TCP1 complex is a molecular chaperone in tubulin biogenesis.

A role in folding of newly translated proteins in the cytosol of eukaryotes has been proposed for t-complex polypeptide-1 (TCP1), although its molecular targets have not yet been identified. Tubulin is a major cytosolic protein whose assembly into microtubules is critical to many cellular processes. Although numerous studies have focused on the expression of tubulin, little is known about the processes whereby newly translated tubulin subunits acquire conformations that enable them to form alpha-beta-heterodimers. We examined the biogenesis of alpha- and beta-tubulin in rabbit reticulocyte lysate, and report here that newly translated tubulin subunits entered a 900K complex in a protease-sensitive conformation. Addition of Mg-ATP, but not nonhydrolysable analogues, released the tubulin subunits as assembly-competent protein with a conformation that was relatively protease-resistant. The 900K complex purified from reticulocyte lysate contained as its major constituent a 58K protein that cross-reacted with a monoclonal antiserum against mouse TCP1. We conclude that TCP1 functions as a cytosolic chaperone in the biogenesis of tubulin.     

T-complex polypeptide-1 is a subunit of a heteromeric particle in the eukaryotic cytosol.

The murine t-complex encodes t-complex polypeptide-1 (TCP1), which is constitutively expressed in almost all cells, and upregulated during spermatogenesis. Mammalian sequences have greater than 96% identity with each other, and greater than 60% identity with Drosophila melanogaster and yeast orthologues. TCP1 is essential in yeast, and is postulated to be the cytosolic mammalian equivalent of groEL. We report here that, in the native state, murine and human TCP1 is distributed throughout the cytosol as an 800K-950K hetero-oligomeric particle in association with four to six unidentified proteins and two Hsp70 heat-shock proteins. Negative-stain electron microscopy indicates that the structure is two stacked rings, 12-16 nm in diameter. Therefore, despite similarities with the chaperonin 60 proteins, these data indicate that TCP1 is biochemically and structurally unique. We suggest that TCP1 may represent one of a family of molecules in the eukaryotic cytosol involved in protein folding and regulated in part by their heteromeric associations.     

Eukaryotic type II chaperonin CCT interacts with actin through specific subunits

Chaperonins assist the folding of other proteins. Type II chaperonins, such as chaperonin containing TCP-1(CCT), are found in archaea and in the eukaryotic cytosol. They are hexadecameric or nonadecameric oligomers composed of one to eight different polypeptides. Whereas type I chaperonins like GroEL are promiscuous, assisting in the folding of many other proteins, only a small number of proteins, mainly actin and tubulin, have been described as natural substrates of CCT. This specificity may be related to the divergence of the eight CCT subunits. Here we have obtained a three-dimensional reconstruction of the complex between CCT and alpha-actin by cryo-electron microscopy and image processing. This shows that alpha-actin interacts with the apical domains of either of two CCT subunits. Immunolabelling of CCT-substrate complexes with antibodies against two specific CCT subunits showed that actin binds to CCT using two specific and distinct interactions: the small domain of actin binds to CCTdelta and the large domain to CCTbeta or CCTepsilon (both in position 1,4 with respect to delta). These results indicate that the binding of actin to CCT is both subunit-specific and geometry-dependent. Thus, the substrate recognition mechanism of eukaryotic CCT may differ from that of prokaryotic GroEL.     

Two contrary modes of chemolithotrophy in the same archaebacterium

Sulphur-dependent archaebacteria, which are found around nearly boiling continental solfataric springs and mud holes, can be assigned to two distinct branches: the aerobic, sulphur-oxidizing Sulfolobales and the strictly anaerobic sulphur-reducing Thermoproteales. Here, we report the isolation of a group of extremely thermophilic solfataric archaebacteria that are able to grow either strictly anaerobically by reduction, or fully aerobically by oxidation of molecular sulphur, depending on the oxygen supply. We have also established that the ability to grow in these two ways is shared by Sulfolobus brierleyi, a well-known less thermophilic sulphur-oxidizing archaebacterium capable of ore-leaching. The phenomenon may be dependent on a fundamental switch in genome expression. These organisms might represent the primitive fore-runners of sulphur-oxidizing archaebacteria, meeting their energy requirements either by oxidation or by reduction of the same element.     

Real-time observation of trigger factor function on translating ribosomes

The contribution of co-translational chaperone functions to protein folding is poorly understood. Ribosome-associated trigger factor (TF) is the first molecular chaperone encountered by nascent polypeptides in bacteria. Here we show, using fluorescence spectroscopy to monitor TF function and structural rearrangements in real time, that TF interacts with ribosomes and translating polypeptides in a dynamic reaction cycle. Ribosome binding stabilizes TF in an open, activated conformation. Activated TF departs from the ribosome after a mean residence time of approximately 10 s, but may remain associated with the elongating nascent chain for up to 35 s, allowing entry of a new TF molecule at the ribosome docking site. The duration of nascent-chain interaction correlates with the occurrence of hydrophobic motifs in translating polypeptides, reflecting a high aggregation propensity. These findings can explain how TF prevents misfolding events during translation and may provide a paradigm for the regulation of nucleotide-independent chaperones.     

Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex

Hsp90 (heat shock protein of 90 kDa) is a ubiquitous molecular chaperone responsible for the assembly and regulation of many eukaryotic signalling systems and is an emerging target for rational chemotherapy of many cancers. Although the structures of isolated domains of Hsp90 have been determined, the arrangement and ATP-dependent dynamics of these in the full Hsp90 dimer have been elusive and contentious. Here we present the crystal structure of full-length yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1. The structure reveals the complex architecture of the 'closed' state of the Hsp90 chaperone, the extensive interactions between domains and between protein chains, the detailed conformational changes in the amino-terminal domain that accompany ATP binding, and the structural basis for stabilization of the closed state by p23/Sba1. Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the chaperone ATPase cycle.     

Hsp90 chaperones protein folding in vitro.

The heat-shock protein Hsp90 is the most abundant constitutively expressed stress protein in the cytosol of eukaryotic cells, where it participates in the maturation of other proteins, modulation of protein activity in the case of hormone-free steroid receptors, and intracellular transport of some newly synthesized kinases. A feature of all these processes could be their dependence on the formation of protein structure. If Hsp90 is a molecular chaperone involved in maintaining a certain subset of cellular proteins in an inactive form, it should also be able to recognize and bind non-native proteins, thereby influencing their folding to the native state. Here we investigate whether Hsp90 can influence protein folding in vitro and show that Hsp90 suppresses the formation of protein aggregates by binding to the target proteins at a stoichiometry of one Hsp90 dimer to one or two substrate molecule(s). Furthermore, the yield of correctly folded and functional protein is increased significantly. The action of Hsp90 does not depend on the presence of nucleoside triphosphates, so it may be that Hsp90 uses a novel molecular mechanism to assist protein folding in vivo.     

The mitochondrial chaperonin hsp60 is required for its own assembly

Heatshock protein 60 (hsp60) in the matrix of mitochondria is essential for the folding and assembly of newly imported proteins. Hsp60 belongs to a class of structurally related chaperonins found in organelles of endosymbiotic origin and in the bacterial cytosol. Hsp60 monomers form a complex arranged as two stacked 7-mer rings. This 14-mer complex binds unfolded proteins at its surface, then seems to catalyse their folding in an ATP-dependent process. The question arises as to how such an assembly machinery is itself folded and assembled. Hsp60 subunits are encoded by a nuclear gene and translated in the cytosol as precursors which are translocated into mitochondria and proteolytically processed. In both intact cells and isolated mitochondria of the hsp60-defective yeast mutant mif4, self-assembly of newly imported wild-type subunits is not observed. Functional pre-existing hsp60 complex is required in order to form new, assembled, 14-mer. Subunits imported in vitro are assembled with a surprisingly fast half-time of 5-10 min, indicative of a catalysed reaction. These findings are further evidence that self-assembly may not be the principal mechanism by which proteins attain their functional conformation in the intact cell.     

Chemolithoautotrophic metabolism of anaerobic extremely thermophilic archaebacteria

Several types of extremely thermophilic archaebacteria have recently been isolated from solfataric water holes, hot springs and hot sea floors. It has been shown that some of them can live using sulphur respiration of reduced carbon substrates as a source of energy, a type of metabolism previously described for the eubacterium Desulfuromonas. We report here that several extremely thermophilic archaebacteria can live with carbon dioxide as their sole carbon source, obtaining energy from the oxidation of hydrogen by sulphur, producing hydrogen sulphide. They are thus capable of a new type of anaerobic, purely chemolithoautotrophic metabolism, a possible primaeval mode of life.     

The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol.

In eukaryotic cells, incorrectly folded proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded by the proteasome. This pathway is co-opted by some viruses. For example, the US11 protein of the human cytomegalovirus targets the major histocompatibility complex class I heavy chain for cytosolic degradation. How proteins are extracted from the ER membrane is unknown. In bacteria and mitochondria, members of the AAA ATPase family are involved in extracting and degrading membrane proteins. Here we demonstrate that another member of this family, Cdc48 in yeast and p97 in mammals, is required for the export of ER proteins into the cytosol. Whereas Cdc48/p97 was previously known to function in a complex with the cofactor p47 (ref. 5) in membrane fusion, we demonstrate that its role in ER protein export requires the interacting partners Ufd1 and Npl4. The AAA ATPase interacts with substrates at the ER membrane and is needed to release them as polyubiquitinated species into the cytosol. We propose that the Cdc48/p97-Ufd1-Npl4 complex extracts proteins from the ER membrane for cytosolic degradation.     

The mechanism of membrane-associated steps in tail-anchored protein insertion

Tail-anchored (TA) membrane proteins destined for the endoplasmic reticulum are chaperoned by cytosolic targeting factors that deliver them to a membrane receptor for insertion. Although a basic framework for TA protein recognition is now emerging, the decisive targeting and membrane insertion steps are not understood. Here we reconstitute the TA protein insertion cycle with purified components, present crystal structures of key complexes between these components and perform mutational analyses based on the structures. We show that a committed targeting complex, formed by a TA protein bound to the chaperone ATPase Get3, is initially recruited to the membrane through an interaction with Get2. Once the targeting complex has been recruited, Get1 interacts with Get3 to drive TA protein release in an ATPase-dependent reaction. After releasing its TA protein cargo, the now-vacant Get3 recycles back to the cytosol concomitant with ATP binding. This work provides a detailed structural and mechanistic framework for the minimal TA protein insertion cycle.     

Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP.

In vitro reconstitution of active ribulose bisphosphate carboxylase (Rubisco) from unfolded polypeptides is facilitated by the molecular chaperones: chaperonin-60 from Escherichia coli (groEL), yeast mitochondria (hsp60) or chloroplasts (Rubisco sub-unit-binding protein), together with chaperonin-10 from E. coli (groES), and Mg-ATP. Because chaperonins are ubiquitous, a conserved Mg-ATP-dependent mechanism exists that uses the chaperonins to facilitate the folding of some other proteins.     

A possible biochemical missing link among archaebacteria

Until recently all archaebacteria isolated conformed to one of three basic phenotypes: they were either methanogens, extreme halophiles, or ('sulphur-dependent') extreme thermophiles. However, a novel phenotype, that fits none of these categories, has recently been described. The organism, strain VC-16 (tentatively called "Archaeoglobus fulgidus") reduces sulphate--the only archaebacterium so far known to do so--and makes very small quantities of methane, although it lacks some of the cofactors normally associated with methanogenesis. These characteristics suggest that strain VC-16 might represent a transition form between an anaerobic thermophilic sulfur-based type of metabolism (which seems to be the ancestral metabolism for archaebacteria and methanogenesis (which somehow then derives from it). We here show that the lineage represented by strain VC-16 arises from the archaebacterial tree precisely where such an interpretation would predict that it would, between the Methanococcus lineage (which is the deepest of the methanogen branchings) and that of Thermococcus (the deepest of all branchings on the methanogen side of the tree).     

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.     

Identification of an actin-binding protein from Dictyostelium as elongation factor 1a

Indirect evidence has implicated an interaction between the cytoskeleton and the protein synthetic machinery. Two recent reports have linked the elongation factor 1a (EF-1a) which is involved in protein synthesis, with the microtubular cytoskeleton. In situ hybridization has, however, revealed that the messages for certain cytoskeletal proteins are preferentially associated with actin filaments. ABP-50 is an abundant actin filament bundling protein of native relative molecular mass 50,000 (50K) isolated from Dictyostelium discoideum. Immunofluorescence studies show that ABP-50 is present in filopodia and other cortical regions that contain actin filament bundles. In addition, ABP-50 binds to monomeric actin in the cytosol of unstimulated cells and the association of ABP-50 with the actin cytoskeleton is regulated during chemotaxis. Through complementary DNA sequencing and subsequent functional analysis, we have identified ABP-50 as D. discoideum EF-1a. The ability of EF-1a to bind reversibly to the actin cytoskeleton upon stimulation could provide a mechanism for spatially and temporally regulated protein synthesis in eukaryotic cells.     

A proteasomal ATPase subunit recognizes the polyubiquitin degradation signal

The 26S proteasome is the chief site of regulatory protein turnover in eukaryotic cells. It comprises one 20S catalytic complex (composed of four stacked rings of seven members) and two axially positioned 19S regulatory complexes (each containing about 18 subunits) that control substrate access to the catalytic chamber. In most cases, targeting to the 26S proteasome depends on tagging of the substrate with a specific type of polyubiquitin chain. Recognition of this signal is followed by substrate unfolding and translocation, which are presumably catalysed by one or more of six distinct AAA ATPases located in the base-a ring-like 19S subdomain that abuts the axial pore of the 20S complex and exhibits chaperone activity in vitro. Despite the importance of polyubiquitin chain recognition in proteasome function, the site of this signal's interaction with the 19S complex has not been identified previously. Here we use crosslinking to a reactive polyubiquitin chain to show that a specific ATPase subunit, S6' (also known as Rpt5), contacts the bound chain. The interaction of this signal with 26S proteasomes is modulated by ATP hydrolysis. Our results suggest that productive recognition of the proteolytic signal, as well as proteasome assembly and substrate unfolding, are ATP-dependent events.     

Molecular chaperones in protein folding and proteostasis

Most proteins must fold into defined three-dimensional structures to gain functional activity. But in the cellular environment, newly synthesized proteins are at great risk of aberrant folding and aggregation, potentially forming toxic species. To avoid these dangers, cells invest in a complex network of molecular chaperones, which use ingenious mechanisms to prevent aggregation and promote efficient folding. Because protein molecules are highly dynamic, constant chaperone surveillance is required to ensure protein homeostasis (proteostasis). Recent advances suggest that an age-related decline in proteostasis capacity allows the manifestation of various protein-aggregation diseases, including Alzheimer's disease and Parkinson's disease. Interventions in these and numerous other pathological states may spring from a detailed understanding of the pathways underlying proteome maintenance.     

分子伴侣及其作用机理的研究概况

分子伴侣为一类与其他蛋白不稳定构象结合并使之稳定的蛋白质,广泛分布于各种生物体内。本文对分子伴侣的最新研究作一综述。     

Phylogenetic analysis based on rRNA sequences supports the archaebacterial rather than the eocyte tree

How many primary lineages of life exist and what are their evolutionary relationships? These are fundamental but highly controversial issues. Woese and co-workers propose that archaebacteria, eubacteria and eukaryotes are the three primary lines of descent and their relationships can be represented by Fig. 1a (the 'archaebacterial tree') if one neglects the root of the tree. In contrast, Lake claims that archaebacteria are paraphyletic, and he groups eocytes (extremely thermophilic, sulphur-dependent bacteria) with eukaryotes, and halobacteria with eubacteria (the 'eocyte tree', Fig. 1b). Lake's view has gained considerable support as a result of an analysis of small subunit ribosomal RNA sequence data by a new approach, the evolutionary parsimony method. Here we report that analysis of small subunit data by the neighbour-joining and maximum parasimony methods favours the archaebacterial tree and that computer simulations using either the archaebacterial or the eocyte tree as a model tree show that the probability of recovering the model tree is very high (greater than 90 per cent) for both the neighbour-joining and maximum parsimony methods but is relatively low for the evolutionary parsimony method. Moreover, analysis of large subunit rRNA sequences by all three methods strongly favours the archaebacterial tree.     

A genomic perspective on membrane compartment organization

Now that whole genome sequences are available for many eukaryotic organisms from yeast to man, we can form broad hypotheses on the basis of the relative expansion of protein families. To investigate the molecular mechanisms responsible for the organization of membrane compartments, we identified members of the SNARE, coat complex, Rab and Sec1 protein families in four eukaryotic genomes. Of these families only the Rab family expanded from the unicellular yeast to the multicellular fly and worm. All families were expanded in humans, where we find 35 SNAREs, 60 Rabs and 53 coat complex subunits. In addition, we were able to resolve the SNARE class of proteins into four distinct subfamilies.