Subcellular localization and targeting mechanisms of arabidopsis endomembrane protein 12 (EMP12).

在酵母和动物细胞中，内膜蛋白（EMP）隶属于进化上保守的九跨膜结构域（TMD）蛋白家族，此类蛋白的共同结构特征是有一个很长的N 末端，紧接着九个跨膜结构域后面连着暴露于胞质的C 末端短肽。在黏菌以及酵母中，EMP 蛋白被发现参与蛋白分泌功能以及细胞的贴壁生长。拟南芥基因组中有12 个EMP 编码基因，关于它们所编码蛋白质的定位以及功能甚少有研究报道。在此项研究中，借助于不同的生化以及细胞生物学手段，包括瞬时表达、共聚焦成像、电子显微镜分析、pull down 相互作用蛋白捕获以及质谱分析，我将主要研究拟南芥中EMP12 蛋白的亚细胞定位以及分选信号和蛋白靶定机理。通过研究我发现：1）在拟南芥植物中，内源性的EMP12 蛋白（通过EMP12 特异性抗体标记）和绿色荧光蛋白标记的GFP-EMP12 蛋白都定位于高尔基体；2）C 末端连接的GFP 导致 EMP12-GFP 融合蛋白错误地定位到后高尔基体细胞器，并最终被运送到液泡而降解；3）EMP12 蛋白的C 末端有两个分选信号：内质网输出信号（FV/Y）和一个新发现的高尔基体滞留信号（KXD/E），这两个分选信号分别和COPII 和COPI 囊泡相互作用从而实现其功能；4）把EMP12 的高尔基体滞留信号连接到其他后高尔基体定位的膜蛋白时可以滞留它们在高尔基体。EMP12 中发掘的内质网输出信号和高尔基体滞留信号在所有的植物EMP 蛋白家族中都是非常保守的，这也预测了植物EMP 蛋白家族都通过类似的分选途径而定位于高尔基体并且预示这些保守的分选信号对于植物EMP 蛋白家族的靶定是非常重要的。

Endomembrane Proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N-terminus, nine transmembrane domains (TMD) and a short cytoplasmic tail (CT). In the slime mold and yeast, it has been reported that EMP family proteins are involved in protein secretion function and cell adhesion growth. The Arabidopsis genome contains 12 EMP members (EMP1 to EMP12) with little information about their protein subcellular localization and function. Here I studied the subcellular localization and targeting mechanisms of EMP12 in Arabidopsis through a combination of biochemical and cell biological approaches including transient expression, confocal observation, electron microscopy, pull down and mass spectrometry. I found that 1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; 2) GFP fusion at the C-terminus of EMP12 caused mis-localization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; 3) EMP12 CT contained dual sorting signals: an ER export motif (FV/Y) and a novel Golgi retention signal (KXD/E) that interacted with COPII and COPI subunits respectively to achieve their ER export or Golgi retention functions; 4) the Golgi-retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all the plant EMP isoforms, thus likely representing a general mechanism for EMP targeting in plant cells.

Detailed summary in vernacular field only.

Gao, Caiji.

Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.

Includes bibliographical references (leaves 86-94).

Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

also in Chinese.

Thesis/Assessment Committee --- p.I

Statement --- p.II

p.III

摘要 --- p.V

Acknowledgements --- p.VI

Table of Contents --- p.VIII

List of Tables --- p.XI

List of Figures --- p.XII

List of Abbreviations --- p.XIV

Chapter Chapter 1 --- General Introduction --- p.1

Chapter 1.1 --- The Plant Secretory Pathway --- p.2

Chapter 1.2 --- Sorting Signals for Membrane Protein Trafficking between ER and Golgi --- p.5

Chapter 1.3 --- Endomembrane Proteins (EMPs) in Non-plant Species --- p.7

Chapter 1.4 --- Endomembrane Proteins (EMPs) in Plants --- p.8

Chapter 1.5 --- Objectives of this Research --- p.11

Chapter Chapter 2 --- Materials and Experimental Procedures --- p.12

Chapter 2.1 --- Plasmid Construction --- p.13

Chapter 2.2 --- Plant Materials and Transient Expression in Protoplasts --- p.17

Chapter 2.3 --- Immunofluorescence Study and Confocal Microscopy --- p.17

Chapter 2.4 --- Electron Microscopy (EM) Study --- p.18

Chapter 2.5 --- Antibodies, Protein Preparation and Western Blot Analysis --- p.21

Chapter 2.6 --- In Vitro Binding Assay of COPI and COPII Coat Proteins to Sorting Motifs --- p.22

Chapter 2.7 --- Mass Spectrometry (MS) Identification of Binding Proteins --- p.23

Chapter 2.8 --- Topology Analysis and Protease Protection Assay --- p.23

Chapter Chapter 3 --- Results --- p.25

Chapter 3.1 --- At EMP12 is a Golgi-localized Multiple TMDs Protein --- p.26

Chapter 3.1.1 --- Trypsin Digestion and Topology Analysis of EMP12 --- p.26

Chapter 3.1.2 --- Golgi Localization of Endogenous EMP12 and GFP-EMP12 Fusion in Arabidopsis Plant --- p.29

Chapter 3.1.3 --- Golgi Localization of GFP-EMP12 Fusion in Arabidopsis Protoplasts --- p.36

Chapter 3.2 --- The Cytosolically Exposed C-terminal Region Contains Essential ER Export Signals for the Trafficking of EMP12 from the ER to the Golgi --- p.38

Chapter 3.2.1 --- C-terminus is Essential for ER export of EMP12 --- p.38

Chapter 3.2.2 --- Identification of FV/Y Residues as the ER Export Signals for EMP12 --- p.40

Chapter 3.2.3 --- The ER Export Signals, FV/Y, Can Interact with COPII Subunit Sec24 --- p.46

Chapter 3.3 --- The C-terminal Fused GFP-tag Causes Mislocalization of EMP12-GFP Fusion to TGN, PVC and Vacuole --- p.48

Chapter 3.4 --- The EMP12 CT Contains a Novel KXD/E Motif for Golgi Retention --- p.51

Chapter 3.4.1 --- Identification of the KXD/E Motif for Retaining the Mislocalized EMP12-GFP in the Golgi Apparatus --- p.51

Chapter 3.4.2 --- The Mislocalized EMP12-GFP Fusions Go to Vacuole via PVC for Degradation --- p.58

Chapter 3.4.3 --- Western Blot Analysis of Protoplasts Expressing various EMP12-GFP fusions --- p.61

Chapter 3.5 --- The KXD/E Motif Shows Similar Golgi Retention Function for Other Plant EMP Homologues --- p.63

Chapter 3.6 --- The KXD/E Motif Interacts with COPI Vesicle to Achieve its Golgi Retention Function --- p.65

Chapter 3.7 --- The RNIKCD Functions as a Golgi Retention Motif for Post-Golgi Membrane Proteins --- p.69

Chapter 3.7.1 --- The RNIKCD Can Retain the SCAMP1-GFP in the Golgi Apparatus --- p.69

Chapter 3.7.2 --- The RNIKCD Motif Causes Partial Golgi Retention of TPK1-GFP --- p.71

Chapter 3.8 --- Localization Patterns of Singly-expressed Various EMP12 Fusions and Their Mutants in Arabidopsis Protoplasts --- p.74

Chapter Chapter 4 --- Discussions, Conclusions and Perspectives --- p.76

Chapter 4.1 --- Discussions --- p.77

Chapter 4.1.1 --- The Position of GFP Tag Affects the Proper Golgi Localization of EMP12 --- p.77

Chapter 4.1.2 --- Multiple Sorting Signals and Proper COPII Vesicle Function are Involved in ER Export of EMP12 --- p.79

Chapter 4.1.3 --- KXD/E Motif and COPI Vesicle Mediate Golgi Localization of EMP12 --- p.80

Chapter 4.2 --- Conclusions and Working Model of EMP12 Trafficking --- p.83

Chapter 4.3 --- Future Perspectives --- p.85

References --- p.86

List of Publications --- p.95