Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria

A nuclear encoded mitochondrial heat-shock protein hsp60 is required for the assembly into oligomeric complexes of proteins imported into the mitochondrial matrix. hsp60 is a member of the 'chaperonin' class of protein factors, which include the Escherichia coli groEL protein and the Rubisco subunit-binding protein of chloroplasts.     

栽培和野生大豆核酮糖-1,5-二磷酸羧化酶的结晶及其大小亚基等电聚焦分析

栽培大豆和野生大豆叶片的蛋白抽提液经硫酸铵盐析、Ｓｅｐｈａｄｅｘ Ｇ－５０柱层析以及浓缩透析等步骤,分别得到核酮糖－１,５－二磷酸羟化酶（简称Ｒｕｂｉｓｃｏ）的结晶．经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ酶标抗体技术鉴定证实为Ｒｕｂｉｓｃｏ．用ＩＥＦ－ＰＡＧＥ方法分析这２种不同进化类型大豆的Ｒｕｂｉｓｃｏ 大小亚基,发现其具有高度同源性     

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.     

Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the fixation of atmospheric CO(2) in photosynthesis, but tends to form inactive complexes with its substrate ribulose 1,5-bisphosphate (RuBP). In plants, Rubisco is reactivated by the AAA(+) (ATPases associated with various cellular activities) protein Rubisco activase (Rca), but no such protein is known for the Rubisco of red algae. Here we identify the protein CbbX as an activase of red-type Rubisco. The 3.0-? crystal structure of unassembled CbbX from Rhodobacter sphaeroides revealed an AAA(+) protein architecture. Electron microscopy and biochemical analysis showed that ATP and RuBP must bind to convert CbbX into functionally active, hexameric rings. The CbbX ATPase is strongly stimulated by RuBP and Rubisco. Mutational analysis suggests that CbbX functions by transiently pulling the carboxy-terminal peptide of the Rubisco large subunit into the hexamer pore, resulting in the release of the inhibitory RuBP. Understanding Rubisco activation may facilitate efforts to improve CO(2) uptake and biomass production by photosynthetic organisms.     

Characteristics of photosynthesis in rice plants transformed with an antisense Rubisco activase gene

Transgenic rice plants with an antisense gene inserted via Agrobacterium tumefaciens were used to explore the impact of the reduction of Rubisco activase (RCA) on Rubisco and photosynthesis. In this study, transformants containing 15% to 35% wild type Rubisco activase were selected, which could survive in ambient CO2 concentration but grew slowly compared with wild type controls. Gas exchange measurements indicated that the rate of photosynthesis decreased sig-nificantly, while stomatal conductance and transpiration rate did not change; and that the intercellular CO2 concentration even increased. Rubisco determination showed that these plants had approximately twice as much Rubisco as the wild types,although they showed 70% lower rate of photosynthesis, whichRubsico activase and/or the reduction in carbamylation.was likely an acclimation response to the reduction in Rubsico activase and/or the reduction in carbamylation.     

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.     

XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6 (ref. 1), a protein which allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. We have identified a protein that is required for the assembly of pre-replication complexes, in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Here we show, using Xenopus in vitro systems, that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase.     

Nucleosome assembly

Histones and DNA can spontaneously associate to form the nucleosome subunits of eukaryotic chromatin, but two proteins which occur in the eukaryotic nucleus can facilitate nucleosome assembly and greatly extend the conditions which permit assembly to occur. Recently several laboratories have reported new experimental systems which help to analyse, reconsruct and exploit the cellular mechanisms of chromatin assembly.     

An eIF-4A-like protein is a suppressor of an Escherichia coli mutant defective in 50S ribosomal subunit assembly

The assembly of ribosomes in bacterial cells is a complex process that remains poorly characterized. The in vitro assembly of active ribosomal subunits from purified RNA and protein components indicates that all of the information for proper assembly resides in the primary sequences of these macromolecules. On the other hand, the in vitro requirement of unphysiological heating steps suggests that this pathway may not accurately reflect the in vivo pathway, and that other proteins may be required. One approach to identify any additional proteins is to isolate second-site revertants of mutants defective in ribosome assembly. Ribosomal protein L24 is essential in the assembly of 50S subunits. We have identified an Escherichia coli gene, srmB, that, when expressed at high copy number, can suppress the effect of a temperature-sensitive lethal mutation in L24. The SrmB amino-acid sequence has sequence identity with mouse translation initiation factor eIF-4A and with the human nuclear protein, p68. The purified SrmB protein is a nucleic acid-dependent ATPase, like eIF-4A, but can also bind RNA in the absence of ATP and other auxiliary protein factors. The RNA dependent ATPase activity of SrmB suggests that like, eIF-4A, it could be involved in specific alterations of RNA secondary structure.     

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.     

细茎大豆（G.gracilis）rbcS基因结构与分子进化分析

从细茎大豆的嫩叶中提取总ＤＮＡ，用ＰＣＲ方法扩增得到包含完整编码区的ｒｂｃＳ基因并将其克隆到ｐＢＬＵＥＳＣＲＩＰＴ载体中。完成全基因１０８９个核苷酸测序后，运用ＰＣＧＥＮＥ进行顺序编辑和同源比较，并应用ＭＥＧＡ１．０２１软件中的Ｎｅｉｇｈｂｏｒ－ｊｏｉｎｉｎｇ方面画出Ｒｕｂｉｓｃｏ小亚基仓的系统进化树。     

Centriole assembly in Caenorhabditis elegans

Centrioles are necessary for flagella and cilia formation, cytokinesis, cell-cycle control and centrosome organization/spindle assembly. They duplicate once per cell cycle, but the mechanisms underlying their duplication remain unclear. Here we show using electron tomography of staged C. elegans one-cell embryos that daughter centriole assembly begins with the formation and elongation of a central tube followed by the peripheral assembly of nine singlet microtubules. Tube formation and elongation is dependent on the SAS-5 and SAS-6 proteins, whereas the assembly of singlet microtubules onto the central tube depends on SAS-4. We further show that centriole assembly is triggered by an upstream signal mediated by SPD-2 and ZYG-1. These results define a structural pathway for the assembly of a daughter centriole and should have general relevance for future studies on centriole assembly in other organisms.     

Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly

Rapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. How the Escherichia coli 16S ribosomal RNA and the 20 proteins that make up the 30S ribosomal subunit can assemble correctly in a few minutes remains a challenging problem, partly because of the lack of real-time data on the earliest stages of assembly. By providing snapshots of individual RNA and protein interactions as they emerge in real time, here we show that 30S assembly nucleates concurrently from different points along the rRNA. Time-resolved hydroxyl radical footprinting was used to map changes in the structure of the rRNA within 20 milliseconds after the addition of total 30S proteins. Helical junctions in each domain fold within 100 ms. In contrast, interactions surrounding the decoding site and between the 5', the central and the 3' domains require 2-200 seconds to form. Unexpectedly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. Although early steps in assembly are linked to intrinsically stable rRNA structure, later steps correspond to regions of induced fit between the proteins and the rRNA.     

人类乙型肝炎样病毒核心蛋白中第7位疏水性氨基酸重复肚段区域的突变可以抑制病毒核衣壳的组装

人类乙型肝炎病毒的核衣壳由核心蛋白的二聚体所组成．但是,核心蛋白亚单位与亚单位之间相互作用的机制至今尚不清楚．研究发现,在人类乙型肝炎样病毒──土拨鼠肝炎病毒（WHV）核心蛋白的氨基端,存在着4个保守的疏水氨基酸残基（氨基酸位置101～102）．它们分别是亮氨酸101,亮氨酸108,缬氨酸115和苯丙氨酸122．这4个疏水氨基酸残基以每隔6个氨基酸残基而重复出现1次．它们被称为“第7位疏水性氨基酸重复肽段（hhr）”．由于蛋白质中的疏水键往往在蛋白质的相互作用中起重要作用,因此就在培养细胞系统中研究WHV核心蛋白的hhr区域在…     

Poly(ADP-ribose) is required for spindle assembly and structure

The mitotic spindle is typically thought of as an array of microtubules, microtubule-associated proteins and motors that self-organizes to align and segregate chromosomes. The major spindle components consist of proteins and DNA, the primary structural elements of the spindle. Other macromolecules including RNA and lipids also associate with spindles, but their spindle function, if any, is unknown. Poly(ADP-ribose) (PAR) is a large, branched, negatively charged polymeric macromolecule whose polymerization onto acceptor proteins is catalysed by a family of poly(ADP-ribose) polymerases (PARPs). Several PARPs localize to the spindle in vertebrate cells, suggesting that PARPs and/or PAR have a role in spindle function. Here we show that PAR is enriched in the spindle and is required for spindle function--PAR hydrolysis or perturbation leads to rapid disruption of spindle structure, and hydrolysis during spindle assembly blocks the formation of bipolar spindles. PAR exhibits localization dynamics that differ from known spindle proteins and are consistent with a low rate of turnover in the spindle. Thus, PAR is a non-proteinaceous, non-chromosomal component of the spindle required for bipolar spindle assembly and function.     

Homologous plant and bacterial proteins chaperone oligomeric protein assembly

An abundant chloroplast protein is implicated in the assembly of the oligomeric enzyme ribulose bisphosphate carboxylase-oxygenase, which catalyses photosynthetic CO2-fixation in higher plants. The product of the Escherichia coli groEL gene is essential for cell viability and is required for the assembly of bacteriophage capsids. Sequencing of the groEL gene and the complementary cDNA encoding the chloroplast protein has revealed that these proteins are evolutionary homologues which we term 'chaperonins'. Chaperonins comprise a class of molecular chaperones that are found in chloroplasts, mitochondria and prokaryotes. Assisted post-translational assembly of oligomeric protein structures is emerging as a general cellular phenomenon.     

Protein folding in the cell.

In the cell, as in vitro, the final conformation of a protein is determined by its amino-acid sequence. But whereas some isolated proteins can be denatured and refolded in vitro in the absence of other macromolecular cellular components, folding and assembly of polypeptides in vivo involves other proteins, many of which belong to families that have been highly conserved during evolution.     

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.     

H_2O_2诱导小麦叶片光合功能衰退的研究

采用不同浓度的H2O2处理小麦,研究H2O2对小麦叶片光合功能的影响.结果表明:1 mmol.L-1H2O2对小麦叶片光合作用基本无影响,10、100、200 mmol.L-1H2O2对小麦离体和连体叶片的光合作用均有不同程度的抑制作用,表现为光合速率、叶绿素含量下降.叶绿体超微结构显示10 mmol.L-1H2O2对其有影响,200 mmol.L-1H2O2处理后基粒明显破坏,类囊体膜无序.核酮糖-1,5-二磷酸羧化酶加氧酶(Rubisco)大小亚基和羧化活性在20 mmol.L-1H2O2逆境下变化小,100 mmol.L-1H2O2处理,Rubisco大小亚基降解明显,羧化活性微弱.说明H2O2诱导小麦叶片光合功能衰退.