共查询到20条相似文献,搜索用时 31 毫秒
1.
Andreas Anderluh Enrico Klotzsch Alexander W. A. F. Reismann Mario Brameshuber Oliver Kudlacek Amy Hauck Newman Harald H. Sitte Gerhard J. Schütz 《The Journal of biological chemistry》2014,289(7):4387-4394
The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. Although there is solid evidence that SERT forms oligomeric complexes, the exact stoichiometry of the complexes and the fractions of different coexisting oligomeric states still remain enigmatic. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of the SERT via brightness analysis of single diffraction-limited fluorescent spots. Heterologously expressed SERT was labeled either with the fluorescent inhibitor JHC 1-64 or via fusion to monomeric GFP. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data are in agreement with a linear aggregation model. Furthermore, oligomerization was found to be independent of SERT surface density, and oligomers remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane. 相似文献
2.
Kirsch F Frielingsdorf S Pohlmann A Ziomkowska J Herrmann A Eitinger T 《Journal of bacteriology》2012,194(17):4505-4512
Energy-coupling factor transporters are a large group of importers for trace nutrients in prokaryotes. The in vivo oligomeric state of their substrate-specific transmembrane proteins (S units) is a matter of debate. Here we focus on the S unit BioY of Rhodobacter capsulatus, which functions as a low-affinity biotin transporter in its solitary state. To analyze whether oligomerization is a requirement for function, a tail-to-head-linked BioY dimer was constructed. Monomeric and dimeric BioY conferred comparable biotin uptake activities on recombinant Escherichia coli. Fluorophore-tagged variants of the dimer were shown by fluorescence anisotropy analysis to oligomerize in vivo. Quantitative mass spectrometry identified biotin in the purified proteins at a stoichiometry of 1:2 for the BioY monomer and 1:4 (referring to single BioY domains) for the dimer. Replacement of the conserved Asp164 (by Asn) and Lys167 (by Arg or Gln) in the monomer and in both halves of the dimer inactivated the proteins. The presence of those mutations in one half of the dimers only slightly affected biotin binding but reduced transport activity to 25% (Asp164Asn and Lys167Arg) or 75% (Lys167Gln). Our data (i) suggest that intermolecular interactions of domains from different dimers provide functionality, (ii) confirm an oligomeric architecture of BioY in living cells, and (iii) demonstrate an essential role of the last transmembrane helix in biotin recognition. 相似文献
3.
Brast S Grabner A Sucic S Sitte HH Hermann E Pavenst?dt H Schlatter E Ciarimboli G 《FASEB journal》2012,26(3):976-986
Human organic cation transporter 2 (hOCT2) is involved in transport of many endogenous and exogenous organic cations, mainly in kidney and brain cells. Because the quaternary structure of transmembrane proteins plays an essential role for their cellular trafficking and function, we investigated whether hOCT2 forms oligomeric complexes, and if so, which part of the transporter is involved in the oligomerization. A yeast 2-hybrid mating-based split-ubiquitin system (mbSUS), fluorescence resonance energy transfer, Western blot analysis, cross-linking experiments, immunofluorescence, and uptake measurements of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium were applied to human embryonic kidney 293 (HEK293) cells transfected with hOCT2 and partly also to freshly isolated human proximal tubules. The role of cysteines for oligomerization and trafficking of the transporter to the plasma membranes was investigated in cysteine mutants of hOCT2. hOCT2 formed oligomers both in the HEK293 expression system and in native human kidneys. The cysteines of the large extracellular loop are important to enable correct folding, oligomeric assembly, and plasma membrane insertion of hOCT2. Mutation of the first and the last cysteines of the loop at positions 51 and 143 abolished oligomer formation. Thus, the cysteines of the extracellular loop are important for correct trafficking of the transporter to the plasma membrane and for its oligomerization. 相似文献
4.
Andrew H A Clayton 《Journal of biosciences》2018,43(3):463-469
Membrane protein structures are highly under-represented relative to water-soluble protein structures in the protein databank. This is especially the case because membrane proteins represent more than 30% of proteins encoded in the human genome yet contribute to less than 10% of currently known structures (Torres et al. in Trends Biol Sci 28:137–144, 2003). Obtaining high-resolution structures of membrane proteins by traditional methods such as NMR and x-ray crystallography is challenging, because membrane proteins are difficult to solubilise, purify and crystallize. Consequently, development of methods to examine protein structure in situ is highly desirable. Fluorescence is highly sensitive to protein structure and dynamics (Lakowicz in Principles of fluorescence spectroscopy, Springer, New York, 2007). This is mainly because of the time a fluorescence probe molecule spends in the excited state. Judicious choice and placement of fluorescent molecule(s) within a protein(s) enables the experimentalist to obtain information at a specific site(s) in the protein (complex) of interest. Moreover, the inherent multi-dimensional nature of fluorescence signals across wavelength, orientation, space and time enables the design of experiments that give direct information on protein structure and dynamics in a biological setting. The purpose of this review is to introduce the reader to approaches to determine oligomeric state or quaternary structure at the cell membrane surface with the ultimate goal of linking the oligomeric state to the biological function. In the first section, we present a brief overview of available methods for determining oligomeric state and compare their advantages and disadvantages. In the second section, we highlight some of the methods developed in our laboratory to address contemporary questions in membrane protein oligomerization. In the third section, we outline our approach to determine the link between protein oligomerization and biological activity. 相似文献
5.
Fluorescently labeled proteins that are found both in the cytoplasm and at the plasma membrane, such as peripheral membrane proteins, create stratified fluorescent layers that present a challenging environment for brightness studies with fluorescence fluctuation spectroscopy. The geometry of each layer along with fluorescence and brightness contributions from adjacent layers generates a convoluted raw brightness that conceals the underlying brightness of each individual layer. Because the brightness at a layer establishes the oligomeric state of the fluorescently labeled protein at said layer, we developed a method that connects the experimental raw brightness with the physical brightness at each layered compartment. The technique determines the oligomerization in each compartment from an axial intensity scan through the sample, followed by a fluorescence fluctuation spectroscopy measurement at each layer. We experimentally verify the technique with H-Ras-EGFP as a model system and determine its oligomeric state at both the plasma membrane and in the cytoplasm. Furthermore, we study the oligomerization of the Gag matrix domain of Human T-lymphotropic virus Type 1. The matrix domain targets the Gag polyprotein to the plasma membrane where, subsequently, viral assembly occurs. We determine the oligomerization of matrix in the cytoplasm and observe the onset of protein-protein interactions at the membrane. These observations shed light on the early assembly steps of the retrovirus. 相似文献
6.
Elizabeth?M. Smith Patrick?J. Macdonald Yan Chen Joachim?D. Mueller 《Biophysical journal》2014,107(1):66-75
Fluorescently labeled proteins that are found both in the cytoplasm and at the plasma membrane, such as peripheral membrane proteins, create stratified fluorescent layers that present a challenging environment for brightness studies with fluorescence fluctuation spectroscopy. The geometry of each layer along with fluorescence and brightness contributions from adjacent layers generates a convoluted raw brightness that conceals the underlying brightness of each individual layer. Because the brightness at a layer establishes the oligomeric state of the fluorescently labeled protein at said layer, we developed a method that connects the experimental raw brightness with the physical brightness at each layered compartment. The technique determines the oligomerization in each compartment from an axial intensity scan through the sample, followed by a fluorescence fluctuation spectroscopy measurement at each layer. We experimentally verify the technique with H-Ras-EGFP as a model system and determine its oligomeric state at both the plasma membrane and in the cytoplasm. Furthermore, we study the oligomerization of the Gag matrix domain of Human T-lymphotropic virus Type 1. The matrix domain targets the Gag polyprotein to the plasma membrane where, subsequently, viral assembly occurs. We determine the oligomerization of matrix in the cytoplasm and observe the onset of protein-protein interactions at the membrane. These observations shed light on the early assembly steps of the retrovirus. 相似文献
7.
Veldhuis G Hink M Krasnikov V van den Bogaart G Hoeboer J Visser AJ Broos J Poolman B 《Protein science : a publication of the Protein Society》2006,15(8):1977-1986
Numerous membrane proteins function as oligomers both at the structural and functional levels. The mannitol transporter from Escherichia coli, EnzymeII(mtl), is a member of the phosphoenolpyruvate-dependent phosphotransferase system. During the transport cycle, mannitol is phosphorylated and released into the cytoplasm as mannitol-1-phosphate. Several studies have shown that EII(mtl) functions as an oligomeric species. However, the oligomerization number and stability of the oligomeric complex during different steps of the catalytic cycle, e.g., substrate binding and/or phosphorylation of the carrier, is still under discussion. In this paper, we have addressed the oligomeric state and stability of EII(mtl) using fluorescence correlation spectroscopy. A functional double-cysteine mutant was site-specifically labeled with either Alexa Fluor 488 or Alexa Fluor 633. The subunit exchange of these two batches of proteins was followed in time during different steps of the catalytic cycle. The most important conclusions are that (1) in a detergent-solubilized state, EII(mtl) is functional as a very stable dimer; (2) the stability of the complex can be manipulated by changing the intermicellar attractive forces between PEG-based detergent micelles; (3) substrate binding destabilizes the complex whereas phosphorylation increases the stability; and (4) substrate binding to the phosphorylated species partly antagonizes the stabilizing effect. 相似文献
8.
Structural and functional analysis of the pre-pore and membrane-inserted pore of Cry1Ab toxin 总被引:1,自引:0,他引:1
Pardo-López L Gómez I Muñoz-Garay C Jiménez-Juarez N Soberón M Bravo A 《Journal of invertebrate pathology》2006,92(3):172-177
Bacillus thuringiensis produces insecticidal Cry proteins that are active against different insect species. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Here, we review the recent advances in the understanding of the structural changes presented by Cry1Ab toxin upon membrane insertion. Based on analysis of the Trp fluorescence of pure monomeric and oligomeric Cry1Ab structures in solution and in membrane-bound state we reported that oligomerization caused 27% reduction of Trp exposed to the solvent. After membrane insertion there is another conformational change that allows an additional rearrangement of the Trp residues resulting in a total protection of these residues from exposure to the solvent. The oligomeric structure is membrane insertion competent since more than 96% of the Cry1Ab oligomer inserts into the membrane as a function of lipid:protein ratio, in contrast to the monomer of which only 5-10%, inserts into the membrane. Finally, analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion and this flexible structure is obtained by toxin oligomerization and by alkaline pH. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab and this domain is inserted into the membrane in the membrane-inserted state. 相似文献
9.
We have prepared and characterized a new fluorescent derivative of murine epidermal growth factor (EGF), Alexa Fluor 594-labeled EGF (A-EGF), for fluorescence studies of EGF-EGF receptor interactions. We describe the synthesis of this derivative and its physical and biological characterization. The significant overlap between the excitation and the emission spectra of A-EGF makes this probe well suited to fluorescence resonance energy homo-transfer. Using time-resolved fluorescence to examine the oligomeric state of the EGF receptor, we have observed resonance energy homo-transfer of A-EGF bound to EGF receptors in cells, but not of A-EGF bound to EGF receptors in membrane vesicles. Our results, interpreted in the context of recent crystallographic studies of the ligand-binding domains of EGF receptors, suggest that observed fluorescence resonance energy transfer does not result from transfer within receptor dimers, but rather results from transfer within higher-order oligomers. Furthermore, our results support a structural model for oligomerization of EGF receptors in which dimers are positioned head-to-head with respect to the ligand-binding site, consistent with the head-to-head interactions observed between adjacent receptor dimers by X-ray crystallography. 相似文献
10.
The sugar binding activity of MR60, a mannose-specific shuttling lectin, requires a dimeric state 总被引:1,自引:0,他引:1
MR60 is an intracellular membrane protein which has been shown to act as a
mannoside specific lectin and to be identical to ERGIC-53, a protein
characteristic of the endoplasmic reticulum-Golgi apparatus- intermediate
compartment, acting as a shuttle. According to its primary sequence, this
MR60/ERGIC-53 protein contains a luminal domain including the carbohydrate
recognition domain, a stem, a transmembrane segment and a cytosolic domain.
The endogenous MR60/ERGIC-53 protein is spontaneously oligomeric, (dimers
and hexamers). In this paper, we study the relationship between the
oligomerization state and the sugar binding capacity by using recombinant
proteins. The expression of the recombinant proteins was evidenced by
immunocytochemistry and by immunoprecipitation followed by SDS-PAGE
analysis. The full size recombinant protein binds mannosides and is
oligomeric, up to the hexameric form. Two truncated proteins lacking the
transmembrane and the cytosolic domains were prepared and characterized. A
long one, containing the cysteine 466 close to the C-terminal end of the
recombinant protein but lacking the cysteine 475, close to the C- terminal
end of the native protein, does bind mannosides and forms dimers but no
higher oligomeric forms. A shorter one, lacking both the cysteines 466 and
475, does not bind mannosides and does not form dimers or higher polymers.
The two cysteines in the carbohydrate recognition domain (C190 and C230)
are not involved in the stabilization of oligomers. In conclusion, this
study shows that the luminal moiety of MR60/ERGIC-53 contains a device
allowing both its oligomeric pattern and its sugar binding capability.
相似文献
11.
Phospholipid-induced monomerization and signal-peptide-induced oligomerization of SecA 总被引:1,自引:0,他引:1
Benach J Chou YT Fak JJ Itkin A Nicolae DD Smith PC Wittrock G Floyd DL Golsaz CM Gierasch LM Hunt JF 《The Journal of biological chemistry》2003,278(6):3628-3638
The SecA ATPase drives the processive translocation of the N terminus of secreted proteins through the cytoplasmic membrane in eubacteria via cycles of binding and release from the SecYEG translocon coupled to ATP turnover. SecA forms a physiological dimer with a dissociation constant that has previously been shown to vary with temperature and ionic strength. We now present data showing that the oligomeric state of SecA in solution is altered by ligands that it interacts with during protein translocation. Analytical ultracentrifugation, chemical cross-linking, and fluorescence anisotropy measurements show that the physiological dimer of SecA is monomerized by long-chain phospholipid analogues. Addition of wild-type but not mutant signal sequence peptide to these SecA monomers redimerizes the protein. Physiological dimers of SecA do not change their oligomeric state when they bind signal sequence peptide in the compact, low temperature conformational state but polymerize when they bind the peptide in the domain-dissociated, high-temperature conformational state that interacts with SecYEG. This last result shows that, at least under some conditions, signal peptide interactions drive formation of new intermolecular contacts distinct from those stabilizing the physiological dimer. The observations that signal peptides promote conformationally specific oligomerization of SecA while phospholipids promote subunit dissociation suggest that the oligomeric state of SecA could change dynamically during the protein translocation reaction. Cycles of SecA subunit recruitment and dissociation could potentially be employed to achieve processivity in polypeptide transport. 相似文献
12.
《Biophysical journal》2020,118(8):1861-1875
Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes. 相似文献
13.
Schmid JA Scholze P Kudlacek O Freissmuth M Singer EA Sitte HH 《The Journal of biological chemistry》2001,276(6):3805-3810
Recent biochemical studies indicate that the serotonin transporter can form oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. For this purpose, we generated fusion proteins of hSERT and spectral variants of the green fluorescent protein (cyan and yellow fluorescent proteins, CFP and YFP, respectively). When expressed in human embryonic kidney 293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were efficiently inserted into the plasma membrane and were functionally indistinguishable from wild-type hSERT. Oligomers were visualized by fluorescence resonance energy transfer microscopy in living cells using two complementary methods, i.e. ratio imaging and donor photobleaching. Interestingly, oligomerization was not confined to hSERT; fluorescence resonance energy transfer was also observed between CFP- and YFP-labeled rat gamma-aminobutyric acid transporter. The bulk of serotonin transporters was recovered as high molecular weight complexes upon gel filtration in detergent solution. In contrast, the monomers of CFP-hSERT and YFP-hSERT were essentially undetectable. This indicates that the homo-oligomeric form is the favored state of hSERT in living cells, which is not significantly affected by coincubation with transporter substrates or blockers. Based on our observations, we conclude that constitutive oligomer formation might be a general property of Na(+)/Cl(-)-dependent neurotransmitter transporters. 相似文献
14.
Heuberger EH Veenhoff LM Duurkens RH Friesen RH Poolman B 《Journal of molecular biology》2002,317(4):591-600
Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented.Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric. 相似文献
15.
Akhtar MW Srinivas V Raman B Ramakrishna T Inobe T Maki K Arai M Kuwajima K Rao ChM 《The Journal of biological chemistry》2004,279(53):55760-55769
Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress. 相似文献
16.
G protein-coupled receptors (GPCRs) can form dimeric or oligomeric complexes in vivo. However, the functions and mechanisms of oligomerization remain poorly understood for most GPCRs, including the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. Here we provide evidence indicating that alpha-factor receptor oligomerization involves a GXXXG motif in the first transmembrane domain (TM1), similar to the transmembrane dimerization domain of glycophorin A. Results of fluorescence resonance energy transfer, fluorescence microscopy, endocytosis assays of receptor oligomerization in living cells, and agonist binding assays indicated that amino acid substitutions affecting the glycine residues of the GXXXG motif impaired alpha-factor receptor oligomerization and biogenesis in vivo but did not significantly impair agonist binding affinity. Mutant receptors exhibited signaling defects that were not due to impaired cell surface expression, indicating that oligomerization promotes alpha-factor receptor signal transduction. Structure-function studies suggested that the GXXXG motif in TM1 of the alpha-factor receptor promotes oligomerization by a mechanism similar to that used by the GXXXG dimerization motif of glycophorin A. In many mammalian GPCRs, motifs related to the GXXXG sequence are present in TM1 or other TM domains, suggesting that similar mechanisms are used by many GPCRs to form dimers or oligomeric arrays. 相似文献
17.
2-Cys peroxiredoxins (Prxs) are a large and diverse family of peroxidases which, in addition to their antioxidant functions, regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers (alpha(2)), utilizing a unique intermolecular redox-active disulfide center for the reduction of peroxides, and are known to form two oligomeric states: individual alpha(2) dimers or doughnut-shaped (alpha(2))(5) decamers. Here we characterize both the oligomerization properties and crystal structure of a bacterial 2-Cys Prx, Salmonella typhimurium AhpC. Analytical ultracentrifugation and dynamic light scattering show that AhpC's oligomeric state is redox linked, with oxidization favoring the dimeric state. The 2.5 A resolution crystal structure (R = 18.5%, R(free) = 23.9%) of oxidized, decameric AhpC reveals a metastable oligomerization intermediate, allowing us to identify a loop that adopts distinct conformations associated with decameric and dimeric states, with disulfide bond formation favoring the latter. This molecular switch contains the peroxidatic cysteine and acts to buttress the oligomerization interface in the reduced, decameric enzyme. A structurally detailed catalytic cycle incorporating these ideas and linking activity to oligomeric state is presented. Finally, on the basis of sequence comparisons, we suggest that the enzymatic and signaling activities of all 2-Cys Prxs are regulated by a redox-sensitive dimer to decamer transition. 相似文献
18.
Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteroid insensitive 1 receptor oligomerization
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Receptor kinases play a key role in the cellular perception of signals. To verify models for receptor activation through dimerization, an experimental system is required to determine the precise oligomerization status of proteins within living cells. Here we show that photon counting histogram analysis and dual-color fluorescence cross correlation spectroscopy are able to monitor fluorescently labeled proteins at the single-molecule detection level in living plant cells. In-frame fusion proteins of the brassinosteroid insensitive 1 (BRI1) receptor and the Arabidopsis thaliana somatic embryogenesis receptor-like kinases 1 and 3 (AtSERK1 and 3) to the enhanced cyan or yellow fluorescent protein were transiently expressed in plant cells. Although no oligomeric structures were detected for AtSERK3, 15% (AtSERK1) to 20% (BRI1) of the labeled proteins in the plasma membrane was found to be present as homodimers, whereas no evidence was found for higher oligomeric complexes. 相似文献
19.
Antigen receptors on T cells, B cells, mast cells, and basophils all have cytoplasmic domains containing one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor engagement in an early and obligatory event in the signaling cascade. How clustering of receptor extracellular domains leads to phosphorylation of cytoplasmic domain ITAMs is not known, and little structural or biochemical information is available for the ITAM-containing cytoplasmic domains. Here we investigate the conformation and oligomeric state of several immune receptor cytoplasmic domains, using purified recombinant proteins and a variety of biophysical and biochemical techniques. We show that all of the cytoplasmic domains of ITAM-containing signaling subunits studied are oligomeric in solution, namely, T cell antigen receptor zeta, CD3epsilon, CD3delta, and CD3gamma, B cell antigen receptor Igalpha and Igbeta, and Fc receptor FcepsilonRIgamma. For zeta(cyt), the oligomerization behavior is best described by a two-step monomer-dimer-tetramer fast dynamic equilibrium with dissociation constants in the order of approximately 10 microM (monomer-dimer) and approximately 1 mM (dimer-tetramer). In contrast to the other ITAM-containing proteins, Igalpha(cyt) forms stable dimers and tetramers even below 10 microM. Circular dichroic analysis reveals the lack of stable ordered structure of the cytoplasmic domains studied, and oligomerization does not change the random-coil-like conformation observed. The random-coil nature of zeta(cyt) was also confirmed by heteronuclear NMR. Phosphorylation of zeta(cyt) and FcepsilonRIgamma(cyt) does not significantly alter their oligomerization behavior. The implications of these results for transmembrane signaling and cellular activation by immune receptors are discussed. 相似文献
20.
Constitutive oligomer formation appears to be the rule for the neurotransmitter:sodium symporter (NSS) family of proteins. The propensity to form oligomers is a prerequisite for NSS proteins to pass the rigid mechanisms of quality control in the endoplasmic reticulum. Moreover, recent findings suggest that correct trafficking to the plasma membrane appears to rely on the interaction of NSS homo-oligomers with components of the COPII-vesicle machinery. The transporters present at the plasma membrane are most likely organized in a tetrameric arrangement, as a dimer of dimers. In this review, we will address ongoing efforts to unravel the underlying mechanisms of oligomer formation at the molecular and cellular levels, and we will discuss oligomerization in terms of transporter function. 相似文献