Distribution of a kinesinrelated protein on Golgi apparatus of tobacco pollen tubes

The conventional kinesin and kinesin-related proteins (KRPs) constitute a large family of microtubule-based molecular motors. Members of this family bind to micro-tubules in an ATP-dependent manner. They all have microtubule-activated ATPaes activities, and play essential roles in the transport of various vesicles and organelles in eukaryotic cells[1―3]. The first plant kinesin-like protein was detected in tobacco pollen tube with an antibody against animal kinesin[4]. Recently, a number …     

Retrograde transport of endocytosed Shiga toxin to the endoplasmic reticulum.

Shiga toxin and some other protein toxins that act on targets in the cytosol have previously been shown to enter the trans-Golgi network. Transport by this route may be necessary for translocation of the toxin to the cytosol and for intoxication, but it is not known whether the enzymatically active part of the toxins actually enters the cytosol from the trans-Golgi network. It has been suggested that such toxins are transported in a retrograde manner to the endoplasmic reticulum and that translocation occurs in this organelle, but retrograde transport of endocytosed material beyond the trans-Golgi network has never been demonstrated. Here we show that in butyric acid-treated A431 cells endocytosed Shiga toxin is not only transported to the trans-Golgi network, but also to all Golgi stacks, to the endoplasmic reticulum and to the nuclear envelope. Furthermore, butyric acid sensitizes the cells to Shiga toxin, which is consistent with the possibility that retrograde transport is required for translocation of the toxin to the cytosol.     

Glutamate-receptor-interacting protein GRIP1 directly steers kinesin to dendrites

In cells, molecular motors operate in polarized sorting of molecules, although the steering mechanisms of motors remain elusive. In neurons, the kinesin motor conducts vesicular transport such as the transport of synaptic vesicle components to axons and of neurotransmitter receptors to dendrites, indicating that vesicles may have to drive the motor for the direction to be correct. Here we show that an AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor subunit--GluR2-interacting protein (GRIP1)--can directly interact and steer kinesin heavy chains to dendrites as a motor for AMPA receptors. As would be expected if this complex is functional, both gene targeting and dominant negative experiments of heavy chains of mouse kinesin showed abnormal localization of GRIP1. Moreover, expression of the kinesin-binding domain of GRIP1 resulted in accumulation of the endogenous kinesin predominantly in the somatodendritic area. This pattern was different from that generated by the overexpression of the kinesin-binding scaffold protein JSAP1 (JNK/SAPK-associated protein-1, also known as Mapk8ip3), which occurred predominantly in the somatoaxon area. These results indicate that directly binding proteins can determine the traffic direction of a motor protein.     

Effects of temperature on the expression of STN7 andphosphorylation of LHCII proteins in Arabidopsis thaliana

In Arabidopsis thaliana, STN7 kinase is required for phosphorylation of LHCII and for state transitions. In this paper, a hydrophilic polypeptide, derived from the amino acid sequence of STN7, was conjugated to a carrier protein, bovine serum albumin (BSA), to obtain the polyclonal antibody. Immunogenicity and specificity of the polyclonal antibody were evaluated by agar gel immunodiffusion (AGID) test and Western blot analysis. The results show that besides the phosphorylation of LHCII proteins, also the expression of STN7 was regulated by temperature conditions. In addition, the change tendency of LHCII proteins phosphorylation was not only coherent with expression of STN7 with respect to increasing temperature, but also closely related to state transitions. These results would provide useful information for studying regulatory mechanism of LHCII proteins phosphorylation and expression of STN7.     

几种植物分生组织细胞高尔基体的超微结构

应用电镜观察石刁柏胚芽、水仙幼叶、根尖和甜菊愈伤组织等分生细胞高尔基体、液泡以及内质网和细胞壁之间的联系,在高尔基体的形成面,由内质网芽生的运输小泡,分布于高尔基潴泡周围,高尔基潴泡末端扩大发展形成高尔基液泡,在分生细胞的大小液泡,是由高尔基潴泡末端扩大形成的,这些液泡合并形成中央液泡.在细胞壁的形成过程中,高尔基小泡沿着细胞壁边缘转移,并且与质膜融合。     

Clathrin is a key regulator of basolateral polarity

Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.     

Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2

Membrane transport between the endoplasmic reticulum and the plasma membrane, which involves the budding and fusion of carrier vesicles, is inhibited during mitosis in animal cells. At the same time, the Golgi complex and the nuclear envelope, as well as the endoplasmic reticulum in some cell types, become fragmented. Fragmentation of the Golgi is believed to facilitate its equal partitioning between daughter cells. In fact, it has been postulated that both the inhibition of membrane traffic and Golgi fragmentation during mitosis are due to an inhibition of vesicle fusion, while vesicle budding continues. Although less is known about the endocytic pathway, internalization and receptor recycling are also arrested during mitosis. We have now used a cell-free assay to show that the fusion of endocytic vesicles from baby hamster kidney cells is reduced in Xenopus mitotic cytosol when compared with interphase cytosol. We reconstituted this inhibition in interphase cytosol by adding a preparation enriched in the starfish homologue of the cdc2 protein kinase. Inhibition was greater than or equal to 90% when the added cdc2 activity was in the range estimated for that in mitotic Xenopus eggs, which indicates that during mitosis the cdc2 kinase mediates an inhibition of endocytic vesicle fusion, and possibly other fusion events in membrane traffic.     

An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells

Circumstantial evidence suggests that intracellular membrane trafficking pathways diversified independently in the plant kingdom, but documented examples are rare. ARF-GEFs (guanine-nucleotide exchange factors for ADP-ribosylation factor GTPases) are essential for vesicular trafficking in all eukaryotic kingdoms, but of the eight ARF-GEF families, only the ancestral BIG and GBF types are found in plants. Whereas fungal and animal GBF proteins perform conserved functions at the Golgi, the Arabidopsis thaliana GBF protein GNOM is thought to act in only the process of recycling from endosomes. We now show that the related Arabidopsis GBF protein GNOM-LIKE1 (GNL1) has an ancestral function at the Golgi but is also required for selective internalization from the plasma membrane in the presence of brefeldin A (BFA). We identified gnl1 mutants that accumulated biosynthetic and recycling endoplasmic reticulum markers in enlarged internal compartments. Notably, in the absence of functional GNL1, Golgi stacks were rendered sensitive to the selective ARF-GEF inhibitor BFA, which caused them to fuse with the endoplasmic reticulum. Furthermore, in BFA-treated gnl1 roots, the internalization of a polar plasma-membrane marker, the auxin efflux carrier PIN2, was selectively inhibited. Thus, GNL1 is a BFA-resistant GBF protein that functions with a BFA-sensitive ARF-GEF both at the Golgi and in selective endocytosis, but not in recycling from endosomes. We propose that the evolution of endocytic trafficking in plants was accompanied by neofunctionalization within the GBF family, whereas in other kingdoms it occurred independently by elaboration of additional ARF-GEF families.     

高尔基体蛋白功能研究进展

高尔基体蛋白是一类定位在高尔基体表面上的螺旋卷曲蛋白家族.目前研究最为广泛的有11种高尔基体蛋白,它们分别定位在高尔基体堆栈不同的部位上.其中有3种定位在高尔基体正面膜囊表面上,高尔基体的反面膜囊表面和高尔基体膜囊边缘表面各定位4种高尔基体蛋白,它们以各自肽链羧基端或以跨膜结构域或与小的GTPase结合锚定到高尔基体膜...     

Effects of temperature on the expression of STN7 and phosphorylation of LHCII proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In Arabidopsis thaliana, STN7 kinase is required for phosphorylation of LHCII and for state transitions. In this paper, a hydrophilic polypeptide, derived from the amino acid sequence of STN7, was conjugated to a carrier protein, bovine serum albumin （BSA）, to obtain the polyclonal antibody. Immunogenicity and specificity of the polyclonal antibody were evaluated by agar gel immunodiffusion （AGID） test and Western blot analysis. The results show that besides the phosphorylation of LHCII proteins, also the expression of STN7 was regulated by temperature conditions. In addition, the change tendency of LHCII proteins phosphorylation was not only coherent with expression of STN7 with respect to increasing temperature, but also closely related to state transitions. These results would provide useful information for studying regulatory mechanism of LHCII proteins phosphorylation and expression of STN7.     

'Coatomer': a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles

Golgi-derived coated vesicles contain a set of coat proteins of relative molecular mass 160,000 (Mr 160K; alpha-COP), 110K (beta-COP), 98K (gamma-COP) and 61K (delta-COP), and several smaller subunits. We have now identified and purified a cytosolic complex containing the same four coat proteins as those of Golgi transport vesicles. We term this complex the Golgi coat promoter or 'coatomer'. The coatomer also contains polypeptides of Mr 36K, 35K and 20K. It represents about 0.2% of soluble cytosolic protein. Gel filtration of unfractionated cytosol indicates that beta-COP resides exclusively in the coatomer complex. The complex seems to be a likely candidate for the unassembled precursor of Golgi coated vesicles, and its purification should help investigations of the role of coat proteins in membrane budding, for which it is necessary to use a refined cell-free system.     

兔抗PEB1多克隆抗体的制备及鉴定

摘要：【目的】利用纯化的空肠弯曲菌表面蛋白PEB1,制备其多克隆抗体。【方法】用PEB1蛋白作为抗原免疫兔,分离血清得到兔抗PEB1多克隆抗体,用ELISA法检测抗体效价,并用Western-Blotting验证。【结果】PEB1蛋白免疫兔后,得到了兔抗PEB1多克隆抗体,间接ELASA法检测表明兔抗PEB 1血清效价达到1：10 000,Western印记检测表明,该抗体能与PEB1蛋白发生特异性结合,其特异性与敏感性均较好。【结论】获得了高效价的PEB1多克隆抗体。     

拟南芥AtRAC在盐胁迫应答中的功能研究

本实验所研究的拟南芥AtRAC含有两个RING结构域和三个蛋白激酶C保守区域1结构域即C1结构域.通过体外泛素化证明了AtRAC具有E3连接酶活性,亚细胞定位实验结果表明AtRAC定位在细胞核中.组织特异性分析表明AtRAC在根中的表达量很高,但是在茎、叶和花中的表达量较低,而且AtRAC的表达受到NaCl胁迫的诱导.进一步对atrac突变体的分析表明,在NaCl处理下,突变体atrac的萌发率降低,侧根数目也降低.初步研究表明拟南芥AtRAC可能是一个与盐胁迫应答相关的基因.     

BIG-3的原核表达、纯化及其多克隆抗体的制备与鉴定

原核表达、纯化骨形态发生蛋白诱导基因（BIG-3,BMP-2-induced gene 3kb）所编码的融合蛋白并制备其多克隆抗体。将BIG-3基因插入原核表达载体PGEX-4T-2的多克隆位点获得pGEX-4T-2-BIG-3重组表达载体,转化大肠杆菌BL21（DE3）菌株,诱导表达,SDS-PAGE鉴定融合蛋白表达情况及Sepharose4B层析柱亲和层析法纯化的融合蛋白;用纯化的融合蛋白免疫新西兰大白兔制备其多克隆抗体,并以Western blot鉴定其特异性。结果在大肠杆菌中获得BIG-3-GST融合蛋白高水平的诱导表达,经Sepharose4B层析柱亲和层析,GST-BIG-3融合蛋白在电泳图片上显示较为清晰的单一条带,所制备的多克隆抗体具有较高的特异性。说明在大肠杆菌中成功表达且以亲和层析法纯化得到了BIG-3-GST融合蛋白,并制备了特异性较高的多克隆抗体。     

A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast

A protein sensitive to N-ethylmaleimide catalyses the fusion of transport vesicles with Golgi cisternae in a mammalian cell-free system. By cloning and sequencing its gene from Chinese hamster ovary cells and by use of in vitro assays, we show that this fusion protein is equivalent to the SEC18 gene product of the yeast Saccharomyces cerevisiae, known to be essential for vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus. The mechanism of vesicular fusion is thus highly conserved, both between species and at different stages of transport.     

Golgi maturation visualized in living yeast

The Golgi apparatus is composed of biochemically distinct early (cis, medial) and late (trans, TGN) cisternae. There is debate about the nature of these cisternae. The stable compartments model predicts that each cisterna is a long-lived structure that retains a characteristic set of Golgi-resident proteins. In this view, secretory cargo proteins are transported by vesicles from one cisterna to the next. The cisternal maturation model predicts that each cisterna is a transient structure that matures from early to late by acquiring and then losing specific Golgi-resident proteins. In this view, secretory cargo proteins traverse the Golgi by remaining within the maturing cisternae. Various observations have been interpreted as supporting one or the other mechanism. Here we provide a direct test of the two models using three-dimensional time-lapse fluorescence microscopy of the yeast Saccharomyces cerevisiae. This approach reveals that individual cisternae mature, and do so at a consistent rate. In parallel, we used pulse-chase analysis to measure the transport of two secretory cargo proteins. The rate of cisternal maturation matches the rate of protein transport through the secretory pathway, suggesting that cisternal maturation can account for the kinetics of secretory traffic.     

Immunochemical identification of gelsolin in maize pollen

Gelsolin is a representative of a type of actin-binding proteins (ABPs) universally found in eukaryotes. It plays role in nucleation, capping and severing of actin filamentsin vitro. In our experiment, gelsolin was purified from pig plasma and the polyclonal antibodies against it were prepared. The crude extracts of maize pollen were immunodetected by Western-blotting with polyclonal antibody and monoclonal antibody respectively. The immunodetection results show that gelsolin exists in maize pollen and its molecular weight is about 91 ku, similar to that of gelsolin found in animal tissues.     

Compartmental specificity of cellular membrane fusion encoded in SNARE proteins

Membrane-enveloped vesicles travel among the compartments of the cytoplasm of eukaryotic cells, delivering their specific cargo to programmed locations by membrane fusion. The pairing of vesicle v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with target membrane t-SNAREs has a central role in intracellular membrane fusion. We have tested all of the potential v-SNAREs encoded in the yeast genome for their capacity to trigger fusion by partnering with t-SNAREs that mark the Golgi, the vacuole and the plasma membrane. Here we find that, to a marked degree, the pattern of membrane flow in the cell is encoded and recapitulated by its isolated SNARE proteins, as predicted by the SNARE hypothesis.