腺嘌呤核苷二磷酸核糖化因子 (ADP-ribosylation factors; Arfs)屬於Ras家族的一員，主要透過結合GTP的活化態構型與結合GDP的失活態構型間循環轉換以調控細胞內囊泡運輸影響其胞器結構和細胞型態。 Arfs與GTP結合後被活化，進而召集鞘蛋白質 (coat protein)或銜接蛋白 (adaptors)幫助囊泡出芽形成，有助於調節胞器間囊泡的運輸。 BIG1和BIG2為受brefeldin A抑制之Arf鳥糞嘌呤核苷酸交換因子(brefeldin A-inhibited Arf guanine nucleotide -exchange factors)，最初發現存在於小牛腦細胞中約670 kDa的蛋白複合物中，BIG1與BIG2具有約200個胺基酸的保存性Sec7結構域，負責加速GDP離開Arfs使GTP進入結合位。BIG1與BIG2主要分布於高基氏體、早期內體、循環內體和細胞膜，據研究指出BIG2參與調控integrin β1再循環的運輸，而BIG1與BIG2皆與細胞遷移的調控有關。 我們的實驗結果顯示BIG1與BIG2能夠和Rab11交互作用，調控內體囊泡的運輸。而最近幾項研究證明小GTP結合酶Arf與Rab透過Rab–ArfGEF– Arf之路徑以協調膜運輸。 我們的實驗證明參與調節內吞囊泡運輸之Rab11蛋白與BIG1和BIG2之間有交互作用。透過酵母菌雙雜交實驗與GST pull down實驗證明BIG1與BIG2的C端片段可與野生型和GTP結合活化型態的Rab11蛋白交互作用。透過共軛焦顯微鏡觀察到內源性BIG1/2與Rab11在細胞核周圍區域位置重疊，並且內源性BIG1與BIG2能夠與Rab11免疫共沉澱，證明細胞內BIG1/2與Rab11的相互作用。 在HeLa細胞中敲除 (knockdown) Rab11會造成細胞內BIG1分布位置的改變，並降低BIG1的蛋白表現量，但並不會使BIG2的位置及蛋白量造成影響。 在HeLa細胞中抑制BIG1及BIG2造成內源性Rab11蛋白量降低。我們利用Balifomycin A1和chloroquine等溶酶體抑制劑來抑制溶酶體降解路徑，抑制敲除BIG1或BIG2造成Rab11的蛋白降解。 CD44是一種細胞表面蛋白，調控細胞遷移 (migration)、細胞粘附 (adhesion)等細胞功能，而BIG1、BIG2或Rab11敲除後造成細胞內源性CD44蛋白表現量下降。 雖然還需要更多實驗來研究BIG1/2是否通過與Rab11的相互作用以調控囊泡內吞作用或是細胞表面蛋白的再循環進而影響細胞遷移，我們實驗結果說明Rab11可能能夠與BIG1和BIG2協同調控囊泡的內吞路徑，進而影響細胞遷移或黏附等細胞功能。

ADP-ribosylation factors (Arfs) belong to the Ras superfamily of small GTPases function through a cycle between active GTP-bound and inactive GDP-bound conformations to regulate intracellular membrane transport, organelle structure, and cell morphology. Activated Arf-GTP proteins recruit coat proteins and/or adaptors to initiate vesicular formation, hence help regulating transportation between organelles. Two brefeldin A-inhibited Arf guanine nucleotide-exchange factors (GEFs), known as BIG1 and BIG2, purified together in ca. 670-kDa multiprotein complexes from bovine brain cytosol, sharing the evolutionary conserved ~ 200 amino-acid Sec7 domain that accelerating the replacement of Arf bound GDP with GTP. BIG1 and BIG2 Localized at Golgi, early endosome, recycling endosome and plasma membrane, and BIG2 was reported to involve in regulating recycling of integrin β1 back to the plasma membrane. Functions of BIG1 and BIG2 are also implicated in regulation of cell migration. Several studies reported recently that Arf and Rab small GTPase act coordinately by Rab-ArfGEF-Arf cascades in regulating membrane trafficking. Here, our data demonstrated that BIG1 and BIG2 interact with Rab11, a protein that participate in regulation of cargo transport back to cell surface in endocytic pathway. Associations of BIG1 or BIG2 with Rab11, wildtype and GTP-bound mutant, but not GDP-bound mutant were confirmed by using yeast two-hybrid and GST pull down assays. The interacting regions were mapped to the C-terminal fragments on BIG1 or BIG2. Apparent overlap of endogenous BIG1/2 with Rab11 in the perinuclear region in confocal microscopy and Rab11 coimmunoprecipitated together with BIG1 or BIG2, suggesting an in vivo interaction of BIG1/2 with Rab11. Knockdown of Rab11 in HeLa cells interfered with BIG1 localization and decreased protein amount of BIG1, but not BIG2. Protein amounts of Rab11 were decreased in BIG1- or BIG2-silencing cells. Inhibition of lysosomal degradation pathway by Balifomycin A1 or chloroquine markedly suppressed Rab11 degradation induced by BIG1 or BIG2 knockdown. Depletion of BIG1, BIG2, or Rab11 resulted to decrease the protein amounts of CD44, a multifunctional cell surface protein with critical roles in cell-cell adhesion, cell migration and progression and metastasis of cancer cells. Although it require more experiments to understand whether BIG1/2 contribute to cell migration through interaction with Rab11 to regulate the endocytic and recycling of cell surface protein, our data here suggest that BIG1/2 may coordinate with Rab11 to regulate the endocytic pathway that may participate in various cell functions such as cell-cell adhesion and cell migration.

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