Data_Sheet_1_Vps28 Is Involved in the Intracellular Trafficking of Awd, the Drosophila Homolog of NME1/2.docx

The Awd (abnormal wing discs) gene is the Drosophila homolog of human NME1 and NME2 metastasis suppressor genes. These genes play a key role in tumor progression. Extensive studies revealed that intracellular NME1/2 protein levels could be related to either favorable or poor prognosis depending on tissue context. More recently, extracellular activities of NME1/2 proteins have also been reported, including a tumor- promoting function. We used Drosophila as a genetic model to investigate the mechanism controlling intra- and extracellular levels of NME1/2. We examined the role of several components of the ESCRT (endosomal sorting complex required for transport) complex in controlling Awd trafficking. We show that the Vps28 component of the ESCRT−I complex is required for maintenance of normal intracellular level of Awd in larval adipocytes. We already showed that blocking of Shibire (Shi)/Dynamin function strongly- lowers Awd intracellular level. To further investigate this down regulative effect, we analyzed the distribution of endosomal markers in wild type and Shi-defective adipocytes. Our results suggest that Awd does not enter CD63-positive endosomes. Interestingly, we found that in fat body cells, Awd partly- colocalizes with the ESCRT accessory component ALiX, the ALG-2 (apoptosis-linked gene 2)-interacting protein X. Moreover, we show that the intracellular levels of both proteins are downregulated by blocking the function of the Dynamin encoded by the shibire gene.

Awd（abnormal wing discs，翅盘异常）基因是人类NME1与NME2肿瘤转移抑制基因的果蝇同源基因。此类基因在肿瘤进展中发挥关键作用。大量研究表明，细胞内NME1/2蛋白水平与良好或不良预后均存在关联，具体取决于组织背景。近年来，亦有研究报道了NME1/2蛋白的胞外活性，包括其促肿瘤功能。本研究以果蝇作为遗传模型，探究调控NME1/2胞内与胞外水平的机制。我们检测了ESCRT（endosomal sorting complex required for transport，内体分选复合物）复合体的多种组分在调控Awd转运过程中的作用。研究证实，ESCRT-I复合体的Vps28组分对于维持幼虫脂肪细胞内Awd的正常胞内水平是必需的。我们此前已证实，阻断Shibire（Shi）/动力蛋白的功能会显著降低Awd的胞内水平。为进一步探究该下调效应，我们分析了野生型与Shi缺陷型脂肪细胞中内体标志物的分布情况。研究结果表明，Awd无法进入CD63阳性内体。值得注意的是，我们在脂肪体细胞中发现，Awd与ESCRT辅助组分ALiX——即ALG-2（凋亡相关基因2）相互作用蛋白X——存在部分共定位。此外，我们证实，阻断shibire基因编码的动力蛋白功能后，这两种蛋白的胞内水平均会出现下调。