BTB-kelch蛋白KLEIP在Golgi complex之特性分析與功能探討

Abstract

KLEIP (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.