains were replaced by the non-natural amino acid 4F-phenylglycine, which serves as a highly sensitive reporter on the structure and dynamics of the peptide backbone. Using a modified CPMG multipulse sequence, the distance between the two 19F-labels was measured from their homonuclear dipolar coupling as 6 A. in good agreement with the known backbone structure of natural gramicidin S in solution. By analyzing the anisotropic chemical shift of the 19F-labels in macroscopically oriented membrane samples, we determined the alignment of the peptide in the bilayer and described its temperature-dependent mobility. In the gel phase, the 19F-labelled gramicidin S is aligned symmetrically with respect to the membrane normal, i.e., with its cyclic beta-sheet backbone lying flat in the plane of the bilayer, which is fully consistent with its amphiphilic character. Upon raising the temperature to the liquid crystalline state, a considerable narrowing of the 19F-NMR chemical shift dispersion is observed, which is attributed the onset of global rotation of the peptide and further wobbling motions. This study demonstrates the potential of the 19F nucleus to describe suitably labelled polypeptides in membranes, requiring only little material and short NMR acquisition times.

Cloning of a cDNA encoding the 22-kDa protein in chloroplasts of spores of Osmunda japonica

Five polypeptides in the soluble fraction of chloroplasts and three polypeptides in the thylakoid membranes in chloroplasts from green spores of the fern Osmunda japonica decreased during spore germination. The 22-kDa protein in thylakoid membranes almost completely disappeared during germination over 48 h. The 22-kDa protein had been purified (Physiologia Plantarum 95: 465. 1995). A protease for the 22-kDa protein also had been separated from chloroplasts. Several fragments appeared after proteolysis of the 22-kDa protein by the protease (Physiologia Plantarum 109: 129. 2000). We have attempted to determine a gene of the 22-kDa protein by PCR using the information of amino-terminal sequences of proteolytic products. The cDNA obtained from the mRNA in the spores shows that the precursor protein is composed of the signal peptide having 16 amino acid residues and the mature protein having 180 amino acid residues. A computer search of the protein database revealed similar features between the 22-kDa protein and LEA 3 homologs of Deinococcus radiodurans, Drosophila melanogaster, Caenorhabditis elegans, and cDNA corresponding to cold hardening-induced Chlorella genes. It seems possible that the 22-kDa protein is a homolog of ABA-inducible proteins, desiccation-related proteins, or a LEA protein. The presence of a chloroplast transit peptide in a sequence of the cDNA supports that the 22-kDa protein is one of the thylakoid membranes. The 22-kDa protein may be a desiccation-tolerant protein in quiescent spores, and a role of the protease is a scavenger for the protein during germination.

Mutational analysis of the binding sites of the phylloquinones in PS I

Due to the symmetry of its heterodimeric core, which is composed of the related PsaA and PsaB membrane polypeptides, photosystem I possesses two cofactor branches that traverse the membrane. There are two phylloquinones in PS I, at least one of which serves as a secondary electron acceptor called A1. Using a combination of site-directed mutagenesis and EPR spectroscopy, we have probed the phylloquinone sites and identified mutants that affect the properties of the A1 cofactor. The PsaA-W693F mutant causes loss of both the photo-accumulated A1- EPR signal and an alteration in the time-resolved P700+ A1- EPR spectrum. Mutation of the corresponding tryptophan in PsaB had no such effect. However, flash absorption spectroscopy of isolated PS I particles revealed that mutation of either tryptophan resulted in an acceleration of the back-reaction from the terminal iron-sulfur clusters. Examination of wild-type Chlamydomonas PS I by in vivo flash spectroscopy demonstrated that reoxidation of A1, as measured by monitoring decay of an absorption increase at 380-400 nm, occurs with biphasic kinetics (half-times of about 13 and 140 ns). The PsaA-W693F mutation specifically affected both the rate of the slower kinetic component (slowed 3-4 fold) and its decay-associated spectrum, while the PsaB-W673F mutation had similar effects on the faster phase. Thus it seems that, although the two phylloquinones of PS I are functionally different, both of them may be used as intermediates in forward and reverse electron transfer through PS I.

Cold stress induced high molecular weight membrane polypeptides are responsible for cold tolerance in Rhizobium DDSS69

Cold stress induces a lag phase in the growth cycle of Rhizobium DDSS69. Two cold sensitive mutants of DDSS69 were generated through Tn5 tagged mutagenesis. These mutants do not grow below 15 degrees C but show a growth curve comparable with the wild type grown at 5 degrees C. There is a rapid induction of two high molecular weight membrane polypeptides of 135 and 119 kDa within 15 min of exposure to 5 degrees C in DDSS69. PAGE membrane protein profiles of stressed and non-stressed cells reveal differential regulation of genes. At 15 degrees C both mutants lack the high molecular weight polypeptides, suggesting a role in alleviation of cold stress.

Identification of porin-like polypeptide(s) in the boundary membrane of oilseed glyoxysomes.

A 36-kDa polypeptide of unknown function was identified by us in the boundary membrane fraction of cucumber seedling glyoxysomes. Evidence is presented in this study that this 36-kDa polypeptide is a glyoxysomal membrane porin. A sequence of 24 amino acid residues derived from a CNBr-cleaved fragment of the 36-kDa polypeptide revealed 72% to 95% identities with sequences in mitochondrial or non-green plastid porins of several different plant species. Immunological evidence indicated that the 36-kDa (and possibly a 34-kDa polypeptide) was a porin(s). Antiserum raised against a potato tuber mitochondrial porin recognized on immunoblots 34-kDa and 36-kDa polypeptides in detergent-solubilized membrane fractions of cucumber seedling glyoxysomes and mitochondria, and in similar glyoxysomal fractions of cotton, castor bean, and sunflower seedlings. The 36-kDa polypeptide seems to be a constitutive component because it was detected also in membrane protein fractions derived from cucumber leaf-type peroxisomes. Compelling evidence that one or both of these polypeptides were authentic glyoxysomal membrane porins was obtained from electron microscopic immunogold analyses. Antiporin IgGs recognized antigen(s) in outer membranes of glyoxysomes and mitochondria. Taken together, the data indicate that membranes of cucumber (and other oilseed) glyoxysomes, leaf-type peroxisomes, and mitochondria possess similar molecular mass porin polypeptide(s) (34 and 36 kDa) with overlapping immunological and amino acid sequence similarities.

VP5, the Nonstructural Polypeptide of Infectious Bursal Disease Virus, Accumulates within the Host Plasma Membrane and Induces Cell Lysis

Infectious bursal disease virus (IBDV) encodes a 17-kDa nonstructural polypeptide known as VP5. This polypeptide is not essential for virus replication in vitro but it plays an important role in in vivo dissemination and pathogenesis. We have characterized the expression of VP5 in three eukaryotic systems: (i) IBDV-infected chicken embryo fibroblasts; (ii) BSC-1 cells infected with a recombinant vaccinia virus vector; and (iii) Cos-1 cells transiently transfected with a plasmid vector. Immunofluorescence analyses showed that upon expression VP5 accumulates within the plasma membrane. This finding was consistent with sequence-based topology predictions, indicating that VP5 is a class II membrane protein with a cytoplasmic N-terminus and an extracellular C-terminal domain. Brefeldin A treatment of VP5-expressing cells prevented the accumulation of this polypeptide in the plasma membrane, thus showing the requirement of an active exocytic pathway to reach that compartment. Expression of VP5 was shown to be highly cytotoxic. Induction of VP5 expression resulted in the alteration of cell morphology, the disruption of the plasma membrane, and a drastic reduction of cell viability. VP5-induced cytotoxicity was prevented by blocking its transport to the membrane with Brefeldin A. Our findings suggest that VP5 plays an important role in the release of the IBDV progeny.

Presenilin-1 and -2 Are Molecular Targets for γ-Secretase Inhibitors

Presenilins are integral membrane protein involved in the production of amyloid beta-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter gamma-secretase cleavage of the beta-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A beta, the 4-kDa beta-peptide. Here, we show that radiolabeled gamma-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A beta formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N- and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of gamma-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective gamma-secretase inhibitors block A beta formation by binding to presenilin-1 and -2.

Ceruminolytic Effects of Docusate Sodium: A Randomized, Controlled Trial

Study Objective: Assessment of the tympanic membrane is often impeded by the presence of cerumen. We compared the ceruminolytic effects of triethanolamine polypeptide and docusate sodium in patients with cerumen. Methods: We conducted a prospective, randomized, controlled, double-blind trial on a convenience sample of cooperative adult and pediatric patients presenting to a university-based emergency department who required removal of cerumen to visualize the tympanic membrane. Structured data collection was performed, and the physician determined whether visualization of the tympanic membrane was partially or totally obscured by cerumen (interobserver agreement, ρ=0.79). Patients received intra-aural instillation of 1mL of either docusate sodium or triethanolamine polypeptide in a liquid form. If not completely cleared within 15 minutes, the external ear canal was irrigated with 50 or 100 mL of normal saline solution and additional attempts to visualize the tympanic membrane were made. The main outcome was the proportion of ears in which the tympanic membrane could be totally visualized after ceruminolytic instillation with or without irrigation. This study had 80% power to detect a 40% difference between groups in the proportion of totally visualized tympanic membranes (χ2 test, α=.05). Results: Of 50 enrolled patients, 23 received triethanolamine polypeptide and 27 received docusate sodium. Mean patient age was 40 years (range 1 to 81 years); 35% were female. Groups were similar in age, sex, and proportion of completely obscured tympanic membranes at presentation (78%). The ability to completely visualize the tympanic membrane was significantly greater after treatment with docusate sodium versus triethanolamine polypeptide (81% versus 35%; difference in proportions 47%; 95% confidence interval [CI], 22 to 71) particularly in children aged 5 or less (90% versus 0%; difference in proportions 89%; 95% CI 50 to 100). Conclusion: Docusate sodium solution is a more effective ceruminolytic than triethanolamine polypeptide, allowing complete or partial visualization of the tympanic membrane in most patients after a single application when followed with irrigation. Use of docusate sodium as a ceruminolytic should be encouraged, particularly in children. [Singer AJ, Sauris E, Viccellio AW. Ceruminolytic effects of docusate sodium: a randomized, controlled trial. Ann Emerg Med. September 2000;36:228-232.]

Functional cybrid plants of Lycopersicon peruvianum var 'dentatum ' with chloroplasts of Lycopersicon esculentum.

Fertile cybrid plants of three subclones, B1A, B3A, B4A were regenerated from the single colony obtained after the fusion of mesophyll protoplasts of plastome chlorophyll-deficient mutant Lycopersicon peruvianum var 'dentatum' (line 3767) and γ-irradiated mesophyll protoplasts of L. esculentum (cv 'Quedlinburger Frühe Liebe'). Cytogenetic, isozyme, RAPD, morphological and restriction analyses all showed that the subclones had the nuclear genome of L. peruvianum var 'dentatum' and plastome genome of L. esculentum, while the mitochondrial genome was altered. No phenotypical traits that could be taken as evidence of plastome-genome incompatibility in the cybrid subclones were observed. Genetic functionality of all subclones was proven by the backcrossing analysis. To study the functionality of the cybrid plants we also carried out an analysis of their photosynthetic system. Data on chlorophyll-a and -b content, analyses of the fluorescence induction curves, intensity of CO2 assimilation, pigment-protein complexes and polypeptides of thylakoid membranes showed the absence of structural and functional abnormalities in the photosynthetic apparatus of the cybrid plants. We concluded that the plastome of L. esculentum is able to effectively interract with the nuclear genome of L. peruvianum var 'dentatum' and together with the recombined chondriome can support genetic functionality of cybrid plants of the peruvianum tomato.

Alterations in the plasma membrane polypeptide pattern of tomato roots (Lycopersicon esculentum) during the development of arbuscular mycorrhiza

Changes induced by arbuscular mycorrhizal (AM) formation in the plasma membrane polypeptide pattern of tomato roots have been assessed by 2D-PAGE analysis. Plasma membrane fractions were isolated by aqueous two-phase partitioning from control and mycorrhizal tomato root microsomes. Analysis of 2D-PAGE gels revealed that AM colonization induces at the plasma membrane level two major changes in protein synthesis: down-regulation of some constitutive polypeptides and synthesis of new polypeptides or endomycorrhizins. A comparison of changes induced by two different levels of AM colonization showed that 16 polypeptides were differentially displayed at both AM colonization stages, while some others were transiently regulated. Five of the differentially displayed plasma membrane polypeptides at both AM colonization stages were selected for N-terminal amino acid sequencing. Reliable sequences were obtained for two of the selected spots. Sequence alignment search indicated that one of the sequenced polypeptides showed 75% identity to the N-terminal sequence of the 69 kDa catalytic subunit of the vacuolar type H(+)-ATPase of several plants. The possible significance of these findings is discussed in relation to the functioning of the AM symbiosis.

Alterations in the plasma membrane polypeptide pattern of tomato roots ( Lycopersicon esculentum ) during the development of arbuscular mycorrhiza

Changes induced by arbuscular mycorrhizal (AM) formation in the plasma membrane polypeptide pattern of tomato roots have been assessed by 2D‐PAGE analysis. Plasma membrane fractions were isolated by aqueous two‐phase partitioning from control and mycorrhizal tomato root microsomes. Analysis of 2D‐PAGE gels revealed that AM colonization induces at the plasma membrane level two major changes in protein synthesis: down‐regulation of some constitutive polypeptides and synthesis of new polypeptides or endomycorrhizins. A comparison of changes induced by two different levels of AM colonization showed that 16 polypeptides were differentially displayed at both AM colonization stages, while some others were transiently regulated. Five of the differentially displayed plasma membrane polypeptides at both AM colonization stages were selected for N‐terminal amino acid sequencing. Reliable sequences were obtained for two of the selected spots. Sequence alignment search indicated that one of the sequenced polypeptides showed 75% identity to the N‐terminal sequence of the 69 kDa catalytic subunit of the vacuolar type H + ‐ATPase of several plants. The possible significance of these findings is discussed in relation to the functioning of the AM symbiosis.

RH blood group system and molecular basis of Rh-deficiency

Rhesus (Rh) antigens are defined by a complex association of membrane polypeptides that are missing or severely deficient from the red cells of rare Rhnullindividuals who suffer a clinical syndrome of varying severity characterized by abnormalities of the red cell shape, cation transport and membrane phospholipid organization. The Rhnullphenotype is an inherited condition that may arise from homozygosity either for a ‘suppressor’ gene unrelated to the RH locus (‘regulator type’) or for a silent allele at the RH locus itself (‘amorph type’).A current model suggests that the proteins of the Rh complex (Rh, RhAG, CD47, LW, GPB) are assembled by non-covalent bonds and that it is not assembled or transported to the cell surface when one subunit is missing. Rh and RhAG proteins belong to the same protein family and are quantitatively the major components that form the core of the complex, which is firmly linked to the membrane skeleton. Molecular analysis of Rhnullindividuals has revealed that abnormalities occur only at the RHAG and RH loci, without alteration of the genes encoding the accessory chains.Mutations of the RHAG gene, but not of RH, occur in all Rhnullindividuals of the regulator type (including Rhmod) investigated so far (13 cases), strongly suggesting thatRHAG mutants act as ‘suppressors’ and not as transcriptional regulators of the RH genes and that variable expression of the RHAG alleles may account for the Rhmodphenotypes (exhibiting weak expression of Rh antigens). Conversely, mutations of the RHCE gene, but not of RHAG, occur in two unrelated Rhnullindividuals of the amorph type, supporting the view that RH mutants result from a ‘silent’ allele at the RH locus. These findings strongly support the Rh complex model since when either the Rh or RhAG protein is missing, the assembly and/or transport of the Rh complex is defective. Transcriptional as well as post-transcriptional mechanisms may account for the molecular abnormalities, but experimental evidence based on expression models is required to test these hypotheses, in the hope that they may help to clarify the biological role of the Rh proteins in the red cell membrane.

A thermostable vacuolar-type membrane pyrophosphatase from the archaeon Pyrobaculum aerophilum: implications for the origins of pyrophosphate-energized pumps

Vacuolar-type H +-translocating pyrophosphatases (V-PPases) have been considered to be restricted to plants, a few species of phototrophic proteobacteria and protists. Here, we describe PVP, a thermostable, sequence-divergent V-PPase from the facultatively aerobic hyperthermophilic archaeon Pyrobaculum aerophilum. PVP shares only 38% sequence identity with both the prototypical V-PPase from Arabidopsis thaliana and the H +-PPi synthase from Rhodospirillum rubrum, yet possesses most of the structural features characteristic of V-PPases. Heterologous expression of PVP in Saccharomyces cerevisiae yields a M r 64 000 membrane polypeptide that specifically catalyzes Mg 2+-dependent PPi hydrolysis. The existence of PVP implies that PPi-energized H +-translocation is phylogenetically more deeply rooted than previously thought.