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Current progress on the Small GTPase Gene Superfamily In plant

Current progress on the Small GTPase Gene Superfamily In plant. —— 植物小 G 蛋白功能的研究进展. 植物小 G 蛋白功能的研究进展. 概述 Rab GTPases Rop GTPases Arf GTPases Ran GTPases 展望.

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Current progress on the Small GTPase Gene Superfamily In plant

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  1. Current progress on the Small GTPase Gene Superfamily In plant ——植物小G蛋白功能的研究进展

  2. 植物小G蛋白功能的研究进展 概述 Rab GTPases Rop GTPases Arf GTPases Ran GTPases 展望

  3. In recent years，small G proteins have become an intensively studied group of regulatory GTP hydrolyses involved in cell signaling． • The small G-protein superfamily includes Ras，Rho，Rab， Arf and Ran homologs, which take part in numerous and diverse cellular processes，such as gene expression，cytoskeletal reorganization，microtubule organization，and vesicular and nuclear transport． • 近年来，小G蛋白的调控途径已经成为人们研究细胞信号转导过程的热点问题。 • 小G蛋白家族包括Ras、Rho、Rab、Arf和Ran亚家族，它们起着许多不同的重要细胞生理作用，例如基因表达、细胞骨架重组装、微管的形成以及囊泡和核孔运输机制。

  4. 小G蛋白(small GTPases)家族成员是单体蛋白，分子量较小，大约20～30 kD，区别于异三聚体G蛋白，人们通常称之为小G蛋白。 • 小G蛋白家族成员都具有几个保守的结构特征，包括四个鸟核苷酸结合区和一个效应器分子结合区（图A）

  5. 小G蛋白的“分子开关”是依靠它的附属蛋白调控的，称之为鸟苷酸交换因子(guanine nucleotide exchange factors，GEFs)，它可以催化小G蛋白转换为GTP结合形式，即活化态。 • 大多数小G蛋白循环是在膜结合形式和胞质结合形式循环中进行的。 • 只有膜结合形式的小G蛋白可以被GEF激活，当小G蛋白处于活化态时，就可以和不同的下游效应器分子相互作用， 进而执行不同的细胞生理功能。胞质形式的小G蛋白可以通过鸟苷酸解离抑制因子（GDI）结合，从而负调控这些小G蛋白的GTPases活性。

  6. 每个小G蛋白亚家族成员之间的序列保守性要高于每个亚家族序列间的保守性。分析果蝇、酵母、拟南芥和人类的基因组序列发现小G蛋白亚家族成员的保守性很高。每个小G蛋白亚家族成员之间的序列保守性要高于每个亚家族序列间的保守性。分析果蝇、酵母、拟南芥和人类的基因组序列发现小G蛋白亚家族成员的保守性很高。

  7. 拟南芥基因组序列预测分析拟南芥存在93个小G蛋白，包括57个Rab GTPase、21个 Arf GTPase 、11个Rho GTPase 和4个Ran GTPase，它们调节着细胞的不同生理过程。有趣的是，从基因组预测分析，拟南芥没有Ras GTPase，也许在植物发育中还存在着特殊的细胞信号调控机制。

  8. Rab GTPases • Rab GTPases make up the largest family of the small GTP-binding protein superfamily. Both in vivo and in vitro experiments have demonstrated the roles of this class of proteins in intracellular membrane trafficking. • Furthermore, given the specific distributions of Rab GTPases to different cellular membranes, it was hypothesized that Rab GTPases would, in conjunction with SNARE proteins, provide specificity for membrane fusion events.

  9. Rab GTPases of the Endocytic Trafficking Pathway（ 参与细胞的胞吞运输途径） • Rab GTPases of the Biosynthetic Trafficking Pathway（参与生物合成运输途径） • Rab GTPases Involved in Polarized Secretion（参与极性分泌过程）

  10. Endocytosis is the primary means by which proteins and macromolecules too large to pass through the plasma membrane enter the cell. During endocytosis, regions of the plasma membrane with associated cargo molecules are invaginated and pinch off into transport vesicles, which then fuse with endocytic compartments. In addition, many proteins targeted to lysosomal and/or vacuolar compartments are sorted in endosomal compartments. • 胞吞是蛋白质和大分子跨膜运输的主要方式，细胞质膜区域与相关的物质大分子共同凹陷，形成运输囊泡，然后与胞内小泡融合，大多数蛋白质分子在内吞小泡中被分选运送到溶酶体或液泡等不同的细胞器。

  11. 与其他真核生物的Rab GTPase相比，拟南芥 AtRab F的3个成员与人类Rab5和酵母Ypt5lp家族成员有非常高的相似性。在哺乳动物中，Rab5 GTPase定位于早期内吞小泡，调控笼形蛋白包被的囊泡与早期内吞小泡的融合。 • 利用细胞吸收荧光标记染料FM4-64的定位实验发现， AtRab F2b定位于运输小泡。 • 植物中，液泡除了有消化作用，还有贮藏细胞器的作用。一个细胞可以含有多个液泡类型，这就增加了在植物中研究庞大的Rab GTPase家族成员的复杂性。

  12. Newly synthesized proteins enter the secretory pathway by translocation through ER membranes. Subsequent transport from the ER to Golgi complexes involves recruitment of cargo proteins into vesicle transport intermediates. • 新合成的蛋白质通过内质网进入到分泌途径，接下来从内质网到高尔基体的转运过程包括蛋白质运送到运输囊泡的中间结构。

  13. 不同的Rab 存在于ER向高尔基体的运输、由高尔基体向质膜的组成型和调节型途径中，以及存在于内吞体之间的运输步骤。

  14. 拟南芥AtRab D亚家族有5个成员与哺乳动物Rabl和酵母Yptlp Rab GTPase有很高的同源性。在哺乳动物中，Rabl 定位于内质网、内质网和高尔基体中间结构及高尔基体，并调节着内质网、高尔基体膜运输过程。 • 酵母中编码两种相关蛋白质的基因YPT1 或SEC4 突变，会阻断运输并使膜泡分别在高尔基体垛叠区及高尔基体和质膜之间积累。 • 植物中，瞬时表达失活的AtRab D2a基因的突变体可以导致分泌型绿色荧光标记蛋白在内质网胞内小室中积累，并且抑制高尔基复合体沿着细胞骨架运动的过程。 • 由此说明， Rab GTPases参与生物合成运输途径。

  15. 拟南芥有5个Rab GTPase与参与极性分泌的酵母、哺乳动物的Rab GTPase有很高同源性。植物中的Rab成员还可能参与细胞的细菌病原反应。 • 利用酵母双杂交系统发现番茄的Rab E亚家族成员(与哺乳动物Rab8有很高同源性)可与无毒的avrPto因子相互作用，该因子可以破坏Rab E同源序列调节的膜运输途径，从而使植物感病。 • Rab GTPase可能参与细菌病原应答中抗菌化合物的极性分泌过程，这一发现的研究还刚刚开始。

  16. Rop GTPases Rho GTPases have been categorized into three major subfamilies:CDC42, RAC, and RHO based on their cellular functions and sequence homology.

  17. In Arabidopsis, all small GTPases that segregate with the Rho GTPases appear to be members of a unique subfamily . Because this subfamily has so far only been identified in plants, they have been namedRop GTPases (for Rho-related proteins from plants). • Analysis of the Arabidopsis genome revealed the existence of 11 ROP GTPases, which we have named AtROP.

  18. Rop GTPases Are Multifunctional Signaling Molecules • Rho GTPases play central roles in a wide range of cellular processes, many of which are associated with the actin cytoskeleton. • Mammalian and fungal Rho GTPases regulate the establishment of cell polarity and influence cell morphogenesis.

  19. In mammals, RAC GTPases control production of reactive oxygen species by directly associating with and regulating the activity of plasma membrane associated NADPH oxidase complexes . • plant ROP GTPases may regulate the plasma membrane NADPH oxidase complex.

  20. AtROP GTPases are also involved in signal transduction pathways mediated by the plant hormone,abscisic acid (ABA). • Plant ROP GTPases may also regulate the accumulation of and/or responses to brassinolide and auxin.

  21. Rop-Interacting Proteins • Several types of Rho GTPase regulators are known in animals: GAPs and GDIs (guanine nucleotide dissociation factors) that act as negative regulators of Rho GTPase function, andGEFs, which activate these GTPases through exchange of GDP for GTP.

  22. plants may have evolved different mechanisms to activate the GDP-bound ROP GTPases. One possible mechanism of activation may be through direct association of ROP GTPases with plant receptor-like kinases (RLKs).

  23. No obvious homologs of animal and yeast Rho effector proteins, except for phosphatidyl-inositolphosphate kinases, are present in the Arabidopsis genome.

  24. Arf GTPases • The Arabidopsis genome (Arabidopsis Genome Initiative, 2000) contains 21 Arf GTPase family members, with isoforms present in both ArfandArl GTPase subfamilies

  25. Arf GTPases Recruit Coat Proteins to Transport Vesicles • Arf GTPases play important roles during membrane trafficking steps in eukaryotic cells . • Arf GTPases recruit COPI and clathrin protein coats to transport vesicles, whereas a specific subset of the Arf GTPase family, the Sar1p GTPases, recruit COP-II coats.

  26. Arl GTPases: Regulatory Proteins with Mystery Functions • In plants, mutation of the TITAN5 gene, which corresponds to AtARLC1, results in dramatic alterations of mitosis and cell cycle control during seed development .The authors speculated that this gene might play a role in membrane trafficking steps necessary for proper cell plate deposition during cytokinesis in developing embryos .

  27. Arf GTPase-Interacting Proteins • Arf GTPase cycle is regulated by GEFs and GAPs.

  28. In animals, ArfGAPs are a diverse family of multidomain proteins (Donaldson, 2000) that contain a zinc-finger motif and a conserved Arg residue within the ArfGAP catalytic domain . • Arabidopsis contains 15 proteins with ArfGAP domains, and we have termed these Arf GAP domain (AGD) proteins. We grouped the AtAGD proteins into four distinct classes based on phylogenetic analysis .

  29. Ran GTPases In animals, Ran GTPases together with their regulatory factors, the Ran-binding proteins (RanBPs), RCC1 (a GEF, RanGEF) and RanGAP (a GAP), play key roles in controlling nuclear processes throughout the cell mitotic cycle. 动物中，Ran GTPase 和它的调控因子, Ran 结合蛋白(RanBPs) 、Ran 鸟核苷酸交换因子RCC1(RanGEF) 和Ran GTPase 激活蛋白RanGAP，在细胞有丝分裂周期中起着重要的调控作用。

  30. Ran GTPases, like other small GTPases, cycle between GDP-and GTP-bound states. However, for Ran GTPases, GTP binding and hydrolysis is linked to transport into or out of the nucleus . Ran GTPase，同其他小G 蛋白一样，也有GDP和GTP-结合态循环方式，而且这种循环方式还与核孔运输有关。

  31. Also, unlike other small GTPases, Ran GTPases are not posttranslationally lipid modified and do not associate with cellular membranes . Four Ran GTPases are present in Arabidopsis . AtRAN1, AtRAN2, and AtRAN3 were identified by sequence similarity . The fourth gene, AtRAN4, is annotated as “salt stress-inducible small GTP-binding protein Ran1-like protein,” but so far no data has been published. 与其他小G 蛋白不同，Ran GTPase 没有转录后脂类修饰的改变，不与细胞膜耦联。拟南芥中存在4 个Ran GTPase ,其中 AtRAN1、AtRAN2 和AtRAN3 有蛋白序列同源性，而AtRAN4 与前3 种序列同源性低，它是作为盐胁迫诱导的类RNA1GTP 结合蛋白而被发现的，有关它的功能研究迄今未见报道。

  32. At the protein level, AtRAN1, AtRAN2, and AtRAN3 are nearly identical (95%–96% of identity) differing only in their C-terminal regions, whereas AtRAN4 is more divergent with only 65% identity to the other AtRAN sequences. 蛋白水平上，AtRAN1-3之间同源性很高(95%-96%)，只是在C 端区域有差异，而AtRAN4 与它们只有65% 的同源性。

  33. Ran GTPases功能 • 动物中Ran GTPases功能 • 拟南芥中Ran GTPases和其相关调控蛋白的功能

  34. 动物中Ran GTPases功能 在动物间期细胞中，RanGTPase 参与核质运输。RanGAP是胞质蛋白，而RanGEF定位在核中，这样的定位方式说明，GTP 结合方式的RanGTPase 存在于核中，而细胞质中是GDP 结合方式的Ran GTPase，这种局限于核中的RanGTP 定位方式是保证核孔运输蛋白和RNA等大分子所必需的 。 ★RanGAP可以激活RanGTPase 的内在GTPase 活性 ★RanGEF可产生RanGTP

  35. RanGTP/RanGDP 浓度梯度驱动蛋白的核输入和核输出过程依赖于一种微弱的蛋白与蛋白间交互作用. Importinβ与RanGTP 和RanGDP 的状态密切关联. 一般情况下,importinβ的入核需要RanGDP , 而出核需要RanGTP. ★Importinβ是核质转运的受体，负责细胞内大部分蛋白质和核酸等生物大分子的跨核膜运输。

  36. RanGTPase 在细胞有丝分裂中也起着重要作用，RCC1与染色质的结合导致RanGTP的产生并定位于染色体附近，促进纺锤体的形成; 在有丝分裂末期，RanGDP和RanGTP的循环可能通过控制囊泡结合和融合诱导核孔复合体的重组装过程。

  37. 拟南芥中Ran GTPases和其相关调控蛋白的功能 植物中RanGTPase 的研究工作很少，对之相互作用的调控蛋白研究进展也刚刚开始。在植物生长和发育中，阐述RanGTPase 和RanBPs 作用的直接证据还没有得到。但通过研究，对植物中RanGTPase功能有以下几个推测。

  38. ①动物Exportin-1是带有核输出信号(NES) 蛋白的核输出受体，它与带有NES 信号的蛋白以及RanGTPase 共同作用形成三聚体，通过核孔从细胞核到细胞质。拟南芥的AtRAN1 可与AtXPO1(Exportin-1 的同源蛋白)、AtRanBP1a( 一种含NES信号的蛋白质)相互作用，这些结果暗示核孔运输机制可能在植物中也很保守。 ②反义表达AtRanBP1c,可以使拟南芥的主根增长和抑制侧根的生长，并且其根系对生长素的反应变得更加敏感，此外，AtRanBP1c在运输抑制生长素反应的核蛋白过程中起着重要作用，并且调控根尖的有丝分裂。近期的实验结果还显示，小麦的TaRAN1基因也在分生组织分裂旺盛的器官中表达水平较高，它在酵母细胞中可以明显影响细胞的形态建成和有丝分裂进程，这可能为研究植物RAN 基因又提供了一个新的证据。

  39. 展望 从这些研究结果可以看出小G蛋白在植物中是重要的多功能调控分子，有许多实验表明，植物的小G蛋白可能与相应的动物和酵母蛋白起着相同的生理作用。除此之外，这些小G蛋白亚家族的GAP和GEF蛋白的发现也进一步证实在真核生物进化中这些小G蛋白调控分子的高度保守性。但是，植物在这些保守的调控机制方面还是存在一些多样性。 小G蛋白功能相似

  40. Arf Rab Rho Ran 在拟南芥中已发现Rab、Rho 、Arf 和Ran GTPase，但是没有发现RasGTPase，这主要是与真核生物调控机制的进化有关。酪蛋白激酶受体在动物细胞中是Ras 的上游调控分子，植物中缺少RasGTPase 是与植物中缺少酪蛋白激酶受体有关。

  41. Plant Rab GTPases segregate into distinct subfamilies that appear to be organized around the types of compartments upon which they are localized. 植物RabGTPase 分为不同的亚家族，分别起着不同的生理作用，这也与它们不同的亚细胞定位相一致。

  42. Plant ArfGTPases and RanGTPases functionally complement mutations of their respective counterparts in yeast. However, plants contain multiple, novel ArfGEFs and ArfGAPs, probably with novel functions. A wealth of knowledge of the mechanisms of action of small GTPases and of some of their important activator proteins (GEFs) and inhibitor proteins (GAPs) will clearly serve as a strong basis for understanding how these conserved regulatory mechanisms have been modified to carry out plant-specific processes. 关于植物Arf和Ran GTPase 生理功能的研究还比较少，但是植物有很多新发现的ArfGEFs和ArfGAPs，它们可能起着与生长素有关新的生理作用。人们对小G 蛋白和它的重要激活蛋白及抑制蛋白的作用机制了解越来越多，将会为研究它在植物中特有的调控机制奠定基础。

  43. 总结: 小G蛋白家族成员 • Rab和Arf 亚家族：在膜转运过程中起着不同的重要作用； • Rho亚家族：调控肌动蛋白重组过程和参与MAP激酶的细 胞信号转导过程； • Ran亚家族：在核孔位置调节着蛋白和RNA分子的运输过 程； • Ras亚家族：在酵母和哺乳动物中调节细胞分化过程；

  44. Thank you !

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