Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein

It has been suggested that newly synthesized proteins are maintained in their unfolded state by cellular ATP-driven factors which may prevent or reverse the formation of misfolded structures or promote the correct assembly of oligomeric proteins or post-translational secretion. Using a photocross-linking approach, we have identified the 20S heat-shock GroEL protein as the major cytosolic component which forms a complex with the unfolded newly synthesized pre-beta-lactamase or chloramphenicol acetyltransferase in Escherichia coli. Dissociation of these complexes is ATP-dependent. The unfolded state of pre-beta-lactamase, maintained by the transient interaction with GroEL, may be essential for the secretion of this protein.     

GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli

Assembly of foreign prokaryotic ribulose bisphosphate carboxylases (Rubiscos) in Escherichia coli requires both heat-shock proteins groEL and groES. GroEL is related to a chloroplast protein implicated in Rubisco assembly. Bacteria and chloroplasts therefore have a conserved mechanism that uses auxiliary proteins to assist in the assembly of Rubisco.     

Assembly reflects evolution of protein complexes

A homomer is formed by self-interacting copies of a protein unit. This is functionally important, as in allostery, and structurally crucial because mis-assembly of homomers is implicated in disease. Homomers are widespread, with 50-70% of proteins with a known quaternary state assembling into such structures. Despite their prevalence, their role in the evolution of cellular machinery and the potential for their use in the design of new molecular machines, little is known about the mechanisms that drive formation of homomers at the level of evolution and assembly in the cell. Here we present an analysis of over 5,000 unique atomic structures and show that the quaternary structure of homomers is conserved in over 70% of protein pairs sharing as little as 30% sequence identity. Where quaternary structure is not conserved among the members of a protein family, a detailed investigation revealed well-defined evolutionary pathways by which proteins transit between different quaternary structure types. Furthermore, we show by perturbing subunit interfaces within complexes and by mass spectrometry analysis, that the (dis)assembly pathway mimics the evolutionary pathway. These data represent a molecular analogy to Haeckel's evolutionary paradigm of embryonic development, where an intermediate in the assembly of a complex represents a form that appeared in its own evolutionary history. Our model of self-assembly allows reliable prediction of evolution and assembly of a complex solely from its crystal structure.     

热休克蛋白的研究进展

在不利的环境中,各种有机体都有其共同对应的分子反应,即正常基因的表达抑制和一组特殊基因——热休克基因的激活和表达,导致热休克蛋白的大量产生,热休克蛋白主要作为分子伴侣而参与蛋白质的折叠、转运及组装等过程,能恢复或加速清除细胞内已变性的蛋白质而稳定细胞结构,细胞产生热耐受。随着对热休克蛋白研究的不断深入,在生物工程和医学等方面的应用前景十分广阔。     

Demonstration by genetic suppression of interaction of GroE products with many proteins

The way in which proteins attain and maintain their final form is of fundamental importance. Recent work has focused on the role of a set of ubiquitous proteins, termed chaperonins, in the assembly of phage and multisubunit proteins. The range of chaperonin action is unknown; they could interact with most cellular polypeptides or have a limited subset of protein partners. Included in the chaperonin family is the essential heat-shock regulated Escherichia coli groEL gene product. Over-expression of the groE operon in E. coli causes enhanced assembly of heterologously expressed ribulose bisphosphate carboxylase subunits and suppresses the heat-sensitive mutant phenotype of several dnaA alleles. It has been inferred that suppression of heat-sensitive mutations is confined to dnaA alleles and that this confinement could reflect an interaction between the groE operon products and a dnaA protein aggregate at the replication origin. We now report that multiple copies of the groE operon suppress mutations in genes encoding several diverse proteins. Our data indicate a general role for the groE operon products, the GroEL and GroES proteins, in the folding-assembly pathways of many proteins.     

Different conformations for the same polypeptide bound to chaperones DnaK and GroEL.

The proteins DnaK (hsp70) and GroEL (cpn60) from Escherichia coli are prototypes of two classes of molecular chaperones conserved throughout evolution. The analysis of transferred nuclear Overhauser effects in two-dimensional NMR spectra is ideally suited to determine chaperone-bound conformations of peptides. The peptide vsv-C (amino-acid sequence KLIGVLSSLFRPK) stimulates the ATPase of BiP and Hsc70 (ref. 3) and the intrinsic ATPase of DnaK. The affinity of the vsv-C peptide for DnaK is greatly reduced in the presence of ATP. Here we analyse transferred nuclear Overhauser effects and show that the peptide is in an extended conformation while bound to DnaK but is helical when bound to GroEL. NMR also indicates that the mobility of the peptide backbone is reduced more by binding to DnaK than by binding to GroEL, whereas the side chains are less mobile when bound to GroEL.     

Internal control of the coated vesicle pp50-specific kinase complex

The polyhedral surface lattice of coated vesicles consists of three-legged hexameric protein complexes called triskelions which constitute the basic assembly unit. The triskelion is a molecular complex of molecular weight 630,000 (Mr 630K) composed of three clathrin heavy chains (subunit 180K) and three light chains (subunits 33K and 36K) (refs 2,3). The presence of additional coated vesicle-specific proteins in the 100-130K and 50-55K range have been reported. We previously described the presence of a cyclic nucleotide- and Ca2+-independent protein kinase activity in coated vesicles which was confirmed by others. This protein kinase specifically phosphorylates the 50K protein (pp50). In this report, we show that the coated vesicle kinase and its 50K protein substrate are part of a stable multimolecular system. In addition we show that the clathrin-light chain complex stimulates the pp50 phosphorylation and only light chains are implicated in this stimulation and that the pp50 phosphorylation does not seem to be affected by the vesicle.     

Modulation of spectrin-actin assembly by erythrocyte adducin

The spectrin-based membrane skeleton, an assembly of proteins tightly associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Spectrin, the most abundant component of this assembly, is an elongated and flexible molecule that, with potentiation by protein 4.1, is cross-linked at its ends by short actin filaments to form a lattice beneath the membrane. These and other proteins stabilize the plasma membrane, organize integral membrane proteins and maintain specialized regions of the cell surface. A membrane-skeleton-associated calmodulin-binding protein of erythrocytes is a major substrate for Ca2+- and phospholipid-dependent protein kinase C (ref. 5), and thus is a target for Ca2+ by two regulatory pathways. Here we demonstrate that this protein, called adducin: (1) binds tightly in vitro to spectrin-actin complexes but with much less affinity either to spectrin or to actin alone; (2) promotes assembly of additional spectrin molecules onto actin filaments; and (3) is inhibited in its ability to induce the binding of additional spectrin molecules to actin by micromolar concentrations of calmodulin and Ca2+. Adducin may be involved in the action of Ca2+ on erythrocyte membrane skeleton and in the assembly of spectrin-actin complexes.     

Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid proteins in E. coli

The use of lacZ gene fusions, producing a hybrid protein containing an amino terminus specified by a target gene fused to the functional carboxy terminus of beta-galactosidase, has facilitated the study of protein targeting in various organisms. One of the best characterized fusions in Escherichia coli is phi(lamB-lacZ)42-1(Hyb), which produces a hybrid protein with the signal sequence and 181 N-terminal amino acids of the exported protein LamB, attached to LacZ. In common with other LacZ hybrids, the LamB-LacZ(42-1) protein is poorly exported from E. coli, conferring a Lac+ phenotype. beta-Galactosidase activity decreases markedly when cells producing the LamB-LacZ protein are grown at 42 degrees C or when a heat-shock response is induced at lower temperatures by overproducing heat-shock factor RpoH3, indicating the LacZ hybrids are being efficiently targeted to the cell envelope. We now report that the heat-shock proteins DnaK and GroEL can, in sufficient amounts, decrease beta-galactosidase activity and facilitate the export of lacZ-hybrid proteins.     

NMR analysis of a 900K GroEL GroES complex

Biomacromolecular structures with a relative molecular mass (M(r)) of 50,000 to 100,000 (50K 100K) have been generally considered to be inaccessible to analysis by solution NMR spectroscopy. Here we report spectra recorded from bacterial chaperonin complexes ten times this size limit (up to M(r) 900K) using the techniques of transverse relaxation-optimized spectroscopy and cross-correlated relaxation-enhanced polarization transfer. These techniques prevent deterioration of the NMR spectra by the rapid transverse relaxation of the magnetization to which large, slowly tumbling molecules are otherwise subject. We tested the resolving power of these techniques by examining the isotope-labelled homoheptameric co-chaperonin GroES (M(r) 72K), either free in solution or in complex with the homotetradecameric chaperonin GroEL (M(r) 800K) or with the single-ring GroEL variant SR1 (M(r) 400K). Most amino acids of GroES show the same resonances whether free in solution or in complex with chaperonin; however, residues 17 32 show large chemical shift changes on binding. These amino acids belong to a mobile loop region of GroES that forms contacts with GroEL. This establishes the utility of these techniques for solution NMR studies that should permit the exploration of structure, dynamics and interactions in large macromolecular complexes.     

Structure of pre-pro-von Willebrand factor and its expression in heterologous cells

Von Willebrand factor (vWF), a multifunctional haemostatic glycoprotein derived from endothelial cells and megakaryocytes, mediates platelet adhesion to injured subendothelium and binds coagulation factor VIII in the circulation. Native vWF is a disulphide-bonded homopolymer; the monomeric subunits, of apparent relative molecular mass (Mr) 220,000 (220K) are derived from an intracellular precursor estimated at 260-275K. Multimer assembly is preceded by the formation of dimers, linked near their C-termini, which then assemble into filamentous polymers. The importance of the removal of the large vWF pro-polypeptide during multimer assembly, and whether this or other stages of the complex post-translational processing require components specific to endothelial cells or megakaryocytes, is unknown. Here we report an analysis of the complete sequence of pre-pro-vWF and expression of the molecule in heterologous cells. The vWF precursor is composed of several repeated subdomains. When expressed in COS and CHO cells, it is cleaved and assembled into biologically active high relative molecular mass disulphide bonded multimers. This suggests that the information for assembly of this complex molecule resides largely within its primary structure.     

Assembly dynamics of microtubules at molecular resolution

Microtubules are highly dynamic protein polymers that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro and in vivo, to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule end-binding proteins regulate microtubule dynamics in living cells.     

Association of a Ras-related protein with cytochrome b of human neutrophils

Activation of the superoxide generating system in human neutrophils is thought to involve the interaction or assembly of cytochrome b with other cytosolic and membrane proteins. We have now co-isolated by conventional purification procedures a protein of relative molecular mass 22,000 with cytochrome b. This Ras-related protein is not a fragment of either of the subunits of cytochrome b, and its primary structure, as determined by the sequencing of its complementary DNA, is identical to that predicted from a recently cloned ras-related gene, rap1 (also termed Krev-1). Immunoaffinity purification on anti-cytochrome and anti-Ras immunoaffinity matrices indicates an association between cytochrome b and the Ras-related protein. The association of a Ras-related GTP-binding protein with cytochrome b of human neutrophils could indicate a role for such a protein in the transduction, regulation or structure of the superoxide generating system.     

分子伴侣GroEL对青霉素G酰化酶亚基折叠的影响

采用Tac启动子控制表达质粒，在不同的宿主细胞中表达了青霉素G酰化酶（PAC），检测这些菌株所表达的PAC活性，分析细胞内分子伴侣GroEL含量，PAC翻译后加工为α，β亚基的状况，以及它们之间的关系，结果表明：质粒pKK-SP在不同宿主中表达时，翻译后加工状况有明显差异，单位质量细胞所表达的PAC活性与翻译后加工效率相关，且与细胞内分子伴侣GroEL在菌体总蛋白中含量正相关，同时也阐明了亚基的折叠成为翻译后加工过程的限制步骤，细胞内分子伴侣GroEL有助于PAC亚基的折叠和稳定。     

Trigger factor and DnaK cooperate in folding of newly synthesized proteins.

The role of molecular chaperones in assisting the folding of newly synthesized proteins in the cytosol is poorly understood. In Escherichia coli, GroEL assists folding of only a minority of proteins and the Hsp70 homologue DnaK is not essential for protein folding or cell viability at intermediate growth temperatures. The major protein associated with nascent polypeptides is ribosome-bound trigger factor, which displays chaperone and prolyl isomerase activities in vitro. Here we show that delta tig::kan mutants lacking trigger factor have no defects in growth or protein folding. However, combined delta tig::kan and delta dnaK mutations cause synthetic lethality. Depletion of DnaK in the delta tig::kan mutant results in massive aggregation of cytosolic proteins. In delta tig::kan cells, an increased amount of newly synthesized proteins associated transiently with DnaK. These findings show in vivo activity for a ribosome-associated chaperone, trigger factor, in general protein folding, and functional cooperation of this protein with a cytosolic Hsp70. Trigger factor and DnaK cooperate to promote proper folding of a variety of E. coli proteins, but neither is essential for folding and viability at intermediate growth temperatures.     

Vanadyl ions binding to GroEL （HSP60） and inducing its depolymerization

Several vanadium compounds have been known for the hypoglycemic and anticancer effects. However, the mechanisms of the pharmacological and toxicological effects were not clear. In this work, we in- vestigated the potential targets of vanadium in mitochondria. Vanadyl ions were found to bind to mi- tochondria from rat liver with a stoichiometry of 244±58 nmol/mg protein and an apparent dissocia- tion constant (Kd) of (2.0±0.8)×10·16 mol/L. Using size exclusion chromatography, a vanadium-binding protein was isolated and identified to be the 60-kDa heat shock protein (HSP60) by mass spectrometry analysis and immunoassays. Additionally, binding of vanadyl ions was found to result in depolymeri- zation of homo-oligomeric HSP60 (GroEL). HSP60 is an indispensable molecular chaperone and in- volved in many kinds of pathogenesis of inflammatory and autoimmune diseases, e.g. type 1 diabetes. Our results suggested that HSP60 could be a novel important target involved in the biological and/or toxicological effects of vanadium compounds.     

Inhibition of mitosis by an antibody to the mitotic calcium transport system

Calcium ions are important in the regulation of mitotic apparatus assembly and in the control of chromosome movement. Changes in intracellular free calcium concentration, [Ca2+]i are achieved by an intracellular calcium-transport system which is highly conserved in different cell types. A membrane-bound protein of relative molecular mass (Mr) 46,000 (46K) is part of this transport system and has been implicated in the regulation of the [Ca2+]i changes associated with the course of mitosis. A monoclonal antibody against this 46K protein inhibits Ca2+-uptake into isolated Ca2+-sequestering membranes and specifically labels membranes associated with the mitotic apparatus of sea urchin embryos. Here we investigate the relationship between the intracellular calcium transport system and mitosis by injection of this monoclonal antibody into living mitotic sea urchin embryos. We find that after injection the intracellular free calcium increases up to 10(-6) M, the mitotic apparatus is rapidly destroyed and the cell is irreversibly blocked in its development.     

Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding.

The main stress proteins of Escherichia coli function in an ordered protein-folding reaction. DnaK (heat-shock protein 70) recognizes the folding polypeptide as an extended chain and cooperates with DnaJ in stabilizing an intermediate conformational state lacking ordered tertiary structure. Dependent on GrpE and ATP hydrolysis, the protein is then transferred to GroEL (heat-shock protein 60) which acts catalytically in the production of the native state. This sequential mechanism of chaperone action may represent an important pathway for the folding of newly synthesized polypeptides.