Molecular characterization of an Arabidopsis SH3 domain-containing protein.

• Other Authors: Zhuang, Xiaohong
• Format: Others
• Language: English; Chinese
• Published: 2013
• Subjects: Autophagic vacuoles; Arabidopsis > Molecular aspects
• Online Access: http://library.cuhk.edu.hk/record=b5934663; http://repository.lib.cuhk.edu.hk/en/item/cuhk-328390

在真核细胞中，细胞自噬是一个将细胞物质吞噬到自噬体降解的保守的代谢过程。自噬体起始于自噬前体结构(PAS) 并由其逐渐扩展和延伸形成其最后的双膜结构，最后和溶酶体（lysosome）或液泡（vacuole）融合得以降解。在酵母和动物细胞中，研究已发现一系列自噬体相关基因（ATG）蛋白参与调控自噬体的形成。自噬体形成的相关研究存在两个主要的未解决的问题，它们包括自噬体的膜来源和膜变形机制。而在植物中，相当一部分关键的自噬体同源蛋白的缺失导致其分子机制研究仍处于初步阶段。在本研究中，我主要通过利用SH3P2，一个N 端含有BAR (Bin-Amphiphysin-Rvs) 结构域及C 端含有SH3（Src homology 3）结构域的蛋白作为探针, 在拟南芥中研究自噬体的形成. 在进一步的研究中，借助了免疫细胞化学技术（抗体），分子技术（萤光蛋白标记），基因技术（RNAi 干扰）以及蛋白作用（酵母双杂交和免疫共沉淀）等不同的生化以及细胞生物学手段，我发现在植物细胞中也存在保守的自噬体的形成模式，而在其过程中, SH3P2 起着重要的调控作用。通过研究我发现：1）在拟南芥植物中，绿色荧光蛋白标记的SH3P2-GFP 蛋白具有对自噬诱导的应答反应；；2）在拟南芥转基因植物和PSBD 悬浮细胞中，SH3P2-GFP 蛋白与自噬体标记蛋白共定位； 3） 在自噬途径中，SH3P2-GFP 活跃地参与在自噬体膜变形过程中并且定位在自噬前体结构包括其扩展结构的膜上； 4）基因敲低SH3P2 在拟南芥植物中是致死的并且抑制自噬体的形成过程；5) SH3P2 能通过它的BAR 结构域互相聚合; 6）SH3P2 可以结合磷脂酰肌醇-3-磷酸（PI3P）并且与磷脂酰肌醇-3-激酶复合体存在联系；7）SH3P2 通过它的SH3 结构域直接与ATG8 结合。综上所述，此项研究发掘了一个新型的膜相关蛋白SH3P2 参与在拟南芥植物自噬途径中，而其与ATG8 的直接相互结合同时也揭示了一个新的自噬形成调控机制。 === In eukaryotic cells, autophagy is a conserved catabolic mechanism by engulfing the cytoplasmic cargoes into a structure termed autophagosome. In general, autophagosome is initiated from a site named PAS (phagophore assembly site preautophagosome structure), which then expands and elongates to form a double membrane structure. Ultimately, the outer membrane of autophagosome will fuse with the lysosome or vacuole membrane and deliver the cargoes for degradation or recycling. In yeast and animal cells, a number of ATGs (autophagy related genes) have been identified to regulate the autophagosome formation. Studies of the autophagosome formation involve two main unsolved questions: the membrane origin and the membrane deformation mechanism. In plants, several key players responsible for autophagosome biogenesis are missing and the molecular mechanisms for the autophagosome formation remain elusive. In this study, I have used SH3P2, which contains a N-terminus BAR (Bin-Amphiphysin-Rvs) domain and C-terminus SH3 (Src homology 3) domain, as a probe, to study the autophagosome formation in plants. Using a combination of immunocytochemical (antibodies), molecular (GFP fusions), genetic (RNAi) and interaction (Yeast two-hybrid and Co-IP) approaches, I have shown that a conserved autophagosome formation model exists in plant cells and SH3P2 plays an essential role in the autophagy pathway in Arabidopsis thaliana. I have found that 1) SH3P2-GFP fusion proteins response to autophagic induction in transgenic Arabidopsis plants; 2) SH3P2-GFP colocalize with the known autophagosome markers in both transgenic Arabidopsis plant and PSBD cells; 3) SH3P2-GFP localizes on the PAS membrane and actively participates in membrane deformation events during autophagosome formation throughout its expansion process via the dynamic and ultra structural analysis; 4) Knock-down of SH3P2 is developmental lethal and suppresses the autophagosome formation and autophagic flux; 5) SH3P2 has a self-interaction via its BAR domain; 6) SH3P2 binds to PI3P (Phosphatidylinositol-3-Phosphate) and associates with the PI3K (Phosphatidylinositol-3-Phosphate Kinase) complex; 7) SH3P2 directly interacts with ATG8 via its SH3 domain. Taken together, this thesis research has identified a novel membrane-associated protein and demonstrated its essential role in autophagy in plant. The demonstration for the direct association between SH3P2 and the ATG8 complex may provide an insightful mechanism for autophagosome regulation in Arabidopsis thaliana. === Detailed summary in vernacular field only. === Zhuang, Xiaohong. === Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. === Includes bibliographical references (leaves 97-104). === Abstracts also in Chinese. === Statement --- p.I === Abstract --- p.II === 摘要 --- p.IV === Acknowledgements --- p.VI === Table of Contents --- p.VIII === List of Tables --- p.X === List of Figures --- p.XI === Chapter Chapter 1 --- General Introduction --- p.1 === Chapter 1.1 --- Introduction of Autophagy --- p.2 === Chapter 1.2 --- Molecular Machinery for Autophagy --- p.5 === Chapter 1.3 --- Membrane Origins of Autophagosome --- p.8 === Chapter 1.4 --- Membrane Sensors for Autophagosome Formation --- p.10 === Chapter 1.4.1 --- ATG14 --- p.10 === Chapter 1.4.2 --- Bif1 (Bax-interacting factor 1) --- p.11 === Chapter 1.5 --- Autophagy in Plants --- p.12 === Chapter 1.6 --- Research Objectives --- p.13 === Chapter Chapter 2 --- SH3P2 Defines a Conserved Autohagosome Formation Process in Arabidopsis --- p.15 === Chapter 2.1 --- Introduction --- p.16 === Chapter 2.2 --- Materials and Methods --- p.19 === Chapter 2.2.1 --- Plasmid Construction --- p.19 === Chapter 2.2.2 --- Plant Materials, Growth and Treatment Conditions --- p.24 === Chapter 2.2.3 --- Transient Expression in Protoplasts and Confocal Imaging --- p.24 === Chapter 2.2.4 --- Antibody Generation, Protein Extraction and Western Blot Analysis --- p.25 === Chapter 2.2.5 --- Immunofluorescence Confocal Study --- p.26 === Chapter 2.2.6 --- Electron Microscopy (EM) Study --- p.26 === Chapter 2.2.7 --- Accession Numbers --- p.27 === Chapter 2.3. --- Results --- p.28 === Chapter 2.3.1. --- SH3P2-GFP Fusion Proteins Response to Autophagic Induction in Arabidopsis --- p.28 === Chapter 2.3.2 --- The SH3P2-GFP Positive Compartments are Overlapped with Autophagosome Markers --- p.36 === Chapter 2.3.3 --- Dynamic Analysis of SH3P2-GFP Positive Compartments in Arabidopsis Transgenic Plants upon Autophagic Induction --- p.42 === Chapter 2.3.4 --- EM Analysis of the subcellular localization of SH3P2 after autophagic induction --- p.44 === Chapter 2.4 --- Discussion --- p.52 === Chapter Chapter 3 --- SH3P2 is Essential for Plant Development and Autophagic Pathway in Arabidopsis --- p.54 === Chapter 3.1 --- Introduction. --- p.55 === Chapter 3.2.1 --- Plasmid Construction --- p.57 === Chapter 3.2.2 --- Plant Materials, Growth and Treatment Conditions --- p.57 === Chapter 3.2.3 --- Transient Expression in Protoplasts and Confocal Imaging --- p.58 === Chapter 3.2.4 --- Protein Extraction and Immunoblot Analysis --- p.58 === Chapter 3.2.5 --- RT-PCR --- p.59 === Chapter 3.3 --- Results --- p.60 === Chapter 3.3.1 --- RNAi Knockdown of SH3P2 is Developmental Lethal --- p.60 === Chapter 3.3.2 --- RNAi Knockdown of SH3P2 Suppresses the Autophagosome Formation and Autophagic Flux --- p.63 === Chapter 3.4 --- Discussion --- p.71 === Chapter Chapter 4 --- SH3P2 is Associated with the ATG Machinery --- p.73 === Chapter 4.1 --- Introduction --- p.74 === Chapter 4.2 --- Materials and Methods --- p.76 === Chapter 4.2.1 --- Plasmid Construction --- p.76 === Chapter 4.2.2 --- Plant Materials, Growth and Treatment Conditions --- p.76 === Chapter 4.2.3 --- Recombinant Protein Expression --- p.77 === Chapter 4.2.4 --- In Vitro Lipid Binding Assay --- p.77 === Chapter 4.2.5 --- Yeast-two Hybrid Analysis --- p.78 === Chapter 4.2.5 --- Immunoprecipitation Analysis --- p.78 === Chapter 4.3 --- Results --- p.80 === Chapter 4.3.1 --- SH3P2 Binds to PI3P --- p.80 === Chapter 4.3.2 --- SH3P2 Has a Strong Self-interaction via the BAR Domain --- p.82 === Chapter 4.3.3 --- SH3P2 is Associated with the PI3K Complex and Interacts with ATG8 --- p.84 === Chapter 4.4 --- Discussion --- p.86 === Chapter Chapter 5 --- Discussions and Perspectives --- p.87 === Chapter 5.1 --- Discussions --- p.88 === Chapter 5.1.1 --- Autophagosome Formation is Conserved in Arabidopsis thaliana --- p.88 === Chapter 5.1.2 --- SH3P2 Interacts with the ATG8 Complex and is Required for the Autophagic Pathway in Arabidopsis thaliana --- p.90 === Chapter 5.1.3 --- A Novel Membrane-associated Regulator for Autophagosome formaiton in Arabidopsis thaliana --- p.92 === Chapter 5.2 --- Working Model of SH3P2 during Autophagosome Formation in Arabidopsis --- p.93 === Chapter 5.3 --- Future Perspectives --- p.96 === References --- p.97 === List of Publications --- p.104