Nanomechanical basis of selective gating by the nuclear pore complex

The nuclear pore complex regulates cargo transport between the cytoplasm and the nucleus. We set out to correlate the governing biochemical interactions to the nanoscopic responses of the phenylalanineglycine (FG)-rich nucleoporin domains, which are involved in attenuating or promoting cargo translocation. We found that binding interactions with the transport receptor karyopherin-beta1 caused the FG domains of the human nucleoporin Nup153 to collapse into compact molecular conformations. This effect was reversed by the action of Ran guanosine triphosphate, which returned the FG domains into a polymer brush-like, entropic barrier conformation. Similar effects were observed in Xenopus oocyte nuclei in situ. Thus, the reversible collapse of the FG domains may play an important role in regulating nucleocytoplasmic transport.     

FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties

Nuclear pore complexes permit rapid passage of cargoes bound to nuclear transport receptors, but otherwise suppress nucleocytoplasmic fluxes of inert macromolecules >/=30 kilodaltons. To explain this selectivity, a sieve structure of the permeability barrier has been proposed that is created through reversible cross-linking between Phe and Gly (FG)-rich nucleoporin repeats. According to this model, nuclear transport receptors overcome the size limit of the sieve and catalyze their own nuclear pore-passage by a competitive disruption of adjacent inter-repeat contacts, which transiently opens adjoining meshes. Here, we found that phenylalanine-mediated inter-repeat interactions indeed cross-link FG-repeat domains into elastic and reversible hydrogels. Furthermore, we obtained evidence that such hydrogel formation is required for viability in yeast.     

Ceramide triggers budding of exosome vesicles into multivesicular endosomes

Intraluminal vesicles of multivesicular endosomes are either sorted for cargo degradation into lysosomes or secreted as exosomes into the extracellular milieu. The mechanisms underlying the sorting of membrane into the different populations of intraluminal vesicles are unknown. Here, we find that cargo is segregated into distinct subdomains on the endosomal membrane and that the transfer of exosome-associated domains into the lumen of the endosome did not depend on the function of the ESCRT (endosomal sorting complex required for transport) machinery, but required the sphingolipid ceramide. Purified exosomes were enriched in ceramide, and the release of exosomes was reduced after the inhibition of neutral sphingomyelinases. These results establish a pathway in intraendosomal membrane transport and exosome formation.     

The structure of the kinesin-1 motor-tail complex reveals the mechanism of autoinhibition

When not transporting cargo, kinesin-1 is autoinhibited by binding of a tail region to the motor domains, but the mechanism of inhibition is unclear. We report the crystal structure of a motor domain dimer in complex with its tail domain at 2.2 angstroms and compare it with a structure of the motor domain alone at 2.7 angstroms. These structures indicate that neither an induced conformational change nor steric blocking is the cause of inhibition. Instead, the tail cross-links the motor domains at a second position, in addition to the coiled coil. This "double lockdown," by cross-linking at two positions, prevents the movement of the motor domains that is needed to undock the neck linker and release adenosine diphosphate. This autoinhibition mechanism could extend to some other kinesins.     

Role of Rab9 GTPase in facilitating receptor recruitment by TIP47

Mannose 6-phosphate receptors (MPRs) deliver lysosomal hydrolases from the Golgi to endosomes and then return to the Golgi complex. TIP47 recognizes the cytoplasmic domains of MPRs and is required for endosome-to-Golgi transport. Here we show that TIP47 also bound directly to the Rab9 guanosine triphosphatase (GTPase) in its active, GTP-bound conformation. Moreover, Rab9 increased the affinity of TIP47 for its cargo. A functional Rab9 binding site was required for TIP47 stimulation of MPR transport in vivo. Thus, a cytosolic cargo selection device may be selectively recruited onto a specific organelle, and vesicle budding might be coupled to the presence of an active Rab GTPase.     

Changes in sodium channel gating produced by point mutations in a cytoplasmic linker

Voltage-gated sodium channels are transmembrane proteins of approximately 2000 amino acids and consist of four homologous domains (I through IV). In current topographical models, domains III and IV are linked by a highly conserved cytoplasmic sequence of amino acids. Disruptions of the III-IV linker by cleavage or antibody binding slow inactivation, the depolarization-induced closed state characteristic of sodium channels. This linker might be the positively charged "ball" that is thought to cause inactivation by occluding the open channel. Therefore, groups of two or three contiguous lysines were neutralized or a glutamate was substituted for an arginine in the III-IV linker of type III rat brain sodium channels. In all cases, inactivation occurred more rapidly rather than more slowly, contrary to predictions. Furthermore, activation was delayed in the arginine to glutamate mutation. Hence, the III-IV linker does not simply act as a charged blocker of the channel but instead influences all aspects of sodium channel gating.     

Transmembrane molecular pump activity of Niemann-Pick C1 protein

Niemann-Pick C1 (NPC1) disease is characterized by cholesterol accumulation in lysosomes and aberrant feedback regulation of cellular cholesterol homeostasis. We provide evidence that the NPC1 protein has homology with the resistance-nodulation-division (RND) family of prokaryotic permeases and may normally function as a transmembrane efflux pump. Studies of acriflavine loading in normal and NPC1 fibroblasts indicated that NPC1 uses a proton motive force to remove accumulated acriflavine from the endosomal/lysosomal system. Expression of NPC1 in Escherichia coli (i) facilitated the transport of acriflavine across the plasma membrane, causing cytosolic accumulation, and (ii) resulted in transport of oleic acid but not cholesterol or cholesterol-oleate across the plasma membrane. These studies establish NPC1 as a eukaryotic member of the RND permease family.     

Structural basis of trans-inhibition in a molybdate/tungstate ABC transporter

Transport across cellular membranes is an essential process that is catalyzed by diverse membrane transport proteins. The turnover rates of certain transporters are inhibited by their substrates in a process termed trans-inhibition, whose structural basis is poorly understood. We present the crystal structure of a molybdate/tungstate ABC transporter (ModBC) from Methanosarcina acetivorans in a trans-inhibited state. The regulatory domains of the nucleotide-binding subunits are in close contact and provide two oxyanion binding pockets at the shared interface. By specifically binding to these pockets, molybdate or tungstate prevent adenosine triphosphatase activity and lock the transporter in an inward-facing conformation, with the catalytic motifs of the nucleotide-binding domains separated. This allosteric effect prevents the transporter from switching between the inward-facing and the outward-facing states, thus interfering with the alternating access and release mechanism.     

Role of importin-beta in coupling Ran to downstream targets in microtubule assembly

The guanosine triphosphatase Ran stimulates assembly of microtubule asters and spindles in mitotic Xenopus egg extracts. A carboxyl-terminal region of the nuclear-mitotic apparatus protein (NuMA), a nuclear protein required for organizing mitotic spindle poles, mimics Ran's ability to induce asters. This NuMA fragment also specifically interacted with the nuclear transport factor, importin-beta. We show that importin-beta is an inhibitor of microtubule aster assembly in Xenopus egg extracts and that Ran regulates the interaction between importin-beta and NuMA. Importin-beta therefore links NuMA to regulation by Ran. This suggests that similar mechanisms regulate nuclear import during interphase and spindle assembly during mitosis.     

Structural evidence for common ancestry of the nuclear pore complex and vesicle coats

Nuclear pore complexes (NPCs) facilitate nucleocytoplasmic transport. These massive assemblies comprise an eightfold symmetric scaffold of architectural proteins and central-channel phenylalanine-glycine-repeat proteins forming the transport barrier. We determined the nucleoporin 85 (Nup85)*Seh1 structure, a module in the heptameric Nup84 complex, at 3.5 angstroms resolution. Structural, biochemical, and genetic analyses position the Nup84 complex in two peripheral NPC rings. We establish a conserved tripartite element, the ancestral coatomer element ACE1, that reoccurs in several nucleoporins and vesicle coat proteins, providing structural evidence of coevolution from a common ancestor. We identified interactions that define the organization of the Nup84 complex on the basis of comparison with vesicle coats and confirmed the sites by mutagenesis. We propose that the NPC scaffold, like vesicle coats, is composed of polygons with vertices and edges forming a membrane-proximal lattice that provides docking sites for additional nucleoporins.     

Functional genomic analysis of RNA interference in C. elegans

RNA interference (RNAi) of target genes is triggered by double-stranded RNAs (dsRNAs) processed by conserved nucleases and accessory factors. To identify the genetic components required for RNAi, we performed a genome-wide screen using an engineered RNAi sensor strain of Caenorhabditis elegans. The RNAi screen identified 90 genes. These included Piwi/PAZ proteins, DEAH helicases, RNA binding/processing factors, chromatin-associated factors, DNA recombination proteins, nuclear import/export factors, and 11 known components of the RNAi machinery. We demonstrate that some of these genes are also required for germline and somatic transgene silencing. Moreover, the physical interactions among these potential RNAi factors suggest links to other RNA-dependent gene regulatory pathways.     

Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells

The signals that direct membrane proteins to the apical or basolateral plasma membrane domains of polarized epithelial cells are not known. Several of the class of proteins anchored in the membrane by glycosyl-phosphatidylinositol (GPI) are expressed on the apical surface of such cells. However, it is not known whether the mechanism of membrane anchorage or the polypeptide sequence provides the sorting information. The conversion of the normally basolateral vesicular stomatitis virus glycoprotein (VSV G) to a GPI-anchored protein led to its apical expression. Conversely, replacement of the GPI anchor of placental alkaline phosphatase with the transmembrane and cytoplasmic domains of VSV G shifted its expression from the apical to the basolateral surface. Thus, the mechanism of membrane anchorage can determine the sorting of proteins to the apical or basolateral surface, and the GPI anchor itself may provide an apical transport signal.     

Golgin tethers define subpopulations of COPI vesicles

Coiled-coil proteins of the golgin family have been implicated in intra-Golgi transport through tethering coat protein complex I (COPI) vesicles. The p115-golgin tether is the best studied, and here we characterize the golgin-84-CASP tether. The vesicles bound by this tether were strikingly different from those bound by the p115-golgin tether in that they lacked members of the p24 family of putative cargo receptors and contained enzymes instead of anterograde cargo. Microinjected golgin-84 or CASP also inhibited Golgi-enzyme transport to the endoplasmic reticulum, further implicating this tether in retrograde transport. These and other golgins may modulate the flow patterns within the Golgi stack.     

Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption

Dietary cholesterol consumption and intestinal cholesterol absorption contribute to plasma cholesterol levels, a risk factor for coronary heart disease. The molecular mechanism of sterol uptake from the lumen of the small intestine is poorly defined. We show that Niemann-Pick C1 Like 1(NPC1L1) protein plays a critical role in the absorption of intestinal cholesterol. NPC1L1 expression is enriched in the small intestine and is in the brush border membrane of enterocytes. Although otherwise phenotypically normal, NPC1L1-deficient mice exhibit a substantial reduction in absorbed cholesterol, which is unaffected by dietary supplementation of bile acids. Ezetimibe, a drug that inhibits cholesterol absorption, had no effect in NPC1L1 knockout mice, suggesting that NPC1L1 resides in an ezetimibe-sensitive pathway responsible for intestinal cholesterol absorption.     

苏云金芽孢杆菌cry1Ea基因的克隆及 生物信息学分析

不同来源的苏云金芽孢杆菌能产生多种多样的晶体(Cry)蛋白.基于这个特性,人们可以通过基因工程的手段向工程菌中转入编码多种Cry毒素的基因来控制虫害.通过DNA重组技术,从BtHZM2菌株中克隆出了cry1Ea基因,对其进行了生物信息学分析,同源比对结果表明.cry1Ea8基因的核苷酸序列与已知cry1Ea的同源性为99.77%～99.91%.对应的氨基酸序列同源性为99.49%～99.74%.对cry1Ea8基因的分析还揭示出了cry1Ea8及其编码蛋白的一些生物和理化性质.结构域预测表明,Cry1Ea8由3个结构域组成,其中N-末端螺旋状结构域与膜插入与孔隙形成有关,而第二和第三个结构域与受体的结合有关.该研究为转基因抗虫植物和微生物杀虫工程菌的构建提供了新的基因来源.     

NFAT binding and regulation of T cell activation by the cytoplasmic scaffolding Homer proteins

T cell receptor (TCR) and costimulatory receptor (CD28) signals cooperate in activating T cells, although understanding of how these pathways are themselves regulated is incomplete. We found that Homer2 and Homer3, members of the Homer family of cytoplasmic scaffolding proteins, are negative regulators of T cell activation. This is achieved through binding of nuclear factor of activated T cells (NFAT) and by competing with calcineurin. Homer-NFAT binding was also antagonized by active serine-threonine kinase AKT, thereby enhancing TCR signaling via calcineurin-dependent dephosphorylation of NFAT. This corresponded with changes in cytokine expression and an increase in effector-memory T cell populations in Homer-deficient mice, which also developed autoimmune-like pathology. These results demonstrate a further means by which costimulatory signals are regulated to control self-reactivity.     

植物WRKY转录因子研究进展

转录因子在植物的生长发育及其对外界环境的反应中起着重要的调控作用。典型的转录因子含有DNA结合域,转录调控域,寡聚化位点和核定位信号,转录因子通过这些结构域与相应的顺式元件相互作用调控基因的表达。WRKY转录因子是植物中特有的N-端含有WRKYGQK高度保守氨基酸序列的一种转录调控因子,它能够与(T)(T)TGAC(C/T)序列(W-box)发生特异性结合,从而调节启动子中含W-box元件的调节基因和/或功能基因的表达,参与植物的各种生理生化反应。文章主要论述近年来植物WRKY转录因子的相关研究进展。     

Chromatin docking and exchange activity enhancement of RCC1 by histones H2A and H2B

The Ran guanosine triphosphatase (GTPase) controls nucleocytoplasmic transport, mitotic spindle formation, and nuclear envelope assembly. These functions rely on the association of the Ran-specific exchange factor, RCC1 (regulator of chromosome condensation 1), with chromatin. We find that RCC1 binds directly to mononucleosomes and to histones H2A and H2B. RCC1 utilizes these histones to bind Xenopus sperm chromatin, and the binding of RCC1 to nucleosomes or histones stimulates the catalytic activity of RCC1. We propose that the docking of RCC1 to H2A/H2B establishes the polarity of the Ran-GTP gradient that drives nuclear envelope assembly, nuclear transport, and other nuclear events.     

Allosteric activation of a spring-loaded natriuretic peptide receptor dimer by hormone

Natriuretic peptides (NPs) are vasoactive cyclic-peptide hormones important in blood pressure regulation through interaction with natriuretic cell-surface receptors. We report the hormone-binding thermodynamics and crystal structures at 2.9 and 2.0 angstroms, respectively, of the extracellular domain of the unliganded human NP receptor (NPR-C) and its complex with CNP, a 22-amino acid NP. A single CNP molecule is bound in the interface of an NPR-C dimer, resulting in asymmetric interactions between the hormone and the symmetrically related receptors. Hormone binding induces a 20 angstrom closure between the membrane-proximal domains of the dimer. In each monomer, the opening of an interdomain cleft, which is tethered together by a linker peptide acting as a molecular spring, is likely a conserved allosteric trigger for intracellular signaling by the natriuretic receptor family.