Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

cDNA clones coding for either full-length or truncated forms of the beta-subunit of the Na+,K+-ATPase from pig kidney were engineered into a transcription vector based on a T7 promotor. In vitro transcription and subsequent translation of the mRNA in the presence of rough microsomes (RM) yielded beta-subunit molecules that were N-glycosylated and correctly inserted into the membrane. The signal peptide was not cleaved off. This membrane integration was found to be dependent on the function of the signal recognition particle (SRP). Several lines of evidence suggest that the hydrophilic aminoterminal domain of 34 amino acid residues preceding the postulated signal sequence is located on the cytoplasmic side whereas the carboxyterminal glycosylated domain is located on the exoplasmic side of the ER (endoplasmic reticulum)-membrane (type II membrane protein).
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PMID:SRP-dependent membrane integration of the beta-subunit of Na+,K+-ATPase. 254 83

Ductin is a highly conserved and polytopic transmembrane protein which is the subunit c component of the vacuolar H(+)-ATPase (V-ATPase) and a component of a connexon channel of gap junctions. Previous studies have suggested that ductin in the V-ATPase has the opposite orientation of ductin in a connexon. Using an in vitro translation system coupled to microsomes derived from the endoplasmic reticulum, we show that ductin is co-translationally inserted into the membrane bilayer, suggesting a dependency on the signal recognition particle for synthesis. By attaching a C-terminal polypeptide derived from beta-lactamase and by using cysteine replacement coupled to chemical labelling, we show that ductin is inserted into the microsomal membrane in both orientations in similar proportions. In contrast, squid rhodopsin appears to be inserted in a single orientation. Changing conserved charged residues at the N-terminus of ductin does not affect the ratio of the two orientations. Once in the microsomal membrane, ductin assembles into an oligomeric complex which contains a pore accessible to a water-soluble probe, reminiscent of the ductin complex found in the V-ATPase and a connexon.
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PMID:Membrane insertion and assembly of ductin: a polytopic channel with dual orientations. 764 80

We have developed two independent assays to study the integration, folding, and intracellular transport of the polytopic plasma membrane H(+)-ATPase in yeast. To follow folding, controlled trypsinolysis was used to distinguish between the E1 conformation of the ATPase (favored in the presence of ADP) and the E2 conformation (favored in the presence of vanadate). By this criterion, wild-type ATPase appears to recognize its ligands and assume distinct conformations within a short time after its biosynthesis. To follow intracellular transport, we have exploited the fact that export of newly synthesized ATPase from the endoplasmic reticulum is accompanied by kinase-mediated phosphorylation, leading to a shift in electrophoretic mobility. Because proper folding is required for transport from the endoplasmic reticulum, the mobility shift also serves as a convenient bioassay for correct folding. As a first step toward identifying cell components important in folding of the nascent ATPase, we have used the dual assays to examine the role of KAR2, encoding the yeast homolog of immunoglobulin heavy chain binding protein/78-kDa glucose-regulated protein, and SEC65, encoding a subunit of the yeast signal recognition particle. Although mutation of KAR2 caused defective translocation of several secretory precursors into the endoplasmic reticulum lumen, ATPase folding and intracellular transport were unperturbed. By contrast, in a sec65 mutant, the folding and intracellular transport of newly synthesized ATPase were delayed. Our data suggest that conformational maturation of the ATPase is a rapid process in wild-type cells and that membrane integration mediated by signal recognition peptide is important for the proper folding of this polytopic protein.
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PMID:Folding and intracellular transport of the yeast plasma-membrane H(+)-ATPase: effects of mutations in KAR2 and SEC65. 851 33

The signal recognition particle 54-kDa subunit (SRP54) binds to the signal sequences of nascent presecretory and transmembrane proteins. Previous studies have shown that signal sequences bind to the C-terminal methionine-rich domain of the protein (M-domain), but have raised the possibility that either the N-terminal domain (N-domain) or the central guanosine triphosphatase module (GTPase-domain) also contribute to signal-sequence-binding activity. We have generated a series of N-domain and GTPase-domain mutants to investigate this issue further. Mutations in a conserved N-domain motif (ALLEADV) produced significant defects in signal sequence binding that correlate with the severity of the mutation. The magnitude of the defect was independent of the preprotein substrate, which suggested that the mutations do not alter the specificity of signal sequence recognition. The N-domain mutants also showed defects in promoting the translocation of presecretory proteins across the membrane of microsomal vesicles, but these defects appeared to be a direct consequence of the reduction in signal-sequence-binding activity and not separate effects of the mutations. By contrast, mutations in the guanosine triphosphatase consensus sequence had no effect on signal sequence binding, but instead severely impaired protein translocation activity. These results indicate that a principal function of the SRP54 N-domain is to promote efficient signal sequence binding. These data also suggest that the SRP54 GTPase regulates the cycle of signal sequence binding and release, perhaps by modulating the relative orientation of the N- and M-domains.
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PMID:The N-domain of the signal recognition particle 54-kDa subunit promotes efficient signal sequence binding. 918 11

The relative rates of change for eight sets of ubiquitous proteins were determined by a test in which anciently duplicated paralogs are used to root the universal tree and distances are calculated between each taxonomic group and the last common ancestor. The sets included ATPase subunits, elongation factors, signal recognition particle and its receptor, three sets of tRNA synthetases, transcarbamoylases, and an internal duplication in carbamoyl phosphate synthase. In each case phylogenetic trees were constructed and the distances determined for all pairs. Taken over the period of time since their last common ancestor, average evolutionary rates are remarkably similar for Bacteria and Eukarya, but Archaea exhibit a significantly slower average rate.
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PMID:Determining the relative rates of change for prokaryotic and eukaryotic proteins with anciently duplicated paralogs. 1094 74

Bacteria use several routes to target their exported proteins to the plasma membrane. The majority are exported through pores formed by SecY and SecE. Two different molecular machineries are used to target proteins to the SecYE translocon. Translocated proteins, synthesized as precursors with cleavable signal sequences, require cytoplasmic chaperones, such as SecB, to remain competent for posttranslational transport. In concert with SecB, SecA targets the precursors to SecY and energizes their translocation by its ATPase activity. The latter function involves a partial insertion of SecA itself into the SecYE translocon, a process that is strongly assisted by a couple of membrane proteins, SecG, SecD, SecF, YajC, and the proton gradient across the membrane. Integral membrane proteins, however, are specifically recognized by a direct interaction between their noncleaved signal anchor sequences and the bacterial signal recognition particle (SRP) consisting of Ffh and 4.5S RNA. Recognition occurs during synthesis at the ribosome and leads to a cotranslational targeting to SecYE that is mediated by FtsY and the hydrolysis of GTP. No other Sec protein is required for integration unless the membrane protein also contains long translocated domains that engage the SecA machinery. Discrimination between SecA/SecB- and SRP-dependent targeting involves the specificity of SRP for hydrophobic signal anchor sequences and the exclusion of SRP from nascent chains of translocated proteins by trigger factor, a ribosome-associated chaperone. The SecYE pore accepts only unfolded proteins. In contrast, a class of redox factor-containing proteins leaves the cell only as completely folded proteins. They are distinguished by a twin arginine motif of their signal sequences that by an unknown mechanism targets them to specific pores. A few membrane proteins insert spontaneously into the bacterial plasma membrane without the need for targeting factors and SecYE. Insertion depends only on hydrophobic interactions between their transmembrane segments and the lipid bilayer and on the transmembrane potential. Finally, outer membrane proteins of Gram-negative bacteria after having crossed the plasma membrane are released into the periplasm, where they undergo distinct folding events until they insert as trimers into the outer membrane. These folding processes require distinct molecular chaperones of the periplasm, such as Skp, SurA, and PpiD.
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PMID:Protein traffic in bacteria: multiple routes from the ribosome to and across the membrane. 1105 63

Prokaryotes constitute a single kingdom, Bacteria, here divided into two new subkingdoms: Negibacteria, with a cell envelope of two distinct genetic membranes, and Unibacteria, comprising the new phyla Archaebacteria and Posibacteria, with only one. Other new bacterial taxa are established in a revised higher-level classification that recognizes only eight phyla and 29 classes. Morphological, palaeontological and molecular data are integrated into a unified picture of large-scale bacterial cell evolution despite occasional lateral gene transfers. Archaebacteria and eukaryotes comprise the clade neomura, with many common characters, notably obligately co-translational secretion of N-linked glycoproteins, signal recognition particle with 7S RNA and translation-arrest domain, protein-spliced tRNA introns, eight-subunit chaperonin, prefoldin, core histones, small nucleolar ribonucleoproteins (snoRNPs), exosomes and similar replication, repair, transcription and translation machinery. Eubacteria (posibacteria and negibacteria) are paraphyletic, neomura having arisen from Posibacteria within the new subphylum Actinobacteria (possibly from the new class Arabobacteria, from which eukaryotic cholesterol biosynthesis probably came). Replacement of eubacterial peptidoglycan by glycoproteins and adaptation to thermophily are the keys to neomuran origins. All 19 common neomuran character suites probably arose essentially simultaneously during the radical modification of an actinobacterium. At least 11 were arguably adaptations to thermophily. Most unique archaebacterial characters (prenyl ether lipids; flagellar shaft of glycoprotein, not flagellin; DNA-binding protein lob; specially modified tRNA; absence of Hsp90) were subsequent secondary adaptations to hyperthermophily and/or hyperacidity. The insertional origin of protein-spliced tRNA introns and an insertion in proton-pumping ATPase also support the origin of neomura from eubacteria. Molecular co-evolution between histones and DNA-handling proteins, and in novel protein initiation and secretion machineries, caused quantum evolutionary shifts in their properties in stem neomura. Proteasomes probably arose in the immediate common ancestor of neomura and Actinobacteria. Major gene losses (e.g. peptidoglycan synthesis, hsp90, secA) and genomic reduction were central to the origin of archaebacteria. Ancestral archaebacteria were probably heterotrophic, anaerobic, sulphur-dependent hyperthermoacidophiles; methanogenesis and halophily are secondarily derived. Multiple lateral gene transfers from eubacteria helped secondary archaebacterial adaptations to mesophily and genome re-expansion. The origin from a drastically altered actinobacterium of neomura, and the immediately subsequent simultaneous origins of archaebacteria and eukaryotes, are the most extreme and important cases of quantum evolution since cells began. All three strikingly exemplify De Beer's principle of mosaic evolution: the fact that, during major evolutionary transformations, some organismal characters are highly innovative and change remarkably swiftly, whereas others are largely static, remaining conservatively ancestral in nature. This phenotypic mosaicism creates character distributions among taxa that are puzzling to those mistakenly expecting uniform evolutionary rates among characters and lineages. The mixture of novel (neomuran or archaebacterial) and ancestral eubacteria-like characters in archaebacteria primarily reflects such vertical mosaic evolution, not chimaeric evolution by lateral gene transfer. No symbiogenesis occurred. Quantum evolution of the basic neomuran characters, and between sister paralogues in gene duplication trees, makes many sequence trees exaggerate greatly the apparent age of archaebacteria. Fossil evidence is compelling for the extreme antiquity of eubacteria [over 3500 million years (My)] but, like their eukaryote sisters, archaebacteria probably arose only 850 My ago. Negibacteria are the most ancient, radiating rapidly into six phyla. Evidence from molecular sequences, ultrastructure, evolution of photosynthesis, envelope structure and chemistry and motility mechanisms fits the view that the cenancestral cell was a photosynthetic negibacterium, specifically an anaerobic green non-sulphur bacterium, and that the universal tree is rooted at the divergence between sulphur and non-sulphur green bacteria. The negibacterial outer membrane was lost once only in the history of life, when Posibacteria arose about 2800 My ago after their ancestors diverged from Cyanobacteria.
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PMID:The neomuran origin of archaebacteria, the negibacterial root of the universal tree and bacterial megaclassification. 1183 18

Sequences and available structures were compared for all the widely distributed representatives of the P-loop GTPases and GTPase-related proteins with the aim of constructing an evolutionary classification for this superclass of proteins and reconstructing the principal events in their evolution. The GTPase superclass can be divided into two large classes, each of which has a unique set of sequence and structural signatures (synapomorphies). The first class, designated TRAFAC (after translation factors) includes enzymes involved in translation (initiation, elongation, and release factors), signal transduction (in particular, the extended Ras-like family), cell motility, and intracellular transport. The second class, designated SIMIBI (after signal recognition particle, MinD, and BioD), consists of signal recognition particle (SRP) GTPases, the assemblage of MinD-like ATPases, which are involved in protein localization, chromosome partitioning, and membrane transport, and a group of metabolic enzymes with kinase or related phosphate transferase activity. These two classes together contain over 20 distinct families that are further subdivided into 57 subfamilies (ancient lineages) on the basis of conserved sequence motifs, shared structural features, and domain architectures. Ten subfamilies show a universal phyletic distribution compatible with presence in the last universal common ancestor of the extant life forms (LUCA). These include four translation factors, two OBG-like GTPases, the YawG/YlqF-like GTPases (these two subfamilies also consist of predicted translation factors), the two signal-recognition-associated GTPases, and the MRP subfamily of MinD-like ATPases. The distribution of nucleotide specificity among the proteins of the GTPase superclass indicates that the common ancestor of the entire superclass was a GTPase and that a secondary switch to ATPase activity has occurred on several independent occasions during evolution. The functions of most GTPases that are traceable to LUCA are associated with translation. However, in contrast to other superclasses of P-loop NTPases (RecA-F1/F0, AAA+, helicases, ABC), GTPases do not participate in NTP-dependent nucleic acid unwinding and reorganizing activities. Hence, we hypothesize that the ancestral GTPase was an enzyme with a generic regulatory role in translation, with subsequent diversification resulting in acquisition of diverse functions in transport, protein trafficking, and signaling. In addition to the classification of previously known families of GTPases and related ATPases, we introduce several previously undetected families and describe new functional predictions.
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PMID:Classification and evolution of P-loop GTPases and related ATPases. 1191 78

The major route of protein translocation in bacteria is the so-called general secretion pathway (Sec-pathway). This route has been extensively studied in Escherichia coli and other bacteria. The movement of preproteins across the cytoplasmic membrane is mediated by a multimeric membrane protein complex called translocase. The core of the translocase consists of a proteinaceous channel formed by an oligomeric assembly of the heterotrimeric membrane protein complex SecYEG and the peripheral adenosine triphosphatase (ATPase) SecA as molecular motor. Many secretory proteins utilize the molecular chaperone SecB for targeting and stabilization of the unfolded state prior to translocation, while most nascent inner membrane proteins are targeted to the translocase by the signal recognition particle and its membrane receptor. Translocation is driven by ATP hydrolysis and the proton motive force. In the last decade, genetic and biochemical studies have provided detailed insights into the mechanism of preprotein translocation. Recent crystallographic studies on SecA, SecB and the SecYEG complex now provide knowledge about the structural features of the translocation process. Here, we will discuss the mechanistic and structural basis of the translocation of proteins across and the integration of membrane proteins into the cytoplasmic membrane.
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PMID:The bacterial translocase: a dynamic protein channel complex. 1461 54

Two structurally homologous guanosine triphosphatase (GTPase) domains interact directly during signal recognition particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The 2.05 angstrom structure of a complex of the NG GTPase domains of Ffh and FtsY reveals a remarkably symmetric heterodimer sequestering a composite active site that contains two bound nucleotides. The structure explains the coordinate activation of the two GTPases. Conformational changes coupled to formation of their extensive interface may function allosterically to signal formation of the targeting complex to the signal-sequence binding site and the translocon. We propose that the complex represents a molecular "latch" and that its disengagement is regulated by completion of assembly of the GTPase active site.
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PMID:Heterodimeric GTPase core of the SRP targeting complex. 1472 91


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