過度表現果蠅高基氏體蛋白GOLPH3, Rotini, 藉由干擾Wntless蛋白而影響Wingless signaling

Rotini（Rti）是果蠅的人類GOLPH3蛋白同源物，已知Rti是一個在高基氏體負責調控EXT蛋白群回送運輸的蛋白質。EXT蛋白群是生合成HSPG支鏈所需要的酵素群，當Rti增量或缺失時皆會導致HSPG表現量減少以及Hh訊息傳遞路徑的失常。果蠅中Wnt/Wingless（Wg）調控翅脈和翅膀周圍剛毛的形成，與Hh同樣都是會受Rti影響表現的morphogen。我們先前的研究中發現在果蠅翅碟上，Rti增量表現時會導致Wg累積於其表現細胞內而無法送至細胞膜，造成Wg訊息傳導的下游目標基因誘發表現量下降，而Rti缺失時Wg染色則沒有明顯改變。因此我們認為Rti增量表現時會影響Wg的分泌。由文獻得知Wntless（Wls）是負責將Wg由高基氏體送往細胞膜的穿膜蛋白，在Wg表現細胞中會特別聚集在高基氏體而造成較強的染色訊號。當Wg移至細胞膜後，Wls會被內吞回細胞內並由retromer回收至高基氏體以利進行下一循環。Wls缺失會導致Wg蛋白累積在分泌細胞內，這樣的突變性狀類似於Rti增量表現時觀察到的情形。因此本研究目的為探討Rti增量表現是否影響Wls功能而造成Wg堆積在其表現細胞內。在Rti增量表現的Wg表現細胞中，Wls染色訊號下降，而且Wls與高基氏體的標示蛋白Galt-GFP重疊面積下降，顯示Rti影響Wg表現細胞中Wls蛋白的座落。在運用hrsD28突變阻礙溶酶體的蛋白降解路徑的實驗條件下，觀察Wg表現細胞中不同Rti表現量時Wls的染色情形，發現在hrsD28突變的組織中Wls染色不變而Wg染色增加；當加上Rti增量表現時，Wls染色恢復成像wild type，另一方面，當Rti缺失時也是Wls染色不變而Wg分泌量增加。綜合以上我們推測Rti可能如同對於EXT蛋白群的影響，以其參與在回送運輸的功能改變Wg表現細胞中Wls位置。

關鍵字

高基氏體蛋白GOLPH3 ； Wg分泌； Wls ；回送運輸

並列摘要

Rotini (Rti), the Drosophila Golgi protein GOLPH3, has been shown to regulate the biosynthesis of Heparan Sulfate Proteoglycans (HSPGs) by modulating the retrograde trafficking of glycosyltransferases, Exostosins (EXTs). Loss of function of Rti shifts the distribution of EXTs to the trans-Golgi, while overexpression of Rti results in EXTs mislocalization toward cis-Golgi. Both situations interfere with EXTs activity and result in incomplete HSPG and perturbation of Hh signaling. In addition to Hh, Drosophila Wnt/Wingless (Wg) has been found affected by Rti. Wg is required for a wide range of patterning events, including defining the wing blade and specifying the wing margin. It has been found that Rti overexpression causes the wing disc atrophied and Wg accumulation inside Wg-producing cells while loss-of-function of Rti appears to have no effect on Wg expression. Therefore, we proposed that Rti may involve in modulation of Wg secretion. According to studies in the modulation of Wg secretion, Wntless (Wls) is known to be a dedicated transporter for Wg protein from trans-Golgi to plasma membrane. In Wg-producing cells, Wls proteins accumulate in Golgi which results in strengthen Wls staining. After Wg is released, Wls is endocytosed form the cell surface, and then retromer-mediated retrieval brings it back to the trans-Golgi network for maintenance of continuous Wnt secretion. In Wls mutant clone, Wg proteins accumulate inside Wg-producing cells, which is similar to the Wg mutant phenotype observed in Rti overexpression. Therefore, the aim of the thesis is to investigate whether Rti overexpression interferes with Wls function, thus causing Wg accumulation inside Wg-producing cells. When Rti is overexpressed, the reduction of Wls staining in Wg-producing cells disrupts the partial colocalization of Wls with Golgi marker Galt-GFP. These results suggest that Rti affects the localization of Wls in Wg-producing cells. When use hrsD28 mutant to block lysosomal degradation and observed the Wls staining in Wg-producing cells under different Rti expression dosage, we found that Wls staining was unaffected while Wg staining was increased in hrsD28 mutant clone. This suggests that the strong Wls staining in Wg producing cells is not caused by increase of protein stability. In addition, the reduction of Wls staining in Rti overexpression tissue is suppressed in hrsD28 clone. As our expected, the expression in hrsD28 rti double mutant clone is similar to that in hrsD28 mutant clone. Conclusively, as its role in EXTs regulation, in Wg-producing cells, Rti affects Wls localization by retrograde trafficking.

並列關鍵字

Golgi protein GOLPH3 ； Wg secretion ； Wls ； retrograde trafficking

參考文獻

Aberle H, Bauer A, Stappert J, Kispert A, Kemler R (1997) beta-catenin is a target for the ubiquitin-proteasome pathway. The EMBO journal 16: 3797-3804

Arighi CN, Hartnell LM, Aguilar RC, Haft CR, Bonifacino JS (2004) Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor. The Journal of cell biology 165: 123-133

Baeg GH, Lin X, Khare N, Baumgartner S, Perrimon N (2001) Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development 128: 87-94

Banziger C, Soldini D, Schutt C, Zipperlen P, Hausmann G, Basler K (2006) Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125: 509-522

Bartscherer K, Pelte N, Ingelfinger D, Boutros M (2006) Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. Cell 125: 523-533

國際替代計量

過度表現果蠅高基氏體蛋白GOLPH3, Rotini, 藉由干擾Wntless蛋白而影響Wingless signaling

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