果蠅高基氏體蛋白GOLPH3,Rotini,與胞吐成員作用的關係探討

The relationship between the Drosophila Golgi protein GOLPH3, Rotini, with exocyst components

莊淋翔(Lin-Hsiang Chuang)

指導教授 ： 周子賓

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10493

摘要

人類的高基氏體硫蛋白三號(Golgi phosphoprotein 3, GOLPH3)，是一個主要分布於高基氏體的細胞質面的膜蛋白，其分子量為34KDa。與其他同為座落於高基氏體基質上的蛋白一樣，GOLPH3參與在物質於高機氏體基質(trans-Golgi matrix)上的順向及逆向運輸。此外也與細胞骨架交互作用以及維持高機氏體的結構。
Rotini (Y. J. Wang et al.) 為GOLPH3在果蠅中的同源物 ，先前的研究已知Rotini 協助硫醣蛋白HSPGs (Heparan Sulfate Proteoglycans)的聚合酵素 Exostosin (EXTs)蛋白在高基氏體的運輸 ，當Rotini基因在細胞中缺失時 ,會造成EXT蛋白的運輸異常，因而造成硫醣鏈(glycosaminoglycans , GAG)的多醣鏈聚合反應不完全，進而影響Hedgehog 型態決定素的表現異常以及下游訊號的缺損 。
在卵子細胞發育過程中，早期階段Rti主要表現在生殖細胞(germ cells)，但到了晚期階段則主要表現在濾泡細胞內，Rti 表現位置會隨發育時期推衍而在生殖細胞以及濾泡細胞間轉換。加上先前研究得知當Rti在生殖細胞內缺失會造成胚胎扭曲及Dorsal 蛋白異常分佈表現。這些結果顯示Rti 調控胚胎背腹軸發育形成，且Rotini 可能在卵母細胞發育的過程中扮演了特定的角色。
為了更了解Rotini這個蛋白在細胞內運輸是否有所扮演其他角色,我們利用酵母菌雙雜交搜尋系統(yeast two-hybrid screen system)，本論文首先證實一個參與細胞胞吐(exocytosis)相關的蛋白Sec6與Rotini蛋白有交互作用。Sec6蛋白是一個胞吐作用成員，參與將物質從高基氏體運往細胞膜的運輸。經由GST 融合蛋白沉澱實驗(GST pull down assay)確定了Rotini蛋白與Sec6蛋白有直接的交互作用。另外，在果蠅S2 細胞染色中觀察到Rotini 蛋白會與胞吐作用的成員Sec5、Sec6及Sec8部份座落在一起，進一步經由免疫沉澱實驗(Co-immunoprecipitation)證明Rotini會與胞吐作用複合體中的Sec5及Sec8有交互作用。
在果蠅卵母細胞(oocyte)中，蛋白酶Gastrulation Defective (Gd)、Snake (Snk)、Easter (Ea)及Spazle(Spz)在果蠅生殖細胞中會在靠近皮層的位置表現。當Rti在生殖細胞缺失時觀察到Snk及Snk-GFP蛋白酶無法被送至貼近皮層的位置而累積在卵母細胞中。而當Rti在生殖細胞缺失時則是觀察到Gd蛋白酶在皮層的表現量有下降的趨勢。此外，當胞吐作用成員Sec5在生殖細胞缺失時亦觀察與Rti缺失時相似的現象，會使Snk蛋白酶累積在卵母細胞中，而Gd蛋白酶則會在皮層部位表現量下降。由此推測Rti可能參與將這些蛋白酶由卵母細胞分泌至皮層的胞吐作用。
GOLPH3與4-磷脂醯肌醇 (phosphatidylinositol 4-phosphate)有直接的相互作用，且參與了細胞內物質運輸。已知4-磷脂醯肌醇主要分布於高基氏體與分泌性囊泡上。在酵母菌中，4-磷脂醯肌醇透過與Sec4p蛋白結合而參與物質由高基氏體向外運輸。因此Rti或許是以輔助者的角色來協助許多物質的運送，由Rti影響背腹軸決定分子的輸送行為，推測Rti可能藉由和4-磷脂醯肌醇的鍵結參與了許多由高基氏體向外的分泌性運輸。

關鍵字

果蠅 ； 高機氏體蛋白 ； 胞吐作用

並列摘要

Golgi phosphoprotein 3 (GOLPH3), which is localized to the cytoplasmic face of the trans-Golgi, is a membrane protein with a molecular weight of 34 kD. GOLPH3 is one of the many proteins in the trans-Golgi matrix and is involved in anterograde and retrograde Golgi traffic, and in interactions with the cytoskeleton and maintenance of the Golgi structure.
Previous studies have proven that Rotini, a Drosophila homologue of human GOLPH3, is necessary for the retrograde trafficking of the exostosin (EXT) proteins in Golgi (Y. J. Wang et al.). EXT proteins are crucial to the glycosaminoglycans (GAG) chains biogenesis of the Heparan Sulfate Proteoglycans (HSPGs). Polymerization of GAG chains is defective when the trafficking of EXT proteins is disturbed in rti mutant cells. The morphogen Hedgehog (Hh) and its downstream signal transduction pathway are also affected in rti mutant cells.
During Drosophila oogenesis, it was observed that rti is expressed in both follicle and germ cells at early stages, but mostly expressed in follicle cells at later stages when the egg chamber developed. In addition, previous data has shown that in rti loss-of-function in germline, the embryos are twisted and the distribution of Dorsal protein is affected. These results indicate that Rti participates in D/V axis formation of embryo, and it is suggested that rti could play a role in Drosophila oogenesis.
To elucidate the possible roles of Rti in endo-membrane trafficking, we used yeast two-hybrid (Y2H) screen system to search for factors interacting with Rti. We found and confirmed that Sec6, an exocyst component, has physical interaction with Rti. Sec6 is involved in vesicles trafficking after post-Golgi to the plasma membrane. It was further proved that Rti has physically interacts wih Sec6 through GST pull down assay. In the staining of Drosophila S2 cell, it was shown that Rti partially co-localized with exocyst components Sec5, Sec6, and Sec8. And it was proven that Rti could be co-immunoprecipitated with Sec5 and Sec8.
Through immune staining, the behaviors of serine protease cascade proteins (Gd, Snk, Ea, and Spz) in D/V development in Drosophila oogenesis was observed, and they were mostly localized in the cortex of germ cell. It was found that rti loss-of-function in germline reduced the stability of Snk and Snk-GFP proteins at the cortex of oocyte, and Snk would even clump over the space of oocyte.
Additionally, It was observed that rti loss-of-function in germline reduced the localization stability of Gd proteins at the cortex of oocyte. These irregular distribution of Snk described above in rti germ-line clone is similar to that of the exocyst component Sec5E13 germ-line clone. It was observed that Sec5E13 loss-of-function in germline reduced the localization stability of Snk proteins at the cortex of oocyte and caused the Snk protein to clump over the space of oocyte. Moreover, in Sec5E13 germline clone oocyte reduced the localization stability of Gd proteins at the cortex of oocyte.
From the above results, it suggests that Rti may participate in the secreted transport of these proteases to the oocyte cortex.
Recently published papers have proven the direct interaction between GOLPH3, Rti homologue in human and phosphatidylinositol 4-phosphate (PtdIn4P). Furthermore, GOLPH3 were found to be involved in different trafficking pathways in the cell. PtdIn4P is specifically enriched on Golgi apparatus and secretory vesicles. In yeast PtdIn4P performs an essential role in secretory pathway through binding with Sec4p to be involved in the exocytosis of cargo from trans-Golgi network (TGN). Thereby Rti may be an auxiliary to participate in multiple trafficking pathways for different protein effectors. In other words, Rti may collaborate with PtdIn4P in the sorting of proteins into transport vesicles for exocytosis.

並列關鍵字

Drosophila ； Golgi protein ； exocytosis

參考文獻

Albertson, R., Riggs, B., & Sullivan, W. (2005). Membrane traffic: a driving force in cytokinesis. [Review]. Trends Cell Biol, 15(2), 92-101.

Andrews, H. K., Zhang, Y. Q., Trotta, N., & Broadie, K. (2002). Drosophila sec10 is required for hormone secretion but not general exocytosis or neurotransmission. Traffic, 3(12), 906-921.

Arlaud, G. J., Volanakis, J. E., Thielens, N. M., Narayana, S. V., Rossi, V., & Xu, Y. (1998). The atypical serine proteases of the complement system. Adv Immunol, 69, 249-307.

Barr, F. A., & Gruneberg, U. (2007). Cytokinesis: placing and making the final cut. [Research Support, Non-U.S. Gov'tReview]. Cell, 131(5), 847-860.

Beronja, S., Laprise, P., Papoulas, O., Pellikka, M., Sisson, J., & Tepass, U. (2005). Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells. J Cell Biol, 169(4), 635-646.