Jaber Nadia, Mohd-Naim Noor, Wang Ziqing, DeLeon Jennifer L, Kim Seong, Zhong Hua, Sheshadri Namratha, Dou Zhixun, Edinger Aimee L, Du Guangwei, Braga Vania M M, Zong Wei-Xing
Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook NY11794, USA.
Molecular Medicine, NHLI, Faculty of Medicine, Imperial College London, London SW7 2AZ, UK.
J Cell Sci. 2016 Dec 1;129(23):4424-4435. doi: 10.1242/jcs.192260. Epub 2016 Oct 28.
The class III phosphoinositide 3-kinase (PI3K) Vps34 (also known as PIK3C3 in mammals) produces phosphatidylinositol 3-phosphate [PI(3)P] on both early and late endosome membranes to control membrane dynamics. We used Vps34-deficient cells to delineate whether Vps34 has additional roles in endocytic trafficking. In Vps34 mouse embryonic fibroblasts (MEFs), transferrin recycling and EEA1 membrane localization were unaffected despite elevated Rab5-GTP levels. Strikingly, a large increase in Rab7-GTP levels, an accumulation of enlarged late endosomes, and decreased EGFR degradation were observed in Vps34-deficient cells. The hyperactivation of Rab7 in Vps34-deficient cells stemmed from the failure to recruit the Rab7 GTPase-activating protein (GAP) Armus (also known as TBC1D2), which binds to PI(3)P, to late endosomes. Protein-lipid overlay and liposome-binding assays reveal that the putative pleckstrin homology (PH) domain in Armus can directly bind to PI(3)P. Elevated Rab7-GTP led to the failure of intraluminal vesicle (ILV) formation and lysosomal maturation. Rab7 silencing and Armus overexpression alleviated the vacuolization seen in Vps34-deficient cells. Taken together, these results demonstrate that Vps34 has a previously unknown role in regulating Rab7 activity and late endosomal trafficking.
III类磷酸肌醇3激酶（PI3K）Vps34（在哺乳动物中也称为PIK3C3）在早期和晚期内体膜上产生磷脂酰肌醇3-磷酸[PI(3)P]，以控制膜动力学。我们使用Vps34缺陷型细胞来确定Vps34在内吞运输中是否具有其他作用。在Vps34基因敲除的小鼠胚胎成纤维细胞（MEF）中，尽管Rab5-GTP水平升高，但转铁蛋白循环和EEA1膜定位未受影响。令人惊讶的是，在Vps34缺陷型细胞中观察到Rab7-GTP水平大幅增加、晚期内体增大并积累，以及表皮生长因子受体（EGFR）降解减少。Vps34缺陷型细胞中Rab7的过度激活源于未能将与PI(3)P结合的Rab7 GTP酶激活蛋白（GAP）Armus（也称为TBC1D2）募集到晚期内体。蛋白质-脂质覆盖分析和脂质体结合分析表明，Armus中假定的普列克底物蛋白同源（PH）结构域可直接与PI(3)P结合。Rab7-GTP升高导致腔内囊泡（ILV）形成失败和溶酶体成熟受阻。Rab7基因沉默和Armus过表达减轻了Vps34缺陷型细胞中出现的空泡化现象。综上所述，这些结果表明Vps34在调节Rab7活性和晚期内体运输中具有先前未知的作用。
