A bifunctional bacterial protein links GDI displacement to Rab1 activation

Rab guanosine triphosphatases (GTPases) regulate vesicle trafficking in eukaryotic cells by reversibly associating with lipid membranes. Inactive Rab GTPases are maintained in the cytosol by binding to GDP-dissociation inhibitor (GDI). It is believed that specialized proteins are required to displace GDI from Rab GTPases before Rab activation by guanosine diphosphate-guanosine 5'-triphosphate (GDP-GTP) exchange factors (GEFs). Here, we found that SidM from Legionella pneumophila could act as both GEF and GDI-displacement factor (GDF) for Rab1. Rab1 released from GDI was inserted into liposomal membranes and was used as a substrate for SidM-mediated nucleotide exchange. During host cell infection, recruitment of Rab1 to Legionella-containing vacuoles depended on the GDF activity of SidM. Thus, GDF and GEF activity can be promoted by a single protein, and GDF activity can coordinate Rab1 recruitment from the GDI-bound pool.     

Magnaporthe Rab5 homologs show distinct functions in nerve growth factor(NGF)-mediated neurite outgrowth and cell differentiation

Nerve growth factor(NGF) binds to TrkA and forms a NGF/TrkA complex at the cell surface,which is then internalized into signaling endosomes and promotes neuronal survival and neurite outgrowth.The small GTPase Rab5 is reported to localize on the plasma membrane and early endosomes,regulating endosome fusion.It was reported that endogenous Rab5 function may need to be suppressed during NGF-induced neurite outgrowth and cell differentiation.Two Rab5 homologs(MoRab5A:MGG_06241 and MoRab5B:MGG_01185) were characterized from the rice blast fungus Magnaporthe oryzae,and MoRab5 B was identified as the Rab5 ortholog promoting early endosomal fusion,while MoRab5 A specialized to perform a non-redundant function in endosomal sorting.In this study,we examined whether MoRab5 A and MoRab5 B play different roles in NGF-induced neurite outgrowth and cell differentiation in PC12 cells(a rat pheochromocytoma cell line).Our data showed that MoRab5 B is a negative regulator of NGF signaling and neurite outgrowth in PC12 cells,similar to human Rab5(hRab5).MoRab5B:WT inhibits NGF signaling-dependent neurite outgrowth while the dominant-negative MoRab5 B mutant(MoRab5B:DN) enhances NGF signaling and neurite outgrowth.In contrast,MoRab5A:WT and MoRab5A:DN both significantly promote NGF-induced neurite outgrowth,indicating that MoRab5 B is more similar to hRab5 than MoRab5 A in the regulation of NGF signal transduction.     

Modulation of postendocytic sorting of G protein-coupled receptors

Recycling of the mu opioid receptor to the plasma membrane after endocytosis promotes rapid resensitization of signal transduction, whereas targeting of the delta opioid receptor (DOR) to lysosomes causes proteolytic down-regulation. We identified a protein that binds preferentially to the cytoplasmic tail of the DOR as a candidate heterotrimeric GTP-binding protein (G protein)-coupled receptor-associated sorting protein (GASP). Disruption of the DOR-GASP interaction through receptor mutation or overexpression of a dominant negative fragment of GASP inhibited receptor trafficking to lysosomes and promoted recycling. The GASP family of proteins may modulate lysosomal sorting and functional down-regulation of a variety of G protein-coupled receptors.     

Recycling endosomes supply AMPA receptors for LTP

Long-term potentiation (LTP) of synaptic strength, the most established cellular model of information storage in the brain, is expressed by an increase in the number of postsynaptic AMPA receptors. However, the source of AMPA receptors mobilized during LTP is unknown. We report that AMPA receptors are transported from recycling endosomes to the plasma membrane for LTP. Stimuli that triggered LTP promoted not only AMPA receptor insertion but also generalized recycling of cargo and membrane from endocytic compartments. Thus, recycling endosomes supply AMPA receptors for LTP and provide a mechanistic link between synaptic potentiation and membrane remodeling during synapse modification.     

Wnt5a control of cell polarity and directional movement by polarized redistribution of adhesion receptors

Mechanisms by which Wnt pathways integrate the organization of receptors, organelles, and cytoskeletal proteins to confer cell polarity and directional cell movement are incompletely understood. We show that acute responses to Wnt5a involve recruitment of actin, myosin IIB, Frizzled 3, and melanoma cell adhesion molecule into an intracellular structure in a melanoma cell line. In the presence of a chemokine gradient, this Wnt-mediated receptor-actin-myosin polarity (W-RAMP) structure accumulates asymmetrically at the cell periphery, where it triggers membrane contractility and nuclear movement in the direction of membrane retraction. The process requires endosome trafficking, is associated with multivesicular bodies, and is regulated by Wnt5a through the small guanosine triphosphatases Rab4 and RhoB. Thus, cell-autonomous mechanisms allow Wnt5a to control cell orientation, polarity, and directional movement in response to positional cues from chemokine gradients.     

A role for the phagosome in cytokine secretion

Membrane traffic in activated macrophages is required for two critical events in innate immunity: proinflammatory cytokine secretion and phagocytosis of pathogens. We found a joint trafficking pathway linking both actions, which may economize membrane transport and augment the immune response. Tumor necrosis factor alpha (TNFalpha) is trafficked from the Golgi to the recycling endosome (RE), where vesicle-associated membrane protein 3 mediates its delivery to the cell surface at the site of phagocytic cup formation. Fusion of the RE at the cup simultaneously allows rapid release of TNFalpha and expands the membrane for phagocytosis.     

Recombinant antibodies to the small GTPase Rab6 as conformation sensors

Here we report an approach, based on antibody phage display, to generate molecular conformation sensors. Recombinant antibodies specific to the guanosine triphosphate (GTP)-bound conformation of the small guanosine triphosphatase (GTPase) Rab6, a regulator of membrane traffic, were generated and used to locate Rab6.GTP in fixed cells, and, after green fluorescent protein (GFP) tagging and intracellular expression, to follow Rab6.GTP in vivo. Rab6 was in its GTP-bound conformation on the Golgi apparatus and transport intermediates, and the geometry of transport intermediates was modulated by Rab6 activity. More generally, the same approach could be applied to other molecules that can be locked in a particular conformation in vitro.     

Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models

Alpha-synuclein (alphaSyn) misfolding is associated with several devastating neurodegenerative disorders, including Parkinson's disease (PD). In yeast cells and in neurons alphaSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following alphaSyn expression in yeast was a block in endoplasmic reticulum (ER)-to-Golgi vesicular trafficking. In a genomewide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab guanosine triphosphatase Ypt1p, which associated with cytoplasmic alphaSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against alphaSyn-induced dopaminergic neuron loss in animal models of PD. Thus, synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.     

陆地棉GhRab7基因鉴定及酵母中盐胁迫响应分析

Rab参与细胞内囊泡运输的调节,是小G蛋白家族里成员数最多的一类亚家族蛋白,拟南芥、哺乳动物和酵母Rab蛋白功能已经研究的比较清楚,但棉花中Rab蛋白的功能还没有详细的报道。本试验从陆地棉TM-1数据库中检索到24个 Rab7基因,基于这些基因的染色体分布、编码氨基酸个数及分子质量大小,鉴定出19个潜在 GhRab7基因。随后,根据系统进化和序列保守性分析克隆一个 GhRab7基因,该基因编码一个207个氨基酸,分子质量为23.2 ku的Rab类小G蛋白。对其进行高盐条件下异源表达酵母生长抗性试验的结果显示,异源表达 GhRab7的酵母表现出盐胁迫敏感的特征。这些结果说明 GhRab7在棉花应对外界盐胁迫响应时可能发挥重要作用。     

小GTPase蛋白家族的研究进展

[目的]综述小G蛋白家族的研究进展,为更深入地解析小G蛋白的结构、功能及其分子作用模式提供参考。[方法]介绍了小G蛋白的结构、作用机制以及分类,着重综述了其各个亚家族的生物学功能。[结果]小G蛋白是真核生物的1个超基因家族,其成员超过100个,被分为Ras、Rho、Rab、Sar/Arf和Ran5个亚家族,分别参与细胞骨架组装、基因表达、细胞壁合成、囊泡运输、核质运输、微管形成、酵母出芽、纺锤体组装及细胞极性生长等诸多生命体活动过程。[结论]小G蛋白家族成员庞大,其分子作用机制及其调控的复杂网络有待进一步研究。     

Structure of Rab GDP-dissociation inhibitor in complex with prenylated YPT1 GTPase

Rab/Ypt guanosine triphosphatases (GTPases) represent a family of key membrane traffic regulators in eukaryotic cells whose function is governed by the guanosine diphosphate (GDP) dissociation inhibitor (RabGDI). Using a combination of chemical synthesis and protein engineering, we generated and crystallized the monoprenylated Ypt1:RabGDI complex. The structure of the complex was solved to 1.5 angstrom resolution and provides a structural basis for the ability of RabGDI to inhibit the release of nucleotide by Rab proteins. Isoprenoid binding requires a conformational change that opens a cavity in the hydrophobic core of its domain II. Analysis of the structure provides a molecular basis for understanding a RabGDI mutant that causes mental retardation in humans.     

Tumor promoters inhibit morphological differentiation in cultured mouse neuroblastoma cells

When added to mouse neuroblastoma cultures, the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) inhibits spontaneous neurite formation as well as that induced in response to serum deprivation, prostaglandin E1, 5-bromo-2'-deoxyuridine, and papaverine. Other tumor-promoting macrocyclic plant diterpenes also inhibit neurite formation, whereas nonpromoting diterpenes do not. Inhibition by TPA was reversible and was unrelated to toxicity.     

De-AMPylation of the small GTPase Rab1 by the pathogen Legionella pneumophila

The bacterial pathogen Legionella pneumophila exploits host cell vesicle transport by transiently manipulating the activity of the small guanosine triphosphatase (GTPase) Rab1. The effector protein SidM recruits Rab1 to the Legionella-containing vacuole (LCV), where it activates Rab1 and then AMPylates it by covalently adding adenosine monophosphate (AMP). L. pneumophila GTPase-activating protein LepB inactivates Rab1 before its removal from LCVs. Because LepB cannot bind AMPylated Rab1, the molecular events leading to Rab1 inactivation are unknown. We found that the effector protein SidD from L. pneumophila catalyzed AMP release from Rab1, generating de-AMPylated Rab1 accessible for inactivation by LepB. L. pneumophila mutants lacking SidD were defective for Rab1 removal from LCVs, identifying SidD as the missing link connecting the processes of early Rab1 accumulation and subsequent Rab1 removal during infection.     

胞外ATP对胡杨细胞囊泡运输的影响

在植物中,胞外ATP(eATP)作为一种重要的信号分子,调控植物的生长、发育及逆境响应等多种生命活动。这些植物细胞的生命活动与囊泡运输密切相关,然而,eATP是否对植物细胞的囊泡运输具有调控作用尚不清楚。本文利用能够标记囊泡运输的荧光染料FM1-43研究了eATP对胡杨细胞囊泡运输的影响。FM1-43染色结果显示,50 mol/L eATP对胡杨细胞胞吞作用不明显,而高浓度的eATP(200、400 mol/L)则会抑制其胞吞作用,其抑制作用与eATP浓度呈正相关。高浓度的eATP(200、400 mol/L)同样抑制胡杨细胞胞吐作用。而不同浓度的ADP(50、200、400 mol/L)处理则对胡杨细胞囊泡运输无明显影响。这说明高浓度eATP对胡杨细胞囊泡运输的抑制作用不是源于eATP的水解产物,而是来自于其本身的信号作用。药理学实验发现,ATP受体抑制剂PPADS(100 mol/L)能抑制高浓度eATP对胡杨细胞囊泡运输的限制作用,说明eATP是通过嘌呤受体介导的信号通路调控囊泡运输过程。但值得注意的是,嘌呤受体的另一种抑制剂suramin(100 mol/L)却对eATP的抑制作用不明显,因为suramin处理胡杨细胞后eATP(200 mol/L)仍能抑制囊泡运输。这说明在胡杨细胞中某一类与P2X同源的受体介导了高浓度eATP对囊泡运输的抑制作用。综上,eATP作为信号分子可调控胡杨细胞的囊泡运输,并且高浓度eATP对胡杨细胞的囊泡运输具有负调控作用。      

Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion

The guanosine triphosphatase Rab1 regulates the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus through interaction with effector molecules, but the molecular mechanisms by which this occurs are unknown. Here, the tethering factor p115 was shown to be a Rab1 effector that binds directly to activated Rab1. Rab1 recruited p115 to coat protein complex II (COPII) vesicles during budding from the endoplasmic reticulum, where it interacted with a select set of COPII vesicle-associated SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) to form a cis-SNARE complex that promotes targeting to the Golgi apparatus. We propose that Rab1-regulated assembly of functional effector-SNARE complexes defines a conserved molecular mechanism to coordinate recognition between subcellular compartments.     

Origin of mountains on Io by thrust faulting and large-scale mass movements

Voyager stereoimages of Euboea Montes, Io, indicate that this mountain formed when a large crustal block was uplifted 10.5 kilometers and tilted by approximately 6 degrees. Uplift triggered a massive slope failure on the northwest flank, forming one of the largest debris aprons in the solar system. This slope failure probably involved relatively unconsolidated layers totaling approximately 2 kilometers in thickness, overlying a rigid crust (or lithosphere) at least 11 kilometers thick. Mountain formation on Io may involve localized deep-rooted thrust faulting and block rotation, due to compression at depth induced during vertical recycling of Io's crust.     

Membrane recycling: vesiculation of the amoeba contractile vacuole at systole

Ultrastructural data on the protozoan Amoeba proteus support a model of membrane recycling. At systole the amoeba contractile vacuole fuses with the cell surface and expels its contents. Observations by electron microscopy indicate that, as the vacuole empties, its bounding membrane transforms into tiny (35 nanometers in diameter) vesicles, identical to the vesicles that segregate fluid and contribute to the diastolic vacuole.     

Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.     

Dissection of the mammalian midbody proteome reveals conserved cytokinesis mechanisms

Cytokinesis is the essential process that partitions cellular contents into daughter cells. To identify and characterize cytokinesis proteins rapidly, we used a functional proteomic and comparative genomic strategy. Midbodies were isolated from mammalian cells, proteins were identified by multidimensional protein identification technology (MudPIT), and protein function was assessed in Caenorhabditis elegans. Of 172 homologs disrupted by RNA interference, 58% displayed defects in cleavage furrow formation or completion, or germline cytokinesis. Functional dissection of the midbody demonstrated the importance of lipid rafts and vesicle trafficking pathways in cytokinesis, and the utilization of common membrane cytoskeletal components in diverse morphogenetic events in the cleavage furrow, the germline, and neurons.     

自动卸膜式残膜回收机捡拾齿和滚筒的优化

为了更好地解决破碎残膜难以回收以及残膜和土壤、根茬分离的问题,通过理论分析和试验对捡拾齿和滚筒进行优化设计。依据自动卸膜式残膜回收机的工作原理与作业要求,基于Adams运动仿真软件分析捡拾齿的运动特性,优化关键工作部件捡拾齿和滚筒的结构与参数,建立捡拾齿和滚筒的运动方程。通过捡拾率和卸膜率的正交试验和方差分析,确定影响捡拾率和卸膜率的因素和水平以及较优因素水平组合。试验结果表明,在滚筒直径800 mm,转动速度120 r/min,捡拾齿入土深度80 mm,轴向距离110 mm及周向个数为16的条件下,捡拾率最高;在卸膜辊转动速度为400 r/min及半径300 mm的条件下,卸膜率最高。该残膜回收机实现自动卸膜,不需停机卸膜,捡拾率达到89%,卸膜率达到95.4%,为新型残膜回收机的研究提供依据。