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1.
Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea
Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise
combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression
of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the
highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant
reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression.
However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that
specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions
between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into
the structural change of ATP synthase during catalysis. 相似文献
2.
Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea
Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding a, p, y, 8 and e subunits of Spinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding p-galactosidase was detected. Of all the combinations, that of y and e subunit genes showed the highest level of reporter gene expression, while those of a and p, a and e, p and e and p and 8 induced stable and significant reporter gene expression. The combination of 8 and e as well as that of 8 and y induced weak and unstable reporter gene expression. However, combinations of a and y, p and y and a and 8 did not induce reporter gene expression. These results suggested that specific and strong interactions between y and e, a and p, a and e, p and e and p and 8 subunits, and weak and transient interactions between 8 and e and 8 and y subunits occurred in the yeast 相似文献
3.
4.
COPI-coated vesicles mediate retrograde transport from the Golgi back to the ER and intra-Golgi transport. The cytosolic precursor of the COPI coat, the heptameric coatomer complex, can be thought of as composed of two subcomplexes. The first consists of the β-, γ-, δ- and ζ-COP subunits which are distantly homologous to AP clathrin adaptor subunits. The second consists of the α-, β'- and ε-COP subunits. Here, we present the structure of the appendage domain of γ-COP and show that it has a similar overall fold as the α-appendage of AP2. Again, like the α-appendage the γ-COP appendage possesses a single protein/protein interaction site on its platform subdomain. We show that in yeast this site binds to the ARFGAP Glo3p, and in mammalian γ-COP this site binds to a Glo3p orthologue, ARFGAP2. On the basis of mutations in the yeast homologue of γ-COP, Sec21p, a second binding site is proposed to exist on the γ-COP appendage that interacts with the α,β',ε COPI subcomplex. 相似文献
5.
Eukaryotic translation initiation factor eIF2B, the guanine nucleotide exchange factor (GEF) for eIF2, catalyzes conversion of eIF2·GDP to eIF2·GTP. The eIF2B is composed of five subunits, α, β, γ, δ and ε, within which the ε subunit is responsible for catalyzing the guanine exchange reaction. Here we present the crystal structure of the C-terminal domain of human eIF2Bε (eIF2Bε-CTD) at 2.0-Å resolution. The structure resembles a HEAT motif and three charge-rich areas on its surface can be identified. When compared to yeast eIF2Bε-CTD, one area involves highly conserved AA boxes while the other two are only partially conserved. In addition, the previously reported mutations in human eIF2Bε-CTD, which are related to the loss of the GEF activity and human VWM disease, have been discussed. Based on the structure, most of such mutations tend to destabilize the HEAT motif. 相似文献
6.
Mutation in splicing consensus sequences causes lack of TCR membrane expression due to exon excision
T-cell antigen receptor (TCR) membrane-negative T-cell mutants can be divided into two groups: 1) those which lack one of
the six TCR polypeptides and 2) those which contain a mutated TCR chain. The present experiments reveal a new mechanism for
the development of TCR membrane-negative T-cell variants: mutations in splicing consensus motifs causing excision or misreading
of an entire exon (exon 3 of the TCRAC or TCRBC genes). C27.15 cells transcribe a TCR α chain consisting of TCRAVJCexon1Cexon2-encoded amino acids plus six new amino acids. The assembly defect seems to be that the truncated α chain does not interact
with CD3 δ molecules; consequently, no TCR αβ/CD3 δεγε complexes are formed. E6.E12 cells transcribe a TCR β chain composed
of TCRBVDJCexon1Cexon2-encoded amino acids plus twenty-seven new amino acids, which seem not to form a transmembrane region. The truncated β chain
does associate with CD3 γε heterodimers, yet no TCR αβ/CD3 δεγε complexes are made. This may be due either to low assembly
of TCR β/CD3 γε trimers or to lack of access of the mutated TCR β/CD3 γε trimers to the TCR α/CD3 δε compartment in the endoplasmic
reticulum.
Received: 25 September 1996 / Revised: 7 November 1996 相似文献
7.
8.
The F1-ATP synthase complex constitutes the catalytic component of F1F0-ATP synthase, the primary ATP synthetic enzyme in the cell. Previous studies indicate that the glacier ice worm, Mesenchytraeus solifugus, maintains unusually high ATP levels that continue to rise as temperatures decline, suggesting that molecular changes within
ice worm F1-ATP synthase subunits may contribute to this energetic anomaly. In this report, we compared ice worm F1-ATP synthase subunits (α, β, γ) with homologues across metazoan phyla (arthropod, chordate, nematode) and among a group of clitellate annelids (Enchytraeus albidus, Enchytraeus buchholzi, Lumbriculus variegatus, Theromyzon tessulatum). Amino acid alignments indicated that ice worm F1-ATP α and F1-ATP β subunits share strong sequence homology with their mesophilic counterparts, respectively, but that ATP γ has diverged more rapidly. Moreover, F1-ATP α and F1-ATP β displayed amino acid compositional changes consistent with trends observed in other cold adapted proteins, while F1-ATP γ diverged in unexpected directions (e.g., gains in size, charged residues). Several ice worm-specific amino acid substitutions
map to positions near the F1-ATP β catalytic site while others occur near subunit contact sites. 相似文献
9.
The FUD17 strain of Chlamydomonas reinhardtii is a photosynthesis-deficient, acetate-requiring mutant with a defect in the chloroplast atpE gene, which codes for the ε subunit of the chloroplast ATP synthase. In this work, the FUD17 mutant was examined in relation
to other known ATP synthase mutants as an initial step toward using this strain to generate altered versions of the atpE gene for site-directed mutagenesis of the ε subunit. The FUD17 strain grows well and is normally pigmented in the dark (heterotrophic
conditions), but cannot grow autotrophically in the light, even when media are supplemented with acetate. Under heterotrophic
conditions, it shows no accumulation of the ε subunit, and much lower levels of the α and β subunits of the chloroplast ATP
synthase. FUD17 shows no light-dependent oxygen evolution and shows a strong, light-dependent alteration in its chlorophyll
fluorescence. These results show that FUD17 possesses similar characteristics to other ATP synthase mutants and fails to express
an assembled ATP synthase complex on its thylakoid membrane. A preliminary attempt at site-directed mutagenesis is described
which produced a slightly truncated form of the ε subunit, which is expressed normally in the cell.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
10.
Abigail S. Hackam Tian-Li Wang William B. Guggino Garry R. Cutting 《Journal of neurochemistry》1998,70(1):40-46
Abstract: Molecular cloning has revealed that there are six classes of subunits capable of forming GABA-gated chloride channel receptors. GABAA receptors are composed of α, β, γ, δ, and ε/χ subunits, whereas GABAC receptors appear to contain ρ subunits. However, retinal cells exhibiting GABAC responses express α, β, and ρ subunits, raising the possibility that GABAC receptors may be a mixture of subunit classes. Using in vitro translated protein, we determined that human GABAA receptor subunits α1, α5, and β1 did not coimmunoprecipitate with full-length ρ1, ρ2, or the N-terminal domain of ρ1 that contains signals for ρ-subunit interaction. To explore the molecular mechanism underlying these apparently exclusive combinations, chimeric subunits were created and tested for interaction with the wild-type subunits. Transfer of the N terminus of β1 to ρ1 created a β1ρ1 chimera that coimmunoprecipitated with the α1 subunit but not with the ρ2 subunit. Furthermore, exchanging the N terminus of the ρ1 subunit with the corresponding region of β1 produced a ρ1β1 chimera that interfered with ρ1 receptor expression in Xenopus oocytes, whereas the full-length β1 subunit had no effect. Together, these results indicate that sequences in the N termini direct assembly of ρ subunits and GABAA subunits into GABAC and GABAA receptors, respectively. 相似文献
11.
Yan-Bin Teng Yong-Liang Jiang Yong-Xing He Wei-Wei He Fu-Ming Lian Yuxing Chen Cong-Zhao Zhou 《BMC structural biology》2009,9(1):67-8
Background
The carbonic anhydrases (CAs) are involved in inorganic carbon utilization. They have been classified into six evolutionary and structural families: α-, β-, γ-, δ-, ε-, ζ- CAs, with β-CAs present in higher plants, algae and prokaryotes. The yeast Saccharomyces cerevisiae encodes a single copy of β-CA Nce103/YNL036W. 相似文献12.
Beauséjour M Noël D Thibodeau S Bouchard V Harnois C Beaulieu JF Demers MJ Vachon PH 《Apoptosis : an international journal on programmed cell death》2012,17(6):566-578
In human intestinal epithelial crypt (HIEC) cells, the PI3-K/Akt-1 pathway is crucial for the promotion of cell survival and
suppression of anoikis. Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit. Three R
(p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known. Herein, we analyzed the expression of PI3-K isoforms
in HIEC cells and determined their roles in cell survival, as well as in the β1 integrin/Fak/Src-mediated suppression of anoikis.
We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition
and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent
apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and
apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than
that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling;
however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but
Src-independent). Hence, HIEC cells selectively express PI3-K isoform complexes, translating into distinct roles in Akt-1
activation and cell survival, as well as in a selective engagement by Fak and/or Src within the context of β1 integrin/Fak/Src-mediated
suppression of anoikis. 相似文献
13.
The ε subunit of the ATP synthase from E. coli undergoes conformational changes while rotating through 360° during catalysis. The conformation of ε was probed in the membrane-bound
ATP synthase by reaction of mono-cysteine mutants with 3-N-maleimidyl-propionyl biocytin (MPB) under resting conditions, during
ATP hydrolysis, and after inhibition by ADP-AlF3. The relative extents of labeling were quantified after electrophoresis and blotting of the partially purified ε subunit.
Residues from the N-terminal β-sandwich domain showed a position-specific pattern of labeling, consistent with prior structural
studies. Some residues near the ε-γ interface showed changes up to two-fold if labeling occurred during ATP hydrolysis or
after inhibition by ADP-AlF3. In contrast, residues found in the C-terminal α-helices were all labeled to a moderate or high level with a pattern that
was consistent with a partially opened helical hairpin. The results indicate that the two C-terminal α-helices do not adopt
a fixed conformation under resting conditions, but rather exhibit intrinsic flexibility. 相似文献
14.
Tomohiro Yamaguchi Youichi Suzuki Ryuichi Katakura Takusaburo Ebina Junkichi Yokoyama Yoshiaki Fujimiya 《Cancer immunology, immunotherapy : CII》1998,47(2):97-103
γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the
activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer
patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from
glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ
or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT
cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations
of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response,
this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies.
Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific
autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific
activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast
to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced
in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic
use of γδT cells in cancer patients.
Received: 23 January 1998 / Accepted: 20 May 1998 相似文献
15.
GABAA receptors composed of α, β and γ subunits display a significantly higher single-channel conductance than receptors comprised
of only α and β subunits. The pore of GABAA receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at
the 6′, 10′ and 13′ positions. At the 2′ position, however, a polar residue is present in the γ subunit but not the α or β
subunits. As residues at the 2′, 6′ and 10′ positions are exposed in the open channel and as such polar channel-lining residues
may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and
the kinetic properties of wild-type α1β1 and α1β1γ2S receptors with two mutant receptors, αβγ(S2′A) and αβγ(S2′V). We found
that the single-channel conductance of both mutant αβγ receptors was significantly decreased with respect to wild-type αβγ,
with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant αβγ receptors
remained larger than wild-type αβ channels. This reduction in the conductance of mutant αβγ receptors was observed at depolarized
potentials only (ECl = −1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the γ2′ residue. The substitutions at the
γ2′ serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the
open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study
show that residues at the 2′ position in M2 of the γ subunit affects both single-channel conductance and receptor kinetics. 相似文献
16.
Huirong Li Yong Yu Wei Luo Yinxin Zeng Bo Chen 《Extremophiles : life under extreme conditions》2009,13(2):233-246
In order to assess bacterial diversity within four surface sediment samples (0–5 cm) collected from the Pacific Arctic Ocean,
16S ribosomal DNA clone library analysis was performed. Near full length 16S rDNA sequences were obtained for 463 clones from
four libraries and 13 distinct major lineages of Bacteria were identified (α, β, γ, δ and ε-Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Actinobacteria, Firmicutes, Planctomycetes, Spirochetes, and Verrucomicrobia). α, γ, and δ-Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria were common phylogenetic groups from all the sediments. The γ-Proteobacteria were the dominant
bacterial lineage, representing near or over 50% of the clones. Over 35% of γ-Proteobacteria clones of four clone library
were closely related to cultured bacterial isolates with similarity values ranging from 94 to 100%. The community composition
was different among sampling sites, which potentially was related to geochemical differences. 相似文献
17.
Indolepyruvate ferredoxin oxidoreductase (IOR) catalyzes the oxidative decarboxylation of arylpyruvates. Gene cloning and
sequencing analysis of the IOR gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was performed. Two genes, iorA and iorB, encoding α and β subunits of IOR were found to be tandemly arranged, which suggests that gene expression is translationaly
coupled. Sequence analysis showed the C-terminal region of the α subunit to have a typical ferredoxin-type [4Fe-4S] cluster
motif (CXXCXXCXXCXXXCP), which is similar to that present in the δ subunits of other oxidoreductases such as pyruvate ferredoxin
oxidoreductase (POR) and 2-ketoisovalerate ferredoxin oxidoreductase (VOR). We suggest that the α subunit of KOD1-IOR has
a mosaic structure composed of features characteristic of the α, β and δ subunits from POR and VOR. KOD1-IOR was overproduced
in anaerobically incubated Escherichia coli cells and the crude enzyme was extracted under anaerobic conditions. The optimal temperature for activity of recombinant
IOR was 70° C and the half-life of this enzyme in the presence of air was 15 min at 25° C.
Received: 25 September 1996 / Accepted: 20 December 1996 相似文献
18.
α, βI, βII, δ, and ε Protein Kinase C Isoforms and Compound Activity in the Sciatic Nerve of Normal and Diabetic Rats 总被引:1,自引:1,他引:0
Isabelle Borghini Aldonza Ania-Lahuerta Romano Regazzi Giovanna Ferrari Asllan Gjinovci Claes B. Wollheim William-F. Pralong 《Journal of neurochemistry》1994,62(2):686-696
Abstract: Defective protein kinase C (PKC) has been implicated in impaired Na+ ,K+ -ATPase activity in the sciatic nerve of streptozotocin-induced diabetic rats. In the present study, α, βI, βII, γ, δ, and ε isoform-specific antibodies were used in parallel to the measurement of compound PKC activity for the characterization of PKC distribution and isoform expression in sciatic nerves of normal and diabetic rats. To distinguish isoform expression between the axonal and glial compartments, PKC isoforms were evaluated in nerves subjected to Wallerian degeneration and in a pure primary Schwann cell culture. α, βI, βII, δ, and ε but no γ isoforms were detected in sciatic nerve. Similar immunoreactivity was observed in degenerated nerves 3–4 days after transection except for diminished βI and ε species; in Schwann cell cultures, only α, βII, δ, and ε were detected. In normal nerves, two-thirds of PKC compound activity was found in the cytosol and 50% of total enzyme activity translocated to the Na+ ,K+ -ATPase-enriched membrane fraction with phorbol myristate acetate. Similar redistribution patterns were observed for the immunoreactivity of all isoforms with the exception of δ, which did not translocate to the membrane with phorbol myristate acetate. No abnormality in compound PKC activity, in the immunoreactive intensity, or in the distribution of PKC isoforms could be detected in rat sciatic nerve after 6–12 weeks of diabetes. Thus, defective activation rather than decreased intrinsic PKC activity may occur in diabetic neuropathy. 相似文献
19.
Janusz M Bujnicki 《BMC evolutionary biology》2002,2(1):3-11