Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “oligomerization”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 865 records · Page 48Linked to original sources
PubMed2002

Oligomerization of G-protein-coupled receptors shown by selective co-immunoprecipitation.

Recent studies have shown that G-protein-coupled receptors (GPCRs) can assemble as high molecular weight homo- and hetero-oligomeric complexes. This can result in altered receptor-ligand binding, signaling, or intracellular trafficking. We have co-transfected HEK-293 cells with differentially epitope-tagged GPCRs from different subfamilies and determined whether oligomeric complexes were formed by co-immunoprecipitation and immunoblot analysis. This gave the surprising result that the 5HT(1A) receptor was capable of forming hetero-oligomers with all GPCRs tested including the 5HT(1B), 5HT(1D), EDG(1), EDG(3), GPR(26), and GABA(B2) receptors. The testing of other GPCR combinations showed similar results with hetero-oligomer formation occurring for the 5HT(1D) with the 5HT(1B) and EDG(1) receptor. Control studies showed that these complexes were present in co-transfected cells before the time of lysis and that the hetero-oligomers were comprised of GPCRs at discrete stoichiometries. These findings suggest that GPCRs have a natural tendency to form oligomers when co-transfected into cells. Future studies should therefore investigate the presence and physiological role of GPCR hetero-oligomers in cells in which they are endogenously expressed.

Cell Line↗
PubMed2002

The minor subunit splice variants, H2b and H2c, of the human asialoglycoprotein receptor are present with the major subunit H1 in different hetero-oligomeric receptor complexes.

The hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic receptor that mediates the internalization of desialylated glycoproteins and their delivery to lysosomes. The human ASGP-R is a hetero-oligomeric complex composed of H1 and H2 subunits. There are three naturally occurring H2 splice variants, designated H2a, H2b, and H2c, although the expression of the H2c protein had not been reported. Following deglycosylation of purified ASGP-R, we detected the H2b and H2c proteins in HepG2 and HuH-7 hepatoma cells, using an antibody directed against a COOH-terminal peptide common to all H2 isoforms (anti-H2-COOH) and another antibody against a 19-amino acid cytoplasmic insert found only in H2b (anti-H2-Cyto19). H1 and both H2b and H2c were co-purified by affinity chromatography, using asialo-orosomucoid (ASOR)-, anti-H1-, or anti-H2-COOH-Sepharose, whereas only H1 and H2b were immunoprecipitated with anti-H2-Cyto19. These results indicate that H2b and H2c are not present in the same ASGP-R complexes with H1. Similar to the H2b isoform, H2c was also palmitoylated, indicating that the 19-residue cytoplasmic insert does not regulate palmitoylation. Stably transfected SK-Hep-1 cell lines expressing ASGP-R complexes containing H1 and either H2b or H2c had similar binding affinities for ASOR and endocytosed and degraded ASOR at similar rates. The pH dissociation profiles of ASOR.ASGP-R complexes were also identical for complexes containing either H2b or H2c. We conclude that the H2b and H2c isoforms are both functional but are not present with H1 in the same hetero-oligomeric ASGP-R complexes. This structural difference between two functional subpopulations of ASGP-Rs may provide a molecular basis for the existence of two different pathways, designated State 1 and State 2, by which several types of recycling receptors mediate endocytosis.

Alternative Splicing↗
PubMed2003

Structural basis of membrane-induced cardiotoxin A3 oligomerization.

Cobra cardiotoxins (CTXs) have previously been shown to induce membrane fusion of vesicles formed by phospholipids such as cardiolipin or sphingomyelin. CTX can also form a pore in membrane bilayers containing a anionic lipid such as phosphatidylserine or phosphatidylglycerol. Herein, we show that the interaction of CTX with negatively charged lipids causes CTX dimerization, an important intermediate for the eventual oligomerization of CTX during the CTX-induced fusion and pore formation process. The structural basis of the lipid-induced oligomerization of CTX A3, a major CTX from Naja atra, is then illustrated by the crystal structure of CTX A3 in complex with SDS; SDS likely mimics anionic lipids of the membrane under micelle conditions at 1.9-A resolution. The crystal packing reveals distinct SDS-free and SDS-rich regions; in the latter two types of interconnecting CTX A3 dimers, D1 and D2, and several SDS molecules can be identified to stabilize D1 and D2 by simultaneously interacting with residues at each dimer interface. When the three CTXSDS complexes in the asymmetric unit are overlaid, the orientation of CTX A3 monomers relative to the SDS molecules in the crystal is strikingly similar to that of the toxin with respect to model membranes as determined by NMR and Fourier transform infrared methods. These results not only illustrate how lipid-induced CTX dimer formation may be transformed into oligomers either as inverted micelles of fusion intermediates or as membrane pore of anionic lipid bilayers but also underscore a potential role for SDS in x-ray diffraction study of protein-membrane interactions in the future.

Amino Acid Sequence↗
PubMed2002

Structure of homo- and hetero-oligomeric meprin metalloproteases. Dimers, tetramers, and high molecular mass multimers.

Meprin A and B, metalloproteases consisting of evolutionarily related alpha and/or beta subunits, are membrane-bound and secreted enzymes expressed by kidney and intestinal epithelial cells, leukocytes, and cancer cells. Previous work established that the multidomain meprin subunits (each approximately 80 kDa) form disulfide-bridged homo- and heterodimers, and differ in substrate and peptide bond specificities. The work herein clearly demonstrates that meprin dimers differ markedly in their ability to oligomerize. Electrophoresis, light scattering, size exclusion chromatography, and electron microscopy were used to characterize quaternary structures of recombinant rat meprins. Meprin B, consisting of meprin beta subunits only, was dimeric under a wide range of conditions. By contrast, meprin alpha homodimers formed heterogeneous multimers (ring-, circle-, spiral-, and tube-like structures) containing up to 100 subunits, with molecular masses at protein peaks ranging from approximately 1.0 to 6.0 MDa. The size of the meprin alpha homo-oligomers was dependent on protein concentration, ionic strength, and activation state. Meprin alphabeta heterodimers tended to form tetramers but not higher oligomers. Thus, the presence of meprin beta, which has a transmembrane domain in vivo, restricts the oligomerization potential of meprin molecules and localizes meprins to the plasma membrane. By contrast, the propensity of secreted meprin alpha homodimers to self-associate concentrates proteolytic potential into high molecular mass multimers and thus allows for autocompartmentalization. The work indicates that different mechanisms exist to localize and concentrate the proteolytic activity of membrane-bound and secreted meprin metalloproteinases.

Animals↗
PubMed2002

Ligand binding promotes the entropy-driven oligomerization of integrin alpha IIb beta 3.

Integrin alpha(IIb)beta(3) clusters on the platelet surface after binding adhesive proteins in a process that regulates signal transduction. However, the intermolecular forces driving integrin self-association are poorly understood. This work provides new insights into integrin clustering mechanisms by demonstrating how temperature and ligand binding interact to affect the oligomeric state of alpha(IIb)beta(3). The ligand-free receptor, solubilized in thermostable octyl glucoside micelles, exhibited a cooperative transition at approximately 43 degrees C, monitored by changes in intrinsic fluorescence and circular dichroism. Both signals changed in a direction opposite to that for global unfolding, and both were diminished upon binding the fibrinogen gamma-chain ligand-mimetic peptide cHArGD. Free and bound receptors also exhibited differential sensitivity to temperature-enhanced oligomerization, as measured by dynamic light scattering, sedimentation velocity, and sedimentation equilibrium. Van't Hoff analyses of dimerization constants for alpha(IIb)beta(3) complexed with cHArGD, cRGD, or eptifibatide yielded large, favorable entropy changes partly offset by unfavorable enthalpy changes. Transmission electron microscopy showed that ligand binding and 37 degrees C incubation enhanced assembly of integrin dimers and larger oligomers linked by tail-to-tail contacts. Interpretation of these images was aided by threading models for alpha(IIb)beta(3) protomers and dimers based on the ectodomain structure of alpha(v)beta(3). We propose that entropy-favorable nonpolar interactions drive ligand-induced integrin clustering and outside-in signaling.

Adult↗
PubMed2003

Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight, nonpeptidyl ligand.

Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.

Animals↗
PubMed2002

Homo- and hetero-oligomerization of thyrotropin-releasing hormone (TRH) receptor subtypes. Differential regulation of beta-arrestins 1 and 2.

G-protein-coupled receptors (GPCRs) are regulated by a complex network of mechanisms such as oligomerization and internalization. Using the GPCR subtypes for thyrotropin-releasing hormone (TRHR1 and TRHR2), the aim of this study was to determine if subtype-specific differences exist in the trafficking process. If so, we wished to determine the impact of homo- and hetero-oligomerization on TRHR subtype trafficking as a potential mechanism for the differential cellular responses induced by TRH. Expression of either beta-arrestin 1 or 2 promoted TRHR1 internalization. In contrast, only beta-arrestin 2 could enhance TRHR2 internalization. The preference for beta-arrestin 2 by TRHR2 was supported by the impairment of TRHR2 trafficking in mouse embryonic fibroblasts (MEFs) from either a beta-arrestin 2 knockout or a beta-arrestin 1/2 knockout, while TRHR1 trafficking was only abolished in MEFs lacking both beta-arrestins. The differential beta-arrestin-dependence of TRHR2 was directly measured in live cells using bioluminescence resonance energy transfer (BRET). Both BRET and confocal microscopy were also used to demonstrate that TRHR subtypes form hetero-oligomers. In addition, these hetero-oligomers have altered internalization kinetics compared with the homo-oligomer. The formation of TRHR1/2 heteromeric complexes increased the interaction between TRHR2 and beta-arrestin 1. This may be due to conformational differences between TRHR1/2 hetero-oligomers versus TRHR2 homo-oligomers as a mutant TRHR1 (TRHR1 C335Stop) that does not interact with beta-arrestins, could also enhance TRHR2/beta-arrestin 1 interaction. This study demonstrates that TRHR subtypes are differentially regulated by the beta-arrestins and also provides the first evidence that the interactions of TRHRs with beta-arrestin may be altered by hetero-oligomer formation.

Animals↗
PubMed2003

Regulation of dopamine D1 receptor trafficking and desensitization by oligomerization with glutamate N-methyl-D-aspartate receptors.

Activation of dopamine D1 receptors is critical for the generation of glutamate-induced long-term potentiation at corticostriatal synapses. In this study, we report that, in striatal neurons, D1 receptors are co-localized with N-methyl-d-aspartate (NMDA) receptors in the postsynaptic density and that they co-immunoprecipitate with NMDA receptor subunits from postsynaptic density preparations. Using modified bioluminescence resonance energy transfer, we demonstrate that D1 and NMDA receptor clustering reflects the existence of direct interactions. The tagged D1 receptor and NR1 subunit cotransfected in COS-7 cells generated a significant bioluminescence resonance energy transfer signal that was insensitive to agonist stimulation and that did not change in the presence of the NR2B subunit, suggesting that the D1 receptor constitutively and selectively interacts with the NR1 subunit of the NMDA channel. Oligomerization with the NR1 subunit substantially modified D1 receptor trafficking. In individually transfected HEK293 cells, NR1 was localized in the endoplasmic reticulum, whereas the D1 receptor was targeted to the plasma membrane. In cotransfected cells, both the D1 receptor and NR1 subunit were retained in cytoplasmic compartments. In the presence of the NR2B subunit, the NR1-D1 receptor complex was translocated to the plasma membrane. These data suggest that D1 and NMDA receptors are assembled within intracellular compartments as constitutive heteromeric complexes that are delivered to functional sites. Coexpression with NR1 and NR2B subunits also abolished agonist-induced D1 receptor cytoplasmic sequestration, indicating that oligomerization with the NMDA receptor could represent a novel regulatory mechanism modulating D1 receptor desensitization and cellular trafficking.

Animals↗
PubMed2003

Oligomerization and pore formation of a sphingomyelin-specific toxin, lysenin.

Lysenin is a novel protein derived from coelomic fluid of the earthworm Eisenia foetida, which specifically recognizes sphingomyelin and induces cytolysis. The mechanism underlying lysenin-induced cell lysis has not been clarified. In this report we studied the interaction of lysenin with red blood cells as well as artificial liposomes. Our results showed that lysenin bound membranes and assembled to SDS-resistant oligomers in a sphingomyelin-dependent manner, leading to the formation of pores with a hydrodynamic diameter of approximately 3 nm. Antibody scanning analysis suggested that the C-terminal region of lysenin was exposed, whereas the N-terminal was hidden in the isolated oligomer complex. Differential scanning calorimetry revealed that lysenin interacted with both hydrophilic head group and hydrophobic hydrocarbon tails of sphingomyelin. Oligomerization but not binding was affected by the amide-linked fatty acid composition of sphingomyelin, suggesting the role of membrane fluidity in the oligomerization step.

Amino Acid Sequence↗
PubMed2003

Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion, Bax translocation, and Bax/Bak oligomerization suppressed.

Bcl-2 family proteins are important regulators of apoptosis. They can be pro-apoptotic (e.g. Bid, Bax, and Bak) or anti-apoptotic (e.g. Bcl-2 and Bcl-x(L)). The current study examined Bid-induced apoptosis and its inhibition by Bcl-2. Transfection of Bid led to apoptosis in HeLa cells. In these cells, Bid was processed into active forms of truncated Bid or tBid. Following processing, tBid translocated to the membrane-bound organellar fraction. Bcl-2 co-transfection inhibited Bid-induced apoptosis but did not prevent Bid processing or tBid translocation. On the other hand, Bcl-2 blocked the release of mitochondrial cytochrome c in Bid-transfected cells, suggesting actions at the mitochondrial level. Alkaline treatment stripped off tBid from the membrane-bound organellar fraction of Bid plus Bcl-2-co-transfected cells, but not from cells transfected with only Bid, suggesting inhibition of tBid insertion into mitochondrial membranes by Bcl-2. Bcl-2 also prevented Bid-induced Bax translocation from cytosol to the membrane-bound organellar fraction. Finally, Bcl-2 diminished Bid-induced oligomerization of Bax and Bak within the membrane-bound organellar fraction, shown by cross-linking experiments. In conclusion, Bcl-2 inhibited Bid-induced apoptosis at the mitochondrial level by blocking cytochrome c release, without suppressing Bid processing or activation. Critical steps blocked by Bcl-2 included tBid insertion, Bax translocation, and Bax/Bak oligomerization in the mitochondrial membranes.

Apoptosis↗
PubMed2003

Tyrosinase maturation and oligomerization in the endoplasmic reticulum require a melanocyte-specific factor.

Tyrosinase is a glycoprotein responsible for the synthesis of melanin in melanocytes. A large number of mutations have been identified in tyrosinase, with many leading to its misfolding, endoplasmic reticulum (ER) retention, and degradation. Here we describe the folding and maturation of human tyrosinase (TYR) using an in vitro translation system coupled with ER-derived microsomes or with semipermeabilized cells, as an intact ER source. TYR remained misfolded as determined by its sensitivity to trypsin digestion and its persistent interaction with the ER resident lectin chaperones calnexin and calreticulin when produced in ER-derived microsomes or nonmelanocytic semipermeabilized cells. However, when TYR was translocated into semipermeabilized melanocytes, chaperone interactions were transient, maturation progressed to a trypsin-resistant state, and a TYR homodimer was formed. The use of semipermeabilized mouse melanocytes defective for tyrosinase or other melanocyte-specific proteins as the ER source indicated that proper TYR maturation and oligomerization were greatly aided by the presence of wild type tyrosinase and tyrosinase-related protein 1. These findings suggested that oligomerization is a step in proper TYR maturation within the ER that requires melanocyte-specific factors.

Animals↗
PubMed2003

Assembly of stable human type I and III collagen molecules from hydroxylated recombinant chains in the yeast Pichia pastoris. Effect of an engineered C-terminal oligomerization domain foldon.

The C-propeptides of the pro alpha chains of type I and type III procollagens are believed to be essential for correct chain recognition and chain assembly in these molecules. We studied here whether the 30-kDa C-propeptides of the human pC alpha 1(I), pC alpha 2(I), and pC alpha 1(III) chains, i.e. pro alpha chains lacking their N-propeptides, can be replaced by foldon, a 29-amino acid sequence normally located at the C terminus of the polypeptide chains in the bacteriophage T4 fibritin. The alpha foldon chains were expressed in Pichia pastoris cells that also expressed the two types of subunit of human prolyl 4-hydroxylase; the foldon domain was subsequently removed by pepsin treatment, which also digests non-triple helical collagen chains, whereas triple helical collagen molecules are resistant to it. The foldon domain was found to be very effective in chain assembly, as expression of the alpha 1(I)foldon or alpha 1(III)foldon chains gave about 2.5-3-fold the amount of pepsin-resistant type I or type III collagen homotrimers relative to those obtained using the authentic C-propeptides. In contrast, expression of chains with no oligomerization domain led to very low levels of pepsin-resistant molecules. Expression of alpha 2(I)foldon chains gave no pepsin-resistant molecules at all, indicating that in addition to control at the level of the C-propeptide other restrictions at the level of the collagen domain exist that prevent the formation of stable [alpha 2(I)]3 molecules. Co-expression of alpha 1(I)foldon and alpha 2(I)foldon chains led to an efficient assembly of heterotrimeric molecules, their amounts being about 2-fold those obtained with the authentic C-propeptides and the alpha 1(I) to alpha 2(I) ratio being 1.91 +/- 0.31 (S.D.). As the foldon sequence contains no information for chain recognition, our data indicate that chain assembly is influenced not only by the C-terminal oligomerization domain but also by determinants present in the alpha chain domains.

Base Sequence↗
PubMed2003

C5a receptor oligomerization. I. Disulfide trapping reveals oligomers and potential contact surfaces in a G protein-coupled receptor.

G protein-coupled receptors (GPCRs), stimulated by hormones and sensory stimuli, act as molecular switches to relay activation to heterotrimeric G proteins. Recent studies suggest that GPCRs form dimeric or oligomeric structures, a phenomenon that has long been established for growth factor receptors. The elucidation of the domains of GPCRs that mediate receptor association is of critical importance for understanding the function of GPCR oligomers. Using a disulfide-trapping strategy to probe the intermolecular contact surfaces, we demonstrate cross-linking of C5a receptors in membranes prepared from both human neutrophils and stably transfected mammalian cells that is mediated by a cysteine in the second intracellular loop. To explore other surfaces that might be involved in the oligomerization of C5a receptors, we constructed receptors with individual cysteines in other intracellular regions. C5a receptors with a cysteine in the first intracellular loop or the carboxyl terminus displayed the fastest kinetics of dimer formation, whereas an intracellular loop 3 cysteine displayed minimal cross-linking. Since the rate of disulfide trapping reflects the proximity of sulfhydryl groups, assuming similar accessibility and flexibility, these results imply a symmetric dimer interface that may involve either transmembrane helices 1 and 2 or helix 4. However, neither model can account for the ability of the native cysteine in the second intracellular loop to mediate efficient crosslinking. Based on these observations, we propose that C5a receptors form higher order oligomers (i.e. tetramers) or clusters in the membrane.

Amino Acid Sequence↗
PubMed2003

Oligomerization of soluble Fas antigen induces its cytotoxicity.

Soluble Fas antigen can protect cells against Fas-mediated apoptosis. High level soluble Fas antigen characteristic for blood of patients with autoimmune disease or cancer is believed to prevent the elimination of autoimmune lymphocytes or tumor cells. Here we first report that human recombinant FasDeltaTM, i.e. soluble Fas generated by alternative splicing of the intact exon 6, is capable of inducing death of transformed cells by "reverse" apoptotic signaling via transmembrane Fas ligand. FasDeltaTM, as well as transmembrane Fas antigen, can be either monomeric or oligomeric, and both its forms are efficient in blocking Fas-mediated apoptosis, although the cytotoxic activity is exhibited solely by the latter. An in vivo analysis of soluble Fas antigen showed that unlike in healthy controls, nearly the total FasDeltaTM present in sera of rheumatoid arthritis patients was oligomeric. This resulted in suppression of cell proliferation in the experimental sera and in its promotion in controls. Thus, oligomerization/depolymerization of soluble Fas antigen can regulate its activity and contribute to the pathogenesis of autoimmune diseases and cancer.

Alternative Splicing↗
PubMed2003

Homo- and hetero-oligomerization of ammonium transporter-1 NH4 uniporters.

In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh). A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts. Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization. In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system. LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a approximately 35-kDa polypeptide by denaturation. To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH4+ uniporter. Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes. Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake.

Amino Acid Sequence↗
PubMed2003

Role of the conserved SRLFDQFFG region of alpha-crystallin, a small heat shock protein. Effect on oligomeric size, subunit exchange, and chaperone-like activity.

Small heat shock proteins (sHsps) are necessary for several cellular functions and in stress tolerance. Most sHsps are oligomers; intersubunit interactions leading to changes in oligomeric structure and exposure of specific regions may modulate their functioning. Many sHsps, including alpha A- and alpha B-crystallin, contain a well conserved SRLFDQFFG sequence motif in the N-terminal region. Sequence-based prediction shows that it exhibits helical propensity with amphipathic character, suggesting that it plays a critical role in the structure and function of alpha-crystallins. In order to investigate the role of this motif in the structure and function of sHsps, we have made constructs deleting this sequence from alpha A- and alpha B-crystallin, overexpressed, purified, and studied these engineered proteins. Circular dichroism spectroscopic studies show changes in tertiary and secondary structure on deletion of the sequence. Glycerol density gradient centrifugation and dynamic light scattering studies show that the multimeric size of the mutant proteins is significantly reduced, indicating a role for this motif in higher order organization of the subunits. Both deletion mutants exhibit similar oligomeric size and increased chaperone-like activity. Urea-induced denaturation study shows that the SRLFDQFFG sequence contributes significantly to the structural stability. Fluorescence resonance energy transfer studies show that the rate of exchange of the subunits in the alpha Adel-crystallin oligomer is higher compared with that in the alpha A-crystallin oligomer, suggesting that this region contributes to the oligomer dynamics in addition to the higher order assembly and structural stability. Thus, our study shows that the SRLFDQFFG sequence is one of the critical motifs in structure-function regulation of alpha A- and alpha B-crystallin.

Amino Acid Sequence↗
PubMed2004

Amino acid substitutions in the C-terminal AAA+ module of Hsp104 prevent substrate recognition by disrupting oligomerization and cause high temperature inactivation.

Hsp104 is an important determinant of thermotolerance in yeast and is an unusual molecular chaperone that specializes in the remodeling of aggregated proteins. The structural requirements for Hsp104-substrate interactions remain unclear. Upon mild heat shock Hsp104 formed cytosolic foci in live cells that indicated co-localization of the chaperone with aggregates of thermally denatured proteins. We generated random amino acid substitutions in the C-terminal 199 amino acid residues of a GFP-Hsp104 fusion protein, and we used a visual screen to identify mutants that remained diffusely distributed immediately after heat shock. Multiple amino acid substitutions were required for loss of heat-inducible redistribution, and this correlated with complete loss of nucleotide-dependent oligomerization. Based on the multiply substituted proteins, several single amino acid substitutions were generated by site-directed mutagenesis. The singly substituted proteins retained the ability to oligomerize and detect substrates. Intriguingly, some derivatives of Hsp104 functioned well in prion propagation and multiple stress tolerance but failed to protect yeast from extreme thermal stress. We demonstrate that these proteins co-aggregate in the presence of other thermolabile proteins during heat treatment both in vitro and in vivo suggesting a novel mechanism for uncoupling the function of Hsp104 in acute severe heat shock from its functions at moderate temperatures.

Adenosine Triphosphatases↗
PubMed2004

Oligomerization of the Na,K-ATPase in cell membranes.

The higher order oligomeric state of the Na,K-ATPase alphabeta heterodimer in cell membranes is the subject of controversy. We have utilized the baculovirus-infected insect cell system to express Na,K-ATPase with alpha-subunits bearing either His(6) or FLAG epitopes at the carboxyl terminus. Each of these constructs produced functional Na,K-ATPase alphabeta heterodimers that were delivered to the plasma membrane (PM). Cells were simultaneously co-infected with viruses encoding alpha-His/beta and alpha-FLAG/beta Na,K-ATPases. Co-immunoprecipitation of the His-tagged alpha-subunit in the endoplasmic reticulum (ER) and PM fractions of co-infected cells by the anti-FLAG antibody demonstrates that protein-protein associations exist between these heterodimers. This suggests the Na,K-ATPase is present in cell membranes in an oligomeric state of at least (alphabeta)(2) composition. Deletion of 256 amino acid residues from the central cytoplasmic loop of the alpha-subunit results in the deletion alpha-4,5-loop-less (alpha-4,5LL), which associates with beta but is confined to the ER. Co-immunoprecipitation demonstrates that when this inactive alpha-4,5LL/beta heterodimer is co-expressed with wild-type alphabeta, oligomers of wild-type alphabeta and alpha-4,5LL/beta form in the ER, but the alpha-4,5LL mutant remains retained in the ER, and the wild-type protein is still delivered to the PM. We conclude that the Na,K-ATPase is present as oligomers of the monomeric alphabeta heterodimer in native cell membranes.

Animals↗