Mesoscopic surfactant organization and membrane protein crystallization

The paucity of detailed X-ray crystallographic structures of integral membrane proteins arises from substantive technical obstacles in the overexpression of multimilligram quantities of protein, and in the crystallization of purified protein-detergent complexes (PDCs). With rare exception, crystal contacts within the lattice are mediated by protein-protein interaction, and the detergent surrounding the protein behaves as a disordered solvent. The addition and use of surfactants that display mesoscopic self-assembly behavior in membrane protein crystallization experiments presents a novel alternative strategy. Well-ordered crystals of the water channel human aquaporin-1 (hAQP1) that diffract to 4 A resolution have been obtained with this approach.     

In vivo requirement of the alpha-syntrophin PDZ domain for the sarcolemmal localization of nNOS and aquaporin-4

alpha-Syntrophin is a scaffolding adapter protein expressed primarily on the sarcolemma of skeletal muscle. The COOH-terminal half of alpha-syntrophin binds to dystrophin and related proteins, leaving the PSD-95, discs-large, ZO-1 (PDZ) domain free to recruit other proteins to the dystrophin complex. We investigated the function of the PDZ domain of alpha-syntrophin in vivo by generating transgenic mouse lines expressing full-length alpha-syntrophin or a mutated alpha-syntrophin lacking the PDZ domain (Delta PDZ). The Delta PDZ alpha-syntrophin displaced endogenous alpha- and beta 1-syntrophin from the sarcolemma and resulted in sarcolemma containing little or no syntrophin PDZ domain. As a consequence, neuronal nitric oxide synthase (nNOS) and aquaporin-4 were absent from the sarcolemma. However, the sarcolemmal expression and distribution of muscle sodium channels, which bind the alpha-syntrophin PDZ domain in vitro, were not altered. Both transgenic mouse lines were bred with an alpha-syntrophin-null mouse which lacks sarcolemmal nNOS and aquaporin-4. The full-length alpha-syntrophin, not the Delta PDZ form, reestablished nNOS and aquaporin-4 at the sarcolemma of these mice. Genetic crosses with the mdx mouse showed that neither transgenic syntrophin could associate with the sarcolemma in the absence of dystrophin. Together, these data show that the sarcolemmal localization of nNOS and aquaporin-4 in vivo depends on the presence of a dystrophin-bound alpha-syntrophin PDZ domain.     

Functional characterization of a novel plasma membrane intrinsic protein2 in barley

Water homeostasis is crucial to the growth and survival of plants. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. We characterized a novel PIP2 gene, HvPIP2;8 in barley (Hordeum vulgare). HvPIP2;8 shared 72–76% identity with other HvPIP2s and 74% identity with rice OsPIP2;8. The gene was expressed in all organs including the shoots, roots and pistil at a similar level. When HvPIP2;8 was transiently expressed in onion epidermal cells, it was localized to the plasma membrane. HvPIP2;8 showed transport activity for water in Xenopus oocytes, however its interaction with HvPIP1;2 was not observed. These results suggest that HvPIP2;8 plays a role in water homeostasis although further functional analysis is required in future.     

Evaluation of new immunological targets in neuromyelitis optica

The detection of reactivity against autoantigens plays a crucial role in the diagnosis of autoimmune diseases. However, only a few autoantibodies are known in each disease, and their precise targets are often not precisely defined. In neuromyelitis optica (NMO), an autoimmune disease of the central nervous system, anti‐aquaporin 4 antibodies are currently the only available immunological markers, although they are not detected in 10–50% of patients. Using enzyme‐linked immunosorbent assays, we evaluated the reactivity against 19 structurally defined peptides in 26 NMO sera compared with 21 healthy subjects. We observed increased levels of IgG against myelin basic protein sequence MBP(156–175), pyruvate dehydrogenase sequence PDH(167–186) and CSF114(Glc), the last of these having a possible correlation with onset of inflammatory relapse. These preliminary results may suggest that the aquaporin 4 is not the unique target in NMO and that the study of reactivity against these peptides would be helpful for the diagnosis and follow‐up of the disease. Complementary studies are however warranted to confirm these results. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

桃花芽自然休眠期间水孔蛋白基因的转录表达

以10年生大田栽培及3年生盆栽曙光油桃花芽为试材,利用荧光定量PCR测定了油桃休眠及休眠解除期间(2009年9月15日-2010年1月15日)曙光油桃水孔蛋白基因δTIP1、PIP1;1的表达量,以及低温胁迫下的转录表达.结果表明:在油桃休眠及休眠解除期间,曙光油桃PIP1;1的转录水平呈现持续增高趋势,且1月的高水平表达使水分通过液泡膜和细胞质膜流出,减少了芽体水分含量,阻止细胞内冰晶的形成,从而抵御冻害;可溶性糖、可溶性蛋白、脯氨酸含量均达到最高,防止细胞的脱水伤害.低温处理2周后高水平表达说明PIP1;1为冷诱导基因.δTIP1的转录水平在休眠期间呈现波动性变化,至休眠解除时大幅度增高,这可能与休眠解除时,其上调表达被休眠解除信号及植物活性的增强所诱导有关.低温处理2周后,其表达没有升高,说明δTIP1并非冷诱导基因.     

叶肉导度的组成、大小及其对环境因素的响应

植物光合作用过程中,大气中的CO2需要克服气孔和叶肉细胞等阻力传输到羧化位点。CO2从气孔下腔传输到羧化位点的阻力称为叶肉阻力,其倒数即为叶肉导度。近十年内,叶肉导度已经成为光合作用研究领域的一个重要方面。本文首先系统地阐述了叶肉导度的组成及各部分所占的比重;然后通过与气孔导度的比较,分析叶肉导度的大小及其对光合作用的影响;最后阐述了叶肉导度对环境变化的响应,并分析了其中可能的原因。     

Phosphorylation influences water and ion channel function of AtPIP2;1

The phosphorylation state of two serine residues within the C-terminal domain of AtPIP2;1 (S280, S283) regulates its plasma membrane localization in response to salt and osmotic stress. Here, we investigated whether the phosphorylation state of S280 and S283 also influence AtPIP2;1 facilitated water and cation transport. A series of single and double S280 and S283 phosphomimic and phosphonull AtPIP2;1 mutants were tested in heterologous systems. In Xenopus laevis oocytes, phosphomimic mutants AtPIP2;1 S280D, S283D, and S280D/S283D had significantly greater ion conductance for Na⁺ and K⁺, whereas the S280A single phosphonull mutant had greater water permeability. We observed a phosphorylation-dependent inverse relationship between AtPIP2;1 water and ion transport with a 10-fold change in both. The results revealed that phosphorylation of S280 and S283 influences the preferential facilitation of ion or water transport by AtPIP2;1. The results also hint that other regulatory sites play roles that are yet to be elucidated. Expression of the AtPIP2;1 phosphorylation mutants in Saccharomyces cerevisiae confirmed that phosphorylation influences plasma membrane localization, and revealed higher Na⁺ accumulation for S280A and S283D mutants. Collectively, the results show that phosphorylation in the C-terminal domain of AtPIP2;1 influences its subcellular localization and cation transport capacity.     

Bundle‐sheath cell regulation of xylem‐mesophyll water transport via aquaporins under drought stress: a target of xylem‐borne ABA?

The hydraulic conductivity of the leaf vascular system (K_leaf) is dynamic and decreases rapidly under drought stress, possibly in response to the stress phytohormone ABA, which increases sharply in the xylem sap (ABA_xyl) during periods of drought. Vascular bundle‐sheath cells (BSCs; a layer of parenchymatous cells tightly enwrapping the entire leaf vasculature) have been hypothesized to control K_leaf via the specific activity of BSC aquaporins (AQPs). We examined this hypothesis and provide evidence for drought‐induced ABA_xyl diminishing BSC osmotic water permeability (P_f) via downregulated activity of their AQPs. ABA fed to the leaf via the xylem (petiole) both decreased K_leaf and led to stomatal closure, replicating the effect of drought. In contrast, smearing ABA on the leaf blade, while also closing stomata, did not decrease K_leaf within 2–3 h of application, demonstrating that K_leaf does not depend entirely on stomatal closure. GFP‐labeled BSCs showed decreased P_f in response to ‘drought’ and ABA treatment, and a reversible decrease with HgCl₂ (an AQP blocker). These P_f responses, absent in mesophyll cells, suggest stress‐regulated AQP activity specific to BSCs, and imply a role for these cells in decreasing K_leaf via a reduction in P_f. Our results support the above hypothesis and highlight the BSCs as hitherto overlooked vasculature sensor compartments, extending throughout the leaf and functioning as ‘stress‐regulated valves’ converting vasculature chemical signals (possibly ABA_xyl) into leaf hydraulic signals.     

The Arabidopsis thaliana aquaporin AtPIP1;2 is a physiologically relevant CO₂ transport facilitator

Cellular exchange of carbon dioxide (CO₂) is of extraordinary importance for life. Despite this significance, its molecular mechanisms are still unclear and a matter of controversy. In contrast to other living organisms, plants are physiologically limited by the availability of CO₂. In most plants, net photosynthesis is directly dependent on CO₂ diffusion from the atmosphere to the chloroplast. Thus, it is important to analyze CO₂ transport with regards to its effect on photosynthesis. A mutation of the Arabidopsis thaliana AtPIP1;2 gene, which was characterized as a non‐water transporting but CO₂ transport‐facilitating aquaporin in heterologous expression systems, correlated with a reduction in photosynthesis under a wide range of atmospheric CO₂ concentrations. Here, we could demonstrate that the effect was caused by reduced CO₂ conductivity in leaf tissue. It is concluded that the AtPIP1;2 gene product limits CO₂ diffusion and photosynthesis in leaves.