The life cycle of a connexin: Gap junction formation,removal, and degradation

Gap junction proteins, connexins, possess many properties that are atypical of other well-characterized integral membrane proteins. Oligomerization of connexins into hemichannels (connexons) has been shown to occur after the protein exits the endoplasmic reticulum. Once delivered to the cell surface, connexons from one cell pair with connexons from a neighboring cell, a process that is facilitated by calcium-dependent cell adhesion molecules. Channels cluster into defined plasma membrane domains to form plaques. Unexpectedly, gap junctions are not stable (half-life <5 h) and are thought to be retrieved back into the cell in the form of double membrane structures when one cell internalizes the entire gap junction through endocytosis. Evidence exists for both proteasomal and lysosomal degradation of gap junctions, and it remains possible that both mechanisms are involved in connexin degradation. In addition to opening and closing of gap junction channels (gating), the formation and removal of gap junctions play an essential role in regulating the level of intercellular communication.     

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca^2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.     

Sar1 GTPase Activity Is Regulated by Membrane Curvature

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5′-[(β,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.     

Inhibitors of Protein Translocation Across the ER Membrane

Protein translocation into the endoplasmic reticulum (ER) constitutes the first step of protein secretion. ER protein import is essential in all eukaryotic cells and is particularly critical in fast‐growing tumour cells. Thus, the process can serve as target both for potential cancer drugs and for bacterial virulence factors. Inhibitors of protein transport across the ER membrane range from broad‐spectrum to highly substrate‐specific and can interfere with virtually any stage of this multistep process, and even with transport of endocytosed antigens into the cytosol for cross‐presentation.      

Control of Intracellular Movement of Connexins by E-Cadherin in Murine Skin Papilloma Cells

The gap junctional intercellular communication-deficient mouse skin papilloma cell line P3/22 expresses Cx43 but not E-cadherin. The E-cadherin gene-transfected cells (P3E1) communicate in a calcium-dependent manner and they were used to study how E-cadherin restores the function of connexins. At low calcium, Cx43 molecules remain in the cytoplasm of P3E1 cells and appear at cell-cell contact areas only in high-calcium medium. While Cx43 is unphosphorylated in P3E1 cells in low-calcium medium, two phosphorylated bands appeared at high calcium. However, when Cx26, which has no C-terminal tail that can undergo phosphorylation, was expressed in P3E1 cells, this connexin also moved to the plasma membrane after the calcium shift and partly colocalized with Cx43, suggesting that C-terminal phosphorylation is not essential for E-cadherin-mediated intracellular transport of connexins. In low calcium, both Cx26 and Cx43 remained and colocalized in the endoplasmic reticulum. As early as 30 min after the shift to high-calcium medium, both Cx43 and Cx26 began to accumulate in the Golgi apparatus. Intracellular movement of connexins to the cytoplasmic membrane at high calcium was effectively blocked by cytochalasin D and brefeldin A. These results suggest that E-cadherin junction formation at high calcium leads to formation of actin cables, which directly or indirectly transport connexins from the cytoplasm to the cell-cell contact membranes via the Golgi apparatus.     

大鼠脑突触质膜糖皮质激素受体的纯化

本文利用抗大鼠肝细胞内糖皮质激素受体的单克隆抗体制备的免疫亲和层析柱,将大鼠脑突触质膜糖皮质激素受体纯化了约1150倍,SDS聚丙烯酰胺簿层梯度凝胶电泳显示,在约67kD处有一较明显的染色条带。     

Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice

The importance of connexins (Cxs) in the cochlear functions has been indicated by the finding that mutations in connexin genes cause a large proportion of sensorineural deafness cases. However, functional roles of connexins in the cochlea are still unclear. In this study, we compared the relative expression levels of 16 different subtypes of mouse connexins in the cochlea. cDNA macroarray hybridizations identified four most prominently expressed connexins (listed in descending order): Cxs 26, 29, 30, and 43. Two of these connexins (Cx26 and Cx30), both belonging to the beta-group, were investigated for their molecular assemblies in the cochlea. Co-immunostaining showed expressions of Cxs 26 and 30 in the same gap junction plaques and their co-assembly was confirmed by co-immunoprecipitation of proteins extracted from the cochlear tissues. The heterologous molecular assembly of connexins is expected to produce gap junctions with biophysical characteristics appropriate for maintaining ionic homeostasis in the cochlea.     

细胞质膜蛋白与肿瘤的相关性研究进展

肿瘤相关研究一直是科研领域的重点与难点。肿瘤的发生、发展和转移与细胞质膜蛋白关系密切,质膜蛋白的过量表达、缺失或修饰,使细胞的信号转导、物质运输、黏附作用、免疫原性等发生改变,从而影响了肿瘤细胞的上述过程。简要综述了相关领域的研究进展。     

大豆叶片质膜蛋白激酶的自身磷酸化反应

利用Ferrell和Martin（1991）设计的测定印迹在PVDF膜上的蛋白激酶活性方法研究大豆叶片质膜蛋白激酶自身磷酸化反应活性,结果表明：与Mg－ATP相比,Mn－ATP是更有效的57KD蛋白激酶自身磷酸化反应底物;钙离子可以促进该激酶的自身磷酸化反应活性,而且EGTA可以显著降低它在SDS电泳中的迁移率,说明57KD蛋白激酶为依赖于钙的蛋白激酶;预磷酸化反应实验证明57KD蛋白激酶具有多个自身磷酸化反应位点,其分子的自身磷酸化状态可调性暗示这一激酶可能具有重要的生理功能。     

Connexin Expression and Cell Coupling Fail to Reverse the v-src Transformed Growth Characteristics of a Cx43-/- Cell

Gap junctions, composed of connexins, have been shown to suppress transformation in a variety of malignancies and transformed cell types. In addition, transforming factors such as the src oncogene have been shown to directly phosphorylate some connexins (e.g., Cx43) and inhibit coupling. To investigate the role of gap junctions in cell transformsation by v-src, we utilized a clonal cell line derived from Cx43 knockout mice (KoA) that was immortalized, but not transformed. Transfection by v-src induced a marked transformed phenotype characterized by growth in low serum and anchorage-independent conditions. Subsequent transfections by Cx43, Cx32 or vector alone were then tested for their effects on growth. Activity of pp60^{v - src} was confirmed in all transfectants as well as the ability of pp60^{v - src} to phosphorylate Cx43 in several clones. Despite the documented effect of pp60^{v - src} on Cx43 channel closure, modest coupling was still retained in many of the Cx43 and Cx32 transfectants. However, none of the four Cx43 transfected clones showed significant inhibitory effects on proliferation in either anchorage-independent or low serum growth conditions. Of the Cx32 clones, only one in five showed effects on growth in both assays, which was the same ratio observed for the control transfectants. Thus, based on the levels of expression achieved, which were comparable to endogenous levels in established cell lines, neither Cx43 nor Cx32 serve as effective suppressors of the transformed growth phenotype of this v-src expressing cell line.     

Characterizaton and Biosynthesis of the Plasma Membrane Proteolipid Protein in Neural Tissue

In this study we have characterized, in brain, the expression of a plasma membrane proteolipid protein (PM-PLP) complex that can form cation-selective channels in lipid bilayers. We isolated PLP fractions from synaptic plasma membrane and glial microsomes and found a high degree of similarity in both size and amino acid composition to the complex we had previously isolated from kidney. Antibodies specific to the kidney PM-PLP were prepared, and, on the basis of immunoblot and immunoprecipitation studies, the PM-PLP complex isolated from neural membranes was shown to be immunologically related to the kidney PM-PLP. These proteolipid proteins exhibited a molecular weight of approximately 14K and contained a high percentage of hydrophobic amino acids with an apparent absence of cysteine. The biogenesis of PM-PLP in brain was studied by in vitro translation of free and bound polysomes and total RNA in a rabbit reticulocyte lysate followed by immunoprecipitation of the translation products. From these studies it is concluded that the PM-PLP complex is synthesized on the rough endoplasmic reticulum. On the basis of the identical electrophoretic mobility of material isolated from plasma membranes and material immunoprecipitated after translation of bound polysomes and isolated RNA, it appears that the PM-PLP does not undergo detectable posttranslational processing between its site of synthesis and its incorporation into the plasma membrane.     

Plasma Membrane Protein Ubiquitylation and Degradation as Determinants of Positional Growth in Plants

Being sessile organisms, plants evolved an unparalleled plasticity in their post-embryonic development, allowing them to adapt and fine-tune their vital parameters to an ever-changing environment. Crosstalk between plants and their environment requires tight regulation of information exchange at the plasma membrane (PM). Plasma membrane proteins mediate such communication, by sensing variations in nutrient availability, external cues as well as by controlled solute transport across the membrane border. Localization and steady-state levels are essential for PM protein function and ongoing research identified cis- and trans-acting determinants, involved in control of plant PM protein localization and turnover. In this overview, we summarize recent progress in our understanding of plant PM protein sorting and degradation via ubiquitylation, a post-translational and reversible modification of proteins. We highlight characterized components of the machinery involved in sorting of ubiquitylated PM proteins and discuss consequences of protein ubiquitylation on fate of selected PM proteins. Specifically, we focus on the role of ubiquitylation and PM protein degradation in the regulation of polar auxin transport (PAT). We combine this regulatory circuit with further aspects of PM protein sorting control, to address the interplay of events that might control PAT and polarized growth in higher plants.     

大豆下胚轴质膜H+-ATPase质子转运的测定

以大豆下胚轴为材料,采用改进的匀浆介质,通过两相法制得具有质子转运活力的高纯度质膜微囊.并且发现冻融处理可以促进质膜微囊的翻转而提高荧光猝灭效率.质子载体和质子转运特性分析表明,由Mg^2＋-ATP引发的荧光猝灭可以被质子载体CCCP恢复,并被质子通道抑制剂DCCD抑制;并且发现质膜H^＋-ATPase专一抑制剂钒酸钠可以完全抑制荧光猝灭,同时发现荧光猝灭依赖于Mg^2＋,并受K^＋刺激,最适pH为6.5.以上证明所测荧光猝灭是由质膜H^＋-ATPase所进行的质子转运引起的.结果同时表明,维持H^＋-ATPase合适构象和提高质膜微囊封闭性是制备具有H^＋转运活力质膜微囊的两个关键因素.     

细胞质膜蛋白质组学研究技术进展

质膜蛋白在细胞中执行着非常重要的功能。随着蛋白质组学的发展,细胞质膜蛋白质组学成为蛋白质组学研究的重要组成部分,它为质膜蛋白的生物功能研究及药物靶标的发现提供了新的途径。然而,质膜蛋白丰度低、疏水性强,对现有蛋白质组学研究技术提出了挑战。简要综述了近年来质膜蛋白质组研究的相关技术进展,包括富集、提取分离鉴定方法及定量和生物信息学研究方法等。     

Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways

Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren’s syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.     

水稻幼苗根细胞质膜和液泡膜微囊Ca2+-ATP酶的特性

水稻幼苗根质膜和液泡膜Ca2 -ATP酶对ATP的Km值分别为7.1和4.5 μ mol·L-1;反应的最适pH分别为8.0和7.0.两者活性均受Na3VO4和曙红B(EB)抑制;CPZ抑制质膜Ca2 -ATP酶活性,但促进液泡膜Ca2 -ATP酶活性.30mmol·L-1CaCl2浸种和CaCl2浸种结合低温锻炼预处理,均可提高此酶的活性和冷稳定性.     

Phospholipid Flippase ATP10A Translocates Phosphatidylcholine and Is Involved in Plasma Membrane Dynamics

We showed previously that ATP11A and ATP11C have flippase activity toward aminophospholipids (phosphatidylserine (PS) and phosphatidylethanolamine (PE)) and ATP8B1 and that ATP8B2 have flippase activity toward phosphatidylcholine (PC) (Takatsu, H., Tanaka, G., Segawa, K., Suzuki, J., Nagata, S., Nakayama, K., and Shin, H. W. (2014) J. Biol. Chem. 289, 33543–33556). Here, we show that the localization of class 5 P4-ATPases to the plasma membrane (ATP10A and ATP10D) and late endosomes (ATP10B) requires an interaction with CDC50A. Moreover, exogenous expression of ATP10A, but not its ATPase-deficient mutant ATP10A(E203Q), dramatically increased PC flipping but not flipping of PS or PE. Depletion of CDC50A caused ATP10A to be retained at the endoplasmic reticulum instead of being delivered to the plasma membrane and abrogated the increased PC flipping activity observed by expression of ATP10A. These results demonstrate that ATP10A is delivered to the plasma membrane via its interaction with CDC50A and, specifically, flips PC at the plasma membrane. Importantly, expression of ATP10A, but not ATP10A(E203Q), dramatically altered the cell shape and decreased cell size. In addition, expression of ATP10A, but not ATP10A(E203Q), delayed cell adhesion and cell spreading onto the extracellular matrix. These results suggest that enhanced PC flipping activity due to exogenous ATP10A expression alters the lipid composition at the plasma membrane, which may in turn cause a delay in cell spreading and a change in cell morphology.     

Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction

As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca²⁺ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[¹²⁵I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca²⁺-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca²⁺-ATPase activity was related to an increase in the apparent affinity for Ca²⁺ and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca²⁺ homeostasis.     

多维色谱-串联质谱联用技术分离和鉴定小鼠肝脏质膜蛋白质

采用自动在线纳流多维液相色谱 串联质谱联用的方法分离和鉴定蔗糖密度梯度离心法分离和富集的小鼠肝脏质膜蛋白质 .以强阳离子交换柱为第一相 ,反相柱为第二相 ,在两相之间连接一预柱脱盐和浓缩肽段 .用含去污剂的溶剂提取细胞质膜中的蛋白质 ,获得的质膜蛋白质经酶解和适当的酸化后通过离子交换柱吸附 ,分别用 10个不同浓度的乙酸铵盐溶液进行分段洗脱 .洗脱物经预柱脱盐和浓缩后进入毛细管反相柱进行反相分离 ,分离后的肽段直接进入质谱仪离子源进行一级和二级质谱分析 .质谱仪采得的数据经计算机处理后用Mascot软件进行蛋白质数据库搜寻 ,共鉴定出 12 6种蛋白质 ,其中 4 1种为膜蛋白 ,包括与膜相关的蛋白质和具有多个跨膜区的整合膜蛋白 ,为建立质膜蛋白质组学研究的适宜方法和质膜蛋白质数据库提供了有价值的基础性研究资料 .