Cloning and expression of DnaJ homolog in carrot somatic embryo

As the co-chaperone of DnaK/Hsp70 protein, DnaJ/Hsp40 protein influences the synthesis and assembly of the protein complex by regulating ATPase activity of DnaK/Hsp70 protein. By employing the modified method of cDNA representational difference analysis, a homologous fragment of DnaJ was isolated from the deregulated carrot somatic embryos, and it was further used as the probe to screen the cDNA library of carrot somatic embryo deregulated for 12 h. As the result, DcJ1 gene, the homologous gene of DnaJ, was isolated from carrot. Sequence analysis showed that its coding region is 1257 bp, which codes 418 amino acids and comprises 3 highly-conserved characteristic domains. Southern blot analysis suggested that the DcJ1 gene seems to be a single copy in the genome, while Northern blot result indicated that DcJ1 expresses only in roots and its degree of expression changes obviously with the regulation-deregulation process. These results suggest that DcJ1 is correlated with the early development of carrot somatic embryo radicle.     

Function of DnaJ and DnaK as chaperones in origin-specific DNA binding by RepA

Heat-shock proteins are normal constituents of cells whose synthesis is increased on exposure to various forms of stress. They are interesting because of their ubiquity and high conservation during evolution. Two families of heat-shock proteins, hsp60s and hsp70s, have been implicated in accelerating protein folding and oligomerization and also in maintaining proteins in an unfolded state, thus facilitating membrane transport. The Escherichia coli hsp70 analogue, DnaK, and two other heat-shock proteins, DnaJ and GrpE, are required for cell viability at high temperatures and are involved in DNA replication of phage lambda and plasmids P1 and F. These three proteins are involved in replication in vitro of P1 DNA along with many host replication proteins and the P1 RepA initiator protein. RepA exists in a stable protein complex with DnaJ containing a dimer each of RepA and DnaJ. We report here that DnaK and DnaJ mediate an alteration in the P1 initiator protein, rendering it much more active for oriP1 DNA binding.     

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.     

Localization and functionality of microsporidian iron-sulphur cluster assembly proteins

Microsporidia are highly specialized obligate intracellular parasites of other eukaryotes (including humans) that show extreme reduction at the molecular, cellular and biochemical level. Although microsporidia have long been considered as early branching eukaryotes that lack mitochondria, they have recently been shown to contain a tiny mitochondrial remnant called a mitosome. The function of the mitosome is unknown, because microsporidians lack the genes for canonical mitochondrial functions, such as aerobic respiration and haem biosynthesis. However, microsporidial genomes encode several components of the mitochondrial iron-sulphur (Fe-S) cluster assembly machinery. Here we provide experimental insights into the metabolic function and localization of these proteins. We cloned, functionally characterized and localized homologues of several central mitochondrial Fe-S cluster assembly components for the microsporidians Encephalitozoon cuniculi and Trachipleistophora hominis. Several microsporidial proteins can functionally replace their yeast counterparts in Fe-S protein biogenesis. In E. cuniculi, the iron (frataxin) and sulphur (cysteine desulphurase, Nfs1) donors and the scaffold protein (Isu1) co-localize with mitochondrial Hsp70 to the mitosome, consistent with it being the functional site for Fe-S cluster biosynthesis. In T. hominis, mitochondrial Hsp70 and the essential sulphur donor (Nfs1) are still in the mitosome, but surprisingly the main pools of Isu1 and frataxin are cytosolic, creating a conundrum of how these key components of Fe-S cluster biosynthesis coordinate their function. Together, our studies identify the essential biosynthetic process of Fe-S protein assembly as a key function of microsporidian mitosomes.     

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.     

Characterization of the yeast HSP60 gene coding for a mitochondrial assembly factor

The hsp60 protein isolated from the protozoan Tetrahymena thermophila is induced in response to heat stress and is a member of an immunologically conserved family represented in Escherichia coli and in mitochondria of plants and animals. We report here the cloning and characterization of a nuclear gene, HSP60, which codes for the hsp60 homologue from the yeast Saccharomyces cerevisiae. Nucleotide sequence analysis revealed that yeast hsp60 is related to the groEL protein of E. coli and the RUBISCO-binding protein (RBP) of chloroplasts. HSP60 was found to be the genetic locus of the conditional-lethal mutation described by Cheng et al., which at non-permissive temperature is defective in the assembly of several different multisubunit complexes in mitochondria. These data are consistent with the hypothesis that the groEL-related proteins serve an evolutionarily conserved function as accessory factors facilitating the folding and/or association of individual subunits of multimeric protein complexes.     

水稻Hsp70基因第一个内含子中snoRNA基因簇的保守性及分布特点

对水稻、野生稻和茭白Ｈｓｐ７０基因第一个内含子进行ＰＣＲ分析及部分序列测定，分析结果表明植物Ｈｓｐ７０基因内含子编码多个ｓｎｏＲＮＡ的基因组织具有一定的保守性和分布范围，揭示了植物内含子编码的ｓｎｏＲＮＡ在进化过程中具有比脊椎动物和酵母更加丰富的多样性和移动性．     

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.     

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.

Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers. Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA. It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated. Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts. Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase eta. This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. We found that all XP-V cells examined carry mutations in their DNA polymerase eta gene. Recombinant human DNA polymerase eta corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA. Together, these results indicate that DNA polymerase eta could be the XPV gene product.     

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.     

拟南芥 BAH1 与大肠杆菌热胁迫耐受的关系分析

拟南芥BAH1含有保守的C3H4型RING结构域,与DnaJ锌指结构类似.利用原核表达纯化的BAH1进行体外泛素化实验证明了BAH1具有E3连接酶活性.然后通过表型回复实验发现BAH1融合J-domain结构域后(JdBAH1)和DnaJ一样能明显弥补danJ突变株MF634的热敏表型,在43℃存活;而转入突变锌指结构的JdBAH1C231S,C234S,C276S,C279S(JdBAH1△Zn1/2)菌株在43℃高温条件下不能存活,说明BAH1在大肠杆菌内具有类似DnaJ锌指结构的功能.因此,BAH1在E.coli中的功能有可能与DnaJ相似,通过锌指结构参与了DnaK/DnaJ伴侣系统发挥功能.     

An essential role for a phospholipid transfer protein in yeast Golgi function

Progression of proteins through the secretory pathway of eukaryotic cells involves a continuous rearrangement of macromolecular structures made up of proteins and phospholipids. The protein SEC14p is essential for transport of proteins from the yeast Golgi complex. Independent characterization of the SEC14 gene and the PIT1 gene, which encodes a phosphatidylinositol/phosphatidylcholine transfer protein in yeast, indicated that these two genes are identical. Phospholipid transfer proteins are a class of cytosolic proteins that are ubiquitous among eukaryotic cells and are distinguished by their ability to catalyse the exchange of phospholipids between membranes in vitro. We show here that the SEC14 and PIT1 genes are indeed identical and that the growth phenotype of a sec14-1ts mutant extends to the inability of its transfer protein to effect phospholipid transfer in vitro. These results therefore establish for the first time an in vivo function for a phospholipid transfer protein, namely a role in the compartment-specific stimulation of protein secretion.     

Evolutionary capacitance as a general feature of complex gene networks

An evolutionary capacitor buffers genotypic variation under normal conditions, thereby promoting the accumulation of hidden polymorphism. But it occasionally fails, thereby revealing this variation phenotypically. The principal example of an evolutionary capacitor is Hsp90, a molecular chaperone that targets an important set of signal transduction proteins. Experiments in Drosophila and Arabidopsis have demonstrated three key properties of Hsp90: (1) it suppresses phenotypic variation under normal conditions and releases this variation when functionally compromised; (2) its function is overwhelmed by environmental stress; and (3) it exerts pleiotropic effects on key developmental processes. But whether these properties necessarily make Hsp90 a significant and unique facilitator of adaptation is unclear. Here we use numerical simulations of complex gene networks, as well as genome-scale expression data from yeast single-gene deletion strains, to present a mechanism that extends the scope of evolutionary capacitance beyond the action of Hsp90 alone. We illustrate that most, and perhaps all, genes reveal phenotypic variation when functionally compromised, and that the availability of loss-of-function mutations accelerates adaptation to a new optimum phenotype. However, this effect does not require the mutations to be conditional on the environment. Thus, there might exist a large class of evolutionary capacitors whose effects on phenotypic variation complement the systemic, environment-induced effects of Hsp90.     

A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol

Elimination of misfolded proteins from the endoplasmic reticulum (ER) by retro-translocation is an important physiological adaptation to ER stress. This process requires recognition of a substrate in the ER lumen and its subsequent movement through the membrane by the cytosolic p97 ATPase. Here we identify a p97-interacting membrane protein complex in the mammalian ER that links these two events. The central component of the complex, Derlin-1, is a homologue of Der1, a yeast protein whose inactivation prevents the elimination of misfolded luminal ER proteins. Derlin-1 associates with different substrates as they move through the membrane, and inactivation of Derlin-1 in C. elegans causes ER stress. Derlin-1 interacts with US11, a virally encoded ER protein that specifically targets MHC class I heavy chains for export from the ER, as well as with VIMP, a novel membrane protein that recruits the p97 ATPase and its cofactor.     

A membrane protein required for dislocation of misfolded proteins from the ER

After insertion into the endoplasmic reticulum (ER), proteins that fail to fold there are destroyed. Through a process termed dislocation such misfolded proteins arrive in the cytosol, where ubiquitination, deglycosylation and finally proteasomal proteolysis dispense with the unwanted polypeptides. The machinery involved in the extraction of misfolded proteins from the ER is poorly defined. The human cytomegalovirus-encoded glycoproteins US2 and US11 catalyse the dislocation of class I major histocompatibility complex (MHC) products, resulting in their rapid degradation. Here we show that US11 uses its transmembrane domain to recruit class I MHC products to a human homologue of yeast Der1p, a protein essential for the degradation of a subset of misfolded ER proteins. We show that this protein, Derlin-1, is essential for the degradation of class I MHC molecules catalysed by US11, but not by US2. We conclude that Derlin-1 is an important factor for the extraction of certain aberrantly folded proteins from the mammalian ER.     

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.