ClpS is an essential component of the N-end rule pathway in Escherichia coli

The N-end rule states that the half-life of a protein is determined by the nature of its amino-terminal residue. Eukaryotes and prokaryotes use N-terminal destabilizing residues as a signal to target proteins for degradation by the N-end rule pathway. In eukaryotes an E3 ligase, N-recognin, recognizes N-end rule substrates and mediates their ubiquitination and degradation by the proteasome. In Escherichia coli, N-end rule substrates are degraded by the AAA + chaperone ClpA in complex with the ClpP peptidase (ClpAP). Little is known of the molecular mechanism by which N-end rule substrates are initially selected for proteolysis. Here we report that the ClpAP-specific adaptor, ClpS, is essential for degradation of N-end rule substrates by ClpAP in bacteria. ClpS binds directly to N-terminal destabilizing residues through its substrate-binding site distal to the ClpS-ClpA interface, and targets these substrates to ClpAP for degradation. Degradation by the N-end rule pathway is more complex than anticipated and several other features are involved, including a net positive charge near the N terminus and an unstructured region between the N-terminal signal and the folded protein substrate. Through interaction with this signal, ClpS converts the ClpAP machine into a protease with exquisitely defined specificity, ideally suited to regulatory proteolysis.     

Structure and mechanism of the hexameric MecA-ClpC molecular machine

Regulated proteolysis by ATP-dependent proteases is universal in all living cells. Bacterial ClpC, a member of the Clp/Hsp100 family of AAA+ proteins (ATPases associated with diverse cellular activities) with two nucleotide-binding domains (D1 and D2), requires the adaptor protein MecA for activation and substrate targeting. The activated, hexameric MecA-ClpC molecular machine harnesses the energy of ATP binding and hydrolysis to unfold specific substrate proteins and translocate the unfolded polypeptide to the ClpP protease for degradation. Here we report three related crystal structures: a heterodimer between MecA and the amino domain of ClpC, a heterododecamer between MecA and D2-deleted ClpC, and a hexameric complex between MecA and full-length ClpC. In conjunction with biochemical analyses, these structures reveal the organizational principles behind the hexameric MecA-ClpC complex, explain the molecular mechanisms for MecA-mediated ClpC activation and provide mechanistic insights into the function of the MecA-ClpC molecular machine. These findings have implications for related Clp/Hsp100 molecular machines.     

Hsp104 is a highly conserved protein with two essential nucleotide-binding sites

Most eukaryotic cells produce proteins with relative molecular masses in the range of 100,000 to 110,000 after exposure to high temperatures. These proteins have been studied only in yeast and mammalian cells. In Saccharomyces cerevisiae, heat-shock protein hsp104 is vital for tolerance to heat, ethanol and other stresses. The mammalian hsp110 protein is nucleolar and redistributes with growth state, nutritional conditions and heat shock. The relationships between hsp110, hsp104 and the high molecular mass heat-shock proteins of other organisms were unknown. We report here that hsp104 is a member of the highly conserved ClpA/ClpB protein family first identified in Escherichia coli and that additional heat-inducible members of this family are present in Schizosaccharomyces pombe and in mammals. Mutagenesis of two putative nucleotide-binding sites in hsp104 indicates that both are essential for function in thermotolerance.     

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.     

A mitochondrial remnant in the microsporidian Trachipleistophora hominis

Microsporidia are obligate intracellular parasites of several eukaryotes. They have a highly complex and unique infection apparatus but otherwise appear structurally simple. Microsporidia are thought to lack typical eukaryotic organelles, such as mitochondria and peroxisomes. This has been interpreted as support for the hypothesis that these peculiar eukaryotes diverged before the mitochondrial endosymbiosis, which would make them one of the earliest offshoots in eukaryotic evolution. But microsporidial nuclear genes that encode orthologues of typical mitochondrial heatshock Hsp70 proteins have been detected, which provides evidence for secondary loss of the organelle or endosymbiont. In addition, gene trees and more sophisticated phylogenetic analyses have recovered microsporidia as the relatives of fungi, rather than as basal eukaryotes. Here we show that a highly specific antibody raised against a Trachipleistophora hominis Hsp70 protein detects the presence, under light and electron microscopy, of numerous tiny ( approximately 50 x 90 nm) organelles with double membranes in this human microsporidial parasite. The finding of relictual mitochondria in microsporidia provides further evidence of the reluctance of eukaryotes to lose the mitochondrial organelle, even when its canonical function of aerobic respiration has been apparently lost.     

Green fluorescent protein （GFP） as a vital marker for studying the interaction of Phytophthora sojae and soybean

Transgenic Phytophthora sojae strains that produce green fluorescent protein （GFP） were obtained after stable DNA integration using the Hsp70 promoter and the Ham34 terminator of Bremia lactucae. The expression of GFP during different developmental stages of P. sojae was observed using fluorescent microscopy. Based on this reporter system, the histopathologic events caused by the pathogen in soybean leaves, hypocotyls and roots were monitored. Meanwhile, the difference in resistance between different soybean cultivars against P. sojae was analyzed microscopically in roots. The results indicate that GFP can be stably expressed in zoosporangia, zoospores, cysts, hyphae and oospores of P. sojae. Using the GFP marker, the infecting pathogens in leaves, hypocotyls and roots of host could be distinctly visualized. The germ tube length of cysts germinating on the roots of resistant cultivar Nannong 8848 was longer than that on the roots of susceptible cultivar Hefeng 35. These results show for the first time that this eukaryotic reporter can be used in P. sojae as a stable and vital marker, allowing the study of genetics of this hemibiotrophic pathogen.     

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.     

Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo

Evolutionarily diverse eukaryotic cells share many conserved proteins of unknown function. Some are essential for cell viability, emphasising their importance for fundamental processes of cell biology but complicating their analysis. We have developed an approach to the large-scale characterization of such proteins, based on conditional and rapid degradation of the target protein in vivo, so that the immediate consequences of bulk protein depletion can be examined. Budding yeast strains have been constructed in which essential proteins of unknown function have been fused to a 'heat-inducible-degron' cassette that targets the protein for proteolysis at 37 degrees C (ref. 4). By screening the collection for defects in cell-cycle progression, here we identify three DNA replication factors that interact with each other and that have uncharacterized homologues in human cells. We have used the degron strains to show that these proteins are required for the establishment and normal progression of DNA replication forks. The degron collection could also be used to identify other, essential, proteins with roles in many other processes of eukaryotic cell biology.     

A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of oncogenic signalling proteins, including HER-2/ErbB2, Akt, Raf-1, Bcr-Abl and mutated p53. Hsp90 inhibitors bind to Hsp90, and induce the proteasomal degradation of Hsp90 client proteins. Although Hsp90 is highly expressed in most cells, Hsp90 inhibitors selectively kill cancer cells compared to normal cells, and the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is currently in phase I clinical trials. However, the molecular basis of the tumour selectivity of Hsp90 inhibitors is unknown. Here we report that Hsp90 derived from tumour cells has a 100-fold higher binding affinity for 17-AAG than does Hsp90 from normal cells. Tumour Hsp90 is present entirely in multi-chaperone complexes with high ATPase activity, whereas Hsp90 from normal tissues is in a latent, uncomplexed state. In vitro reconstitution of chaperone complexes with Hsp90 resulted in increased binding affinity to 17-AAG, and increased ATPase activity. These results suggest that tumour cells contain Hsp90 complexes in an activated, high-affinity conformation that facilitates malignant progression, and that may represent a unique target for cancer therapeutics.     

A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi

Mitochondria use transport proteins of the eukaryotic mitochondrial carrier family (MCF) to mediate the exchange of diverse substrates, including ATP, with the host cell cytosol. According to classical endosymbiosis theory, insertion of a host-nuclear-encoded MCF transporter into the protomitochondrion was the key step that allowed the host cell to harvest ATP from the enslaved endosymbiont. Notably the genome of the microsporidian Encephalitozoon cuniculi has lost all of its genes for MCF proteins. This raises the question of how the recently discovered microsporidian remnant mitochondrion, called a mitosome, acquires ATP to support protein import and other predicted ATP-dependent activities. The E. cuniculi genome does contain four genes for an unrelated type of nucleotide transporter used by plastids and bacterial intracellular parasites, such as Rickettsia and Chlamydia, to import ATP from the cytosol of their eukaryotic host cells. The inference is that E. cuniculi also uses these proteins to steal ATP from its eukaryotic host to sustain its lifestyle as an obligate intracellular parasite. Here we show that, consistent with this hypothesis, all four E. cuniculi transporters can transport ATP, and three of them are expressed on the surface of the parasite when it is living inside host cells. The fourth transporter co-locates with mitochondrial Hsp70 to the E. cuniculi mitosome. Thus, uniquely among eukaryotes, the traditional relationship between mitochondrion and host has been subverted in E. cuniculi, by reductive evolution and analogous gene replacement. Instead of the mitosome providing the parasite cytosol with ATP, the parasite cytosol now seems to provide ATP for the organelle.     

A preliminary study on functional domains of small heat shock protein Hsp16.3

Hsp16.3, the small heat shock protein (sHSP) from Mycobacterium tuberculosis, was originally identified as an immunodominant antigen,which possesses three functional domains typical of sHSP family, namely the N-terminal hydrophobic region, α-crystallin domain and a short non-conserved C-terminal extension.To further understand the functional assignment of these independent regions, the three functional domains of Hsp16.3 were defined and the two N- or C-terminal truncated Hsp16.3 remnants were successfully cloned, expressed and purified.In the far and near circular dichroism analysis, the results showed that these remnants expressed similar secondary and tertiary structures to that of wild-type protein.During the reassembly of wild-type nonamer, the C-terminal truncated remnant could interact with the wild-type protein to form hetero-oligomers.When trypsin is used to digest the wild-type Hsp16.3, its α-crystallin domain could resist such degradation.Taken together, these results indicate that the stable secondary and tertiary structures of Hsp16.3 are mainly kept by its α-crystallin domain.     

Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines

Hexameric ring-shaped ATPases of the AAA + (for ATPases associated with various cellular activities) superfamily power cellular processes in which macromolecular structures and complexes are dismantled or denatured, but the mechanisms used by these machine-like enzymes are poorly understood. By covalently linking active and inactive subunits of the ATPase ClpX to form hexamers, here we show that diverse geometric arrangements can support the enzymatic unfolding of protein substrates and translocation of the denatured polypeptide into the ClpP peptidase for degradation. These studies indicate that the ClpX power stroke is generated by ATP hydrolysis in a single subunit, rule out concerted and strict sequential ATP hydrolysis models, and provide evidence for a probabilistic sequence of nucleotide hydrolysis. This mechanism would allow any ClpX subunit in contact with a translocating polypeptide to hydrolyse ATP to drive substrate spooling into ClpP, and would prevent stalling if one subunit failed to bind or hydrolyse ATP. Energy-dependent machines with highly diverse quaternary architectures and molecular functions could operate by similar asymmetric mechanisms.     

Chaperone-like activity of the AAA domain of the yeast Yme1 AAA protease

The AAA domain, a conserved Walker-type ATPase module, is a feature of members of the AAA family of proteins, which are involved in many cellular processes, including vesicular transport, organelle biogenesis, microtubule rearrangement and protein degradation. The function of the AAA domain, however, has not been explained. Membrane-anchored AAA proteases of prokaryotic and eukaryotic cells comprise a subfamily of AAA proteins that have metal-dependent peptidase activity and mediate the degradation of non-assembled membrane proteins. Inactivation of an orthologue of this protease family in humans causes neurodegeneration in hereditary spastic paraplegia. Here we investigate the AAA domain of the yeast protein Yme1, a subunit of the iota-AAA protease located in the inner membrane of mitochondria. We show that Yme1 senses the folding state of solvent-exposed domains and specifically degrades unfolded membrane proteins. Substrate recognition and binding are mediated by the amino-terminal region of the AAA domain. The purified AAA domain of Yme1 binds unfolded polypeptides and suppresses their aggregation. Our results indicate that the AAA domain of Ymel has a chaperone-like activity and suggest that the AAA domains of other AAA proteins may have a similar function.     

The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaeon that thrives at 59 degrees C and pH 2, which was isolated from self-heating coal refuse piles and solfatara fields. Species of the genus Thermoplasma do not possess a rigid cell wall, but are only delimited by a plasma membrane. Many macromolecular assemblies from Thermoplasma, primarily proteases and chaperones, have been pivotal in elucidating the structure and function of their more complex eukaryotic homologues. Our interest in protein folding and degradation led us to seek a more complete representation of the proteins involved in these pathways by determining the genome sequence of the organism. Here we have sequenced the 1,564,905-base-pair genome in just 7,855 sequencing reactions by using a new strategy. The 1,509 open reading frames identify Thermoplasma as a typical euryarchaeon with a substantial complement of bacteria-related genes; however, evidence indicates that there has been much lateral gene transfer between Thermoplasma and Sulfolobus solfataricus, a phylogenetically distant crenarchaeon inhabiting the same environment. At least 252 open reading frames, including a complete protein degradation pathway and various transport proteins, resemble Sulfolobus proteins most closely.     

Generation of beta-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli

High-level expression of many eukaryotic genes has proved difficult to achieve even when a strong promoter and the ribosome binding sequence from highly expressed Escherichia coli genes have been placed in front of the coding sequences. To overcome this problem, many eukaryotic proteins have been efficiently produced as hybrids after fusion of their genes with a coding sequence of E. coli genes. However, such hybrid proteins are not suitable for functional studies or clinical use unless the authentic protein sequence can be released by specific cleavage. Here, we have inserted the sequence Ile-Glu-Gly-Arg between the 31 amino-terminal residues of lambda cII protein and Val 1 of human beta-globin, and produced this hybrid in high yield in E. coli. We then cleaved the hybrid specifically at the single arginine, using blood coagulation factor Xa and thus liberated the authentic beta-globin chain. As factor Xa is specific for the tetrapeptide Ile-Glu-Gly-Arg, which is rare in protein sequences, our expression/cleavage system is applicable to the efficient production of many eukaryotic proteins.     

A homologue of the bacterial heat-shock gene DnaJ that alters protein sorting in yeast

Heat-shock proteins have been implicated in assembly of protein complexes, correct protein folding and uptake of proteins into organelles. In Escherichia coli, the heat-shock protein DnaJ and the Hsp70 homologue, DnaK, act together to disassemble a protein complex involved in bacteriophage lambda replication. We report the identification of SCJ1, a gene in the yeast Saccharomyces cerevisiae that encodes a homologue of the bacterial DnaJ protein. SCJ1 was identified by a genetic screen in which increased expression of candidate genes results in missorting of a nuclear-targeted test protein. The predicted amino-acid sequence of SCJ1 is 37% identical to the entire E. coli DnaJ protein. Hybridization experiments indicate that there is a family of yeast genes related to SCJ1. These findings suggest that the Hsp70 DnaK-DnaJ interaction is general to eukaryotes.     

棉铃虫泛素基因的克隆及序列分析

泛素介导的泛素-蛋白酶体通路(Ubiquitin-proteasome pathway,UPP)是真核细胞内依赖ATP的非溶酶体蛋白质降解途径,该途径对细胞内蛋白的选择性降解起着重要作用.设计一对简并引物,从棉铃虫Helicoverpa armigera卵巢细胞Ha831中克隆了泛素基因的编码区,GenBank登录号AY456195.序列分析表明,该编码区的长度为228bp,编码76个氨基酸、其编码蛋白的相对分子质量为8 560,等电点为6.56.同源性比较发现,棉铃虫泛素基因与其他真核生物泛素基因在氨基酸水平上具有96%以上的相似性,而与棉铃虫核多角体病毒泛素的相似性为76%,所有已知的泛素关键功能位点在该泛素蛋白中均保守存在.     

Xenopus oocytes can secrete bacterial beta-lactamase

Most secretory proteins are synthesized as precursor polypeptides carrying N-terminal, hydrophobic sequences which, by means of a signal recognition particle (SRP), trigger the membrane transfer of the polypeptide and are subsequently cleaved off. The signal sequences appear to be interchangeable between prokaryotes and eukaryotes. In bacteria, secretion only involves the crossing of a membrane, whereas in eukaryotes the secretory process can be separated into two distinct phases: translocation across the membrane of the rough endoplasmic reticulum and subsequent intraluminal transport by processes involving vesicle budding and fusion. Since secretory proteins must be distinguished from other soluble proteins destined for various sites in the reticular system, it is conceivable that eukaryotic secretory proteins possess additional markers distinct from the signal peptide to guide the polypeptide after its transfer through the membrane. Proteins are secreted at different rates from a eukaryotic cell, suggesting a role in intracellular transport for receptors with differing affinities for some topogenic features in secretory proteins. We have tested this possibility by introducing into the lumen of eukaryotic rough endoplasmic reticulum a prokaryotic protein which, by virtue of its origin, had not been adapted to the eukaryotic secretory pathway. We reasoned that secretion of the bacterial protein would indicate that after membrane transfer no topogenic signal(s) and corresponding recognition system(s) are required. We report here that this is indeed the case.