探討DPP-IV穿膜區域的N端和C端鄰近序列對於其嵌入內質網的影響

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研究生：	蔡政翰 Tsai, Cheng-Han
論文名稱：	探討DPP-IV穿膜區域的N端和C端鄰近序列對於其嵌入內質網的影響 Contribution of N- and C-terminal region flanking DPP-IV transmembrane domain for ER Translocation
指導教授：	陳新 Chen, Xin 陳令儀 Chen, Linyi
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2009
畢業學年度：	97
語文別：	英文
論文頁數：	50
中文關鍵詞：	穿膜、內質網
外文關鍵詞：	DPP-IV, transmembrane domain, ER, SRP, translocon
相關次數：	點閱：2 下載：0
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Membrane proteins targeting to the plasma membrane is mediated by the translocon on the endoplasmic reticulum (ER) through a co-translational translocation process. For type II membrane proteins, protein transport is initiated by the interaction of transmembrane domain (TM) with the signal recognition particle, followed by insertion of TM into the translocon, partition of TM into the lipid, and the transportation of the C-terminal region of TM through the translocon. In this study, we used type II membrane protein DPP-IV as a model protein to study the importance of sequence flanking TM segment. DPP-IV has a short amino terminus located in the cytoplasm, a TM segment anchored to the membrane, and the extracellular C-terminal catalytic domain. We found that introducing proline mutation to TM results in failure of some mutant proteins to translocate to ER, leading to the degradation of the proteins in the cytoplasm. Moreover, we discovered the translocational defect of these mutant DPP-IVs could be rescued by replacing the extracellular C terminal domain with a GFP molecule. Our results indicated that the charge difference flanking TM segment and dimerization have no correlation with the expression of GFP-fused proteins. It is the property of fusion protein that plays a crucial role for protein translocation. Surprisingly, we found GFP and MBP has secretory tendency which promote fusion proteins across membrane.

膜蛋白被運送到細胞膜上是經由co-translational translocation的過程，此過程的發生只要與內質網上的translocon有關。對於第二類型膜蛋白來說，新生蛋白的運送主要是由蛋白的穿膜區域和訊號辨識分子(SRP)的交互作用而起始的，在作用之後，新生蛋白的穿膜區域嵌入translocon並且側向平移到膜上，然後穿膜區域的C端部份再經由translocon送到內質網內腔。在這次的研究中，我們使用DPP-IV為模式蛋白去研究穿膜區域兩側序列對於蛋白穿膜的重要性。DPP-IV有一段很短的N端序列在細胞質內並以一個穿膜片段嵌在細胞膜上，而其具有催化活性的C端則是位於細胞外。在我們的研究中發現，將穿膜區域以proline做點突變，在某些位置上的proline突變會導致突變蛋白無法移動到內質網，進而導致在細胞質內被降解。此外，我們發現可以藉由用GFP去置換DPP-IV的 C端區域進而使這些運送缺失的突變蛋白可以正常表現。我們的結果指出在穿膜片段附近的帶電分佈以及蛋白本身是否為二聚物都與GFP融合蛋白的表現無關，而是融合蛋白本身的特性對於蛋白的移動扮演重要的角色。令人驚訝的，我們發現GFP和MBP本身具有被送到細胞外的傾向，這樣可以促使這些融合蛋白穿過膜。

誌謝...............................................................................................................................I
Abstract......................................................................................................................III
中文摘要.....................................................................................................................IV
Contents.......................................................................................................................V
Introduction..................................................................................................................1
    Co-translational translocation............................................................................1
ER translocon.......................................................................................................3
Contribution of charges for the topology of transmembrane proteins...........5
DPP-IV is a type II membrane protein with dimeric structure.......................8
Materials and Methods..............................................................................................11
Materials.............................................................................................................11
Plasmid construction..........................................................................................11
Cell culture..........................................................................................................17
Quantification of Protein concentration...........................................................17
Western blot analysis.........................................................................................18
Cell Surface Biotinylation..................................................................................19
Results.........................................................................................................................21
DPP-IV with L17P, V18P or I20P TM mutation is degraded rapidly in the cytosol..........................................................................................................21
Changing N-terminal charged residues could not rescue the translocational defects of proline mutants.........................................................................22
The translocational defect of DPP-IV TM mutants could be rescued by replacing the extracellular C terminal domain with GFP......................23
C-terminal region of DPP-IV did not contribute to rescue protein translocation...............................................................................................24
Dimerization has no correlation with protein translocation..........................25
The translocational defects of TM mutants were rescued by replacing the extracellular C-terminal domain with MBP, but not CD13...................26
GFP and MBP could be secreted to extracellular space by themselves...................................................................................................27
Conclusion...................................................................................................................28
Discussions..................................................................................................................30
References...................................................................................................................33
Figures.........................................................................................................................37
Figure 1. Proline mutation on the TM affects the DPP IV production..................................................................................37
Figure 2. The change of N-terminal charged residues of TM segment cannot rescue the translocational defect of TM mutants........39
Figure 3. The translocational defect of DPP-IV TM mutants are rescued by replacing the extracellular C terminal domain with a GFP molecule.......................................................................................41
Figure 4. The lengthening TM-fusions did not affect the localization of mutants........................................................................................42
Figure 5. Dimerization of DPP-IV is not the course of translocational defect of TM mutants.................................................................44
Figure 6. The property of protein affects the translocation of fusion proteins........................................................................................45
Figure 7. GFP and MBP could be secreted to extracellular space by themselves...................................................................................46
Tables...........................................................................................................................47
Table 1. TM proline mutation primer list..................................................47
Table 2. The list of lengthening TM primer...............................................48
Table 3. The list of lengthen TM-GFP-fused proteins..............................49
Supplementary............................................................................................................50
S1. Proline mutation on the TM affects the DPP IV production.............50

                                

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