陳瑞華臺灣大學：分子醫學研究所袁維謙Yuan, Wei-ChienWei-ChienYuan2007-11-262018-07-092007-11-262018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/51376KLEIP (kelch-like ECT2 interacting protein) 包含一個BTB區塊、一個BACK區塊及六個kelch重複片段。在之前的研究中，我們證實了KLEIP、死亡相關蛋白激酶(DAPK)及Cul3三者會形成一E3泛素結合酶複合體，並得以藉由泛素化的作用機制來調控死亡相關蛋白激酶的穩定程度。在本篇論文中，我們找出了KLEIP在細胞中的所在位置。KLEIP位於細胞與細胞相接處及高基氏體，更進一步我們以蔗糖梯度證明KLEIP位於高基氏體。KLEIP聚集於高基氏體需要actin的聚合作用及Arf1的活性以參與actin的聚合作用。KLEIP對於維持高基氏體的結構是很重要的，像是利用小片段干擾核甘酸(siRNA)抑制KLEIP的表現會導致細胞中的高基氏體出現片段化及腫脹的現象。除此之外，抑制KLEIP的表現也會導致小囊泡從高基氏體送去細胞膜的速度變慢，但是不會影響小囊泡從細胞膜送去高基氏體的速度。除了位於高基氏體之外，在PML蛋白及類泛素(SUMO)大量表現時，KLEIP亦會聚集於PML核體，我們推論KLEIP可能會被類泛素修飾或是與類泛素相互作用。總而言之，我們證明KLEIP會參與傳輸機制中的順行傳輸(anterograde)。我們假設KLEIP影響和actin相關的傳輸步驟。再者，KLEIP在細胞中存在於許多地方也暗示它扮演著其他與傳輸無關的功能。KLEIP (kelch-like ECT2 interacting protein) contains one BTB/POZ domain, one BACK and six kelch repeats. Previous study in our laboratory demonstrated that KLEIP, DAPK, and Cul3 form an E3 ubiquitin ligase complex to regulate the stability of DAPK by mediating its ubiquitination. In this thesis, we identify the subcellular localizations of KLEIP. KLEIP is present in both cell-cell contact and Golgi apparatus. The Golgi localization of KLEIP was further confirmed by the cofractionation of KLEIP with Golgi proteins. The recruitment of KLEIP to Golgi is dependent on actin polymerization and the activity of Arf1, which is involved in actin polymerization at Golgi. In line with its Golgi residence, KLEIP is crucial for maintaining Golgi architecture, as the ultrastructure of Golgi complex is fragmented with shortened and swollen cisternae in KLEIP knockdown cells. In addition, knockdown of KLEIP leads to the delay of post-Golgi transport to plasma membrane without affecting retrograde traffiking from plasma membrane to Golgi. In addition to localizing in Golgi, KLEIP can also be recruited to PML nuclear body when PML or SUMO is overexpressed, suggesting that KLEIP may be modified or associated with SUMO. Together, this study uncovers a role of KLEIP in the anterograde trafficking process. We hypothesize that KLEIP affects an actin-dependent trafficking step. Furthermore, the existence of KLEIP in multiple subcellular compartments implies its involvement in trafficking-unrelated cellular functions.Table of content 2 中文摘要 5 Abstract 6 Introduction 7 The kelch-repeat superfamily 7 BTB-kelch protein 8 KLEIP 10 Golgi complex 11 Golgi complex and actin 13 Material and method 18 Generation and purification of KLEIP antibody 18 Cell culture and transient transfection 18 Establishment of KLEIP knockdown cell by lentivirus system 19 Antibodies and Reagents 19 RT-PCR and semi-quantitative PCR analysis 20 Western blotting and immunoprecipitation 20 Immunofluorescence and microscopy 21 Sucrose gradient 22 Transmission electron microscope 22 VSVG and cholera toxin B transport assay 23 Calcium switch 23 Result 24 Generation and characterization of polyclonal KLEIP antiserum 24 Identification of a novel form of KLEIP at 130 kDa 25 Identification of endogenous KLEIP expression in various cell lines 26 KLEIP is localized at Golgi complex and cell-cell contact, but not colocalized with stress fiber and ER 26 KLEIP associates with Golgi complex 27 KLEIP is recruited to adheren junction during the process of calcium-induced cell-cell adhesion. 28 KLEIP is localized in PML nuclear body under PML or SUMO overexpression 28 BFA treatment leads to KLEIP redistribution 29 Cytochalasin D treatment results in KLEIP redistribution to cytoplasm where it is partially colocalized with actin filaments and patches. 30 Knockdown of KLEIP in HeLa cells alters the Golgi morphology. 31 Overexpression of KLEIP does not affect the anterogrde transport 32 Knockdown of KLEIP expression leads to the delay of anterograde transport process. 33 Re-expression of BTB-BACK segment of KLEIP in KLEIP knockdown cells rescues the defect of anterograde transport process. 34 KLEIP overexpression or KLEIP knockdown dose not affect retrograde transport process 35 Discussion 37 References 42 Figures 52 Figure 1. Characterization of anti-KLEIP polyclonal antibody. 52 Figure 2. Purification of anti-KLEIP polyclonal antibody. 53 Figure 3. The 130 kDa protein detected by purified KLEIP antibody represents a form of KLEIP. 54 Figure 4. The 130 kDa form of KLEIP is resistant to high concentrations of SDS and reducing agent. 55 Figure 5. The 130 kDa KLEIP is not an ubiquitinated form. 56 Figure 6. Identification of endogenous KLEIP expressed in various cell lines 57 Figure 7. Colocalization of KLEIP and Golgi complex in Cos1 cells 58 Figure 8. KLEIP localizes at cell-cell contact, but not colocalizes with stress fiber. 59 Figure 9. Cofractionation of KLEIP with Golgi proteins 60 Figure 10. KLEIP is recruited to adheren junction during calcium-induced cell-cell adhesion 61 Figure 10. (continuous) KLEIP is recruited to adheren junction under the presence of calcium 62 Figure 11. Exogenous KLEIP localizes in PML nuclear body upon PML or SUMO overexpression 63 Figure 12. Endogenous KLEIP localizes in PML nuclear body upon PML overexpression 64 Figure 13. BFA treatment leads to KLEIP redistribution 65 Figure 14. Cytochalasin D treatment leads to KLEIP redistribution 66 Figure 15. Cytochalasin D treatment results in KLEIP redistribution to cytoplasm where it partially colocalizes with actin filaments and patches. 67 Figure 16. Knockdown of KLEIP in HeLa cells alters the morphology of Golgi. 68 Figure 17. Knockdown of KLEIP in HeLa cells leads to fragmentation and swelling of Golgi cisternae 69 Figure 18. Overexpression of KLEIP dose not affect the anterogrde transport 70 Figure 19. Knockdown of KLEIP leads the delay of anterograde transport process 72 Figure 20. Re-expression of flag-KLEIP in KLEIP knockdown cells rescues the defect of anterograde transport process 74 Figure 21. Re-expression of BTB-BACK segment of KLEIP in KLEIP knockdown cells rescues the defect of anterograde transport process. 75 Figure 22. The quantitative results of anterograde transport assays 76 Figure 23. Trafficking defect in KLEIP knockdown cells is not due to glycosylation deficiency. 77 Figure 24. KLEIP overexpression dose not affect retrograde transport process of cholera toxin B 78 Figure 25. KLEIP knockdown dose not affect retrograde transport process of cholera toxin B 80 Figure 26. The quantitative results of retrograde transport assay in knockdown cells 82 Appendix 83 Appendix I. Colocalization of BTB-BACK segment of KLEIP and Golgi complex in Cos1 cells 833580812 bytesapplication/pdfen-US高基氏體運輸類泛素化KLEIPGolgitraffickingSUMOotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51376/1/ntu-96-R94448003-1.pdf