Surface expression and distribution of voltage-gated potassium channels in neurons (Review)

The last decade has witnessed an exponential increase in interest in one of the great mysteries of nerve cell biology: Specifically, how do neurons know where to place the ion channels that control their excitability? Many of the most important insights have been gleaned from studies on the voltage-gated potassium channels (Kvs) which underlie the shape, duration and frequency of action potentials. In this review, we gather recent evidence on the expression, trafficking and maintenance mechanisms which control the surface density of Kvs in different subcellular compartments of neurons and how these may be regulated to control cell excitability.     

Structural and dynamic properties of bacterial Type IV secretion systems (Review)

The type IV secretion systems (T4SS) are widely distributed among the Gram-negative and –positive bacteria. These systems mediate the transfer of DNA and protein substrates across the cell envelope to bacterial or eukaryotic cells generally through a process requiring direct cell-to-cell contact. Bacteria have evolved T4SS for survival during establishment of pathogenic or symbiotic relationships with eukaryotic hosts. The Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation machines serve as models for detailed mechanistic studies aimed at elucidating the nature of translocation signals, machine assembly pathways and architectures, and the dynamics of substrate translocation. The A. tumefaciens VirB/D4 T4SS are polar-localized organelles composed of a secretion channel and an extracellular T pilus. These T4SS are assembled from 11 or more subunits. whose membrane topologies, intersubunit contacts and, in some cases, 3-dimensional structures are known. Recently, powerful in vivo assays have identified C-terminal translocation signals, defined for the first time the translocation route for a DNA substrate through a type IV secretion channel, and supplied evidence that ATP energy consumption contributes to a late stage of machine morphogenesis. Together, these recent findings describe the mechanics of type IV secretion in unprecedented detail.     

Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP

Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB_313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF₃ support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF₃ through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.     

C-terminal truncation of a bovine B12 trafficking chaperone enhances the sensitivity of the glutathione-regulated thermostability

The human B₁₂ trafficking chaperone hCblC is well conserved in mammals and non-mammalian eukaryotes. However, the C-terminal ∼40 amino acids of hCblC vary significantly and are predicted to be deleted by alternative splicing of the encoding gene. In this study, we examined the thermostability of the bovine CblC truncated at the C-terminal variable region (t-bCblC) and its regulation by glutathione. t-bCblC is highly thermolabile (T_m = ∼42℃) similar to the full-length protein (f-bCblC). However, t-bCblC is stabilized to a greater extent than f-bCblC by binding of reduced glutathione (GSH) with increased sensitivity to GSH. In addition, binding of oxidized glutathione (GSSG) destabilizes t-bCblC to a greater extent and with increased sensitivity as compared to f-bCblC. These results indicate that t-bCblC is a more sensitive form to be regulated by glutathione than the full-length form of the protein. [BMB Reports 2013; 46(3): 169-174]     

植物细胞与细胞间物质转运及其对生长发育的调控

细胞与细胞之间的物质运输和信号传递对于多细胞生物的生长发育非常重要．一些内源的大分子物质如蛋白质、核酸或核酸蛋白质复合体可以选择性地通过植物特有的亚细胞结构即胞间连丝（PD）在细胞之间运输．小分子物质主要以被动的形式在细胞间通过PD进行扩散．PD对蛋白质和核酸的运输具有选择性,这种运输受到严格调控．大分子物质在细胞间的运输对植物的生长和发育有极其重要的调控作用．KN1,STM,SHR,TRY和WER等转录因子在细胞之间的转运对于维持植物的茎尖分生组织、根尖分生组织和表皮细胞功能起重要作用．另外,某些小分子RNA也能够在植物细胞间进行选择性运输,并通过在不同细胞中降解或抑制靶mRNA的翻译来调节植物组织的生长发育．     

NtGNL1基因通过调控囊泡运输影响烟草根毛的极性生长

极性生长是植物生长发育中的常见现象,但囊泡运输与极性生长的关系还未完全明确。花粉管和根毛是植物细胞极性生长的典型模式。早期研究显示NtGNL1(Nicotiana tabacum GNOM-LIKE 1)通过调节囊泡的后高尔基体转运来影响烟草的花粉管生长。本文以NtGNL1 RNAi转基因植株为材料,研究NtGNL1基因在根毛生长中的作用。结果表明,NtGNL1 RNAi转基因植株的根毛生长明显滞后于野生型,且其根毛出现膨大、弯折、扭曲等形态,与NtGNL1 RNAi转基因植株的花粉管异常形态类似。q RT-PCR检测RNAi转基因株系根毛中PIN1、PIN2、GL2、ROP6、RHD6基因的m RNA表达量,显示PIN2和GL2的表达量显著下调,PIN1、ROP6和RHD6的表达量变化不明显。FM4-64染色表明烟草根表皮细胞和根毛的囊泡分布都受到影响,即NtGNL1基因也影响根毛中的囊泡运输。BFA处理加剧了囊泡的聚集程度,提示根毛尖端还存在其它对BFA敏感并调控囊泡运输的基因。以上证据显示,NtGNL1基因通过囊泡运输途径影响烟草根毛的极性生长,NtGNL1基因的表达下调也影响了PIN2和GL2的表达,从而间接影响根毛的极性生长。     

Transgenic mice recapitulate the phenotypic heterogeneity of genetic prion diseases without developing prion infectivity: Role of intracellular PrP retention in neurotoxicity

Genetic prion diseases are degenerative brain disorders caused by mutations in the gene encoding the prion protein (PrP). Different PrP mutations cause different diseases, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) syndrome and fatal familial insomnia (FFI). The reason for this variability is not known. It has been suggested that prion strains with unique self-replicating and neurotoxic properties emerge spontaneously in individuals carrying PrP mutations, dictating the phenotypic expression of disease. We generated transgenic mice expressing the FFI mutation, and found that they developed a fatal neurological illness highly reminiscent of FFI, and different from those of similarly generated mice modeling genetic CJD and GSS. Thus transgenic mice recapitulate the phenotypic differences seen in humans. The mutant PrPs expressed in these mice are misfolded but unable to self-replicate. They accumulate in different compartments of the neuronal secretory pathway, impairing the membrane delivery of ion channels essential for neuronal function. Our results indicate that conversion of mutant PrP into an infectious isoform is not required for pathogenesis, and suggest that the phenotypic variability may be due to different effects of mutant PrP on intracellular transport.     

A mutational analysis of the cytosolic domain of the tomato Cf‐9 disease‐resistance protein shows that membrane‐proximal residues are important for Avr9‐dependent necrosis

The tomato Cf‐9 gene encodes a membrane‐anchored glycoprotein that imparts race‐specific resistance against the tomato leaf mould fungus Cladosporium fulvum in response to the avirulence protein Avr9. Although the N‐terminal half of the extracellular leucine‐rich repeat (eLRR) domain of the Cf‐9 protein determines its specificity for Avr9, the C‐terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf‐9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in Avr9‐dependent necrosis triggered by Cf‐9. Our results indicate that the membrane‐proximal region of the cytosolic domain of Cf‐9 plays an important role in Cf‐9‐mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf‐9 function. An alanine mutation of T835 had no effect on Cf‐9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf‐9 function. We therefore postulate that phosphorylation/de‐phosphorylation of T835 could act as a molecular switch to determine whether Cf‐9 is in a primed or inactive state. Yeast two‐hybrid analysis was used to show that the cytosolic domain of Cf‐9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline‐induced kink in the membrane‐proximal region of the cytosolic domain of Cf‐9 may be important for interaction with VAP27, which may, in turn, be important for Cf‐9 function.