Gene silencing reveals a specific function of hVps34 phosphatidylinositol 3-kinase in late versus early endosomes
Gene silencing reveals a specific function of hVps34 phosphatidylinositol 3-kinase in late versus early endosomes
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DOI：
10.1242/jcs.02833
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发表时间：
2006-04-01
影响因子：
4
通讯作者：
Maltese, WA
中科院分区：
文献类型：
--
作者：
Johnson, EE;Overmeyer, JH;Maltese, WA
人 III 型磷脂酰肌醇 3-激酶 hVps34 将磷脂酰肌醇 (PtdIns) 转化为磷脂酰肌醇 3-磷酸 [PtdIns(3)P]。使用磷脂酰肌醇 3-激酶抑制剂的研究表明，PtdIns(3)P 的产生对于各种囊泡介导的运输事件非常重要，包括胞吞作用、多囊核内体中的受体分选以及溶酶体酶从反式高尔基体网络 (TGN) 到核内体和溶酶体的运输。本研究利用小干扰 (si)RNA 介导的基因沉默来定义 hVps34 在人 U-251 胶质母细胞瘤细胞中发挥作用的特定运输途径。 hVps34 表达的抑制降低了细胞生长速率，并导致含有溶酶体膜蛋白 LAMP1 和 LGP85 的大酸性相透明液泡的显着积累。通过电子显微镜对这些结构的分析表明，它们代表了肿胀的晚期内体，这些内体已经失去了向内囊泡形成的能力，但保留了与溶酶体融合的能力。晚期内涵体区室的形态扰动伴随着组织蛋白酶 D 的内涵体中间形式加工成成熟溶酶体形式的速率降低。配体刺激后表皮生长因子受体 (EGFR) 去磷酸化和降解的速率也有所降低，这与 EGFR 保留在扩大的晚期内涵体的限制膜上一致。相比之下，hVps34表达的抑制并不能阻止组织蛋白酶D在TGN和晚期内体之间的运输，或液相标记物的内吞摄取，或PtdIns(3)P结合蛋白EEA1与早期内体的关联。根据其无法结合 PtdIns(3)P 探针 GFP-2xFYVE 的能力来判断，hVps34 敲低细胞中的 LAMP1 阳性液泡耗尽了 PtdIns(3)P。相比之下，LAMP1 阴性囊泡在敲低细胞中继续结合 GFP-2xFYVE。总体而言，这些发现表明 hVps34 在生成 PtdIns(3)P 并促进多囊泡/晚期内体中内囊泡形成方面发挥着重要作用。这些发现还出乎意料地表明其他渥曼青霉素敏感激酶和/或多磷酸肌醇磷酸酶可能能够补偿 hVps34 的损失并维持早期内吞途径或 TGN 中囊泡运输所需的 PtdIns(3)P 水平。
The human type III phosphatidylinositol 3-kinase, hVps34, converts phosphatidylinositol (PtdIns) to phosphatidylinositol 3-phosphate [PtdIns(3)P]. Studies using inhibitors of phosphatidylinositide 3-kinases have indicated that production of PtdIns(3)P is important for a variety of vesicle-mediated trafficking events, including endocytosis, sorting of receptors in multivesicular endosomes, and transport of lysosomal enzymes from the trans-Golgi network (TGN) to the endosomes and lysosomes. This study utilizes small interfering (si)RNA-mediated gene silencing to define the specific trafficking pathways in which hVps34 functions in human U-251 glioblastoma cells. Suppression of hVps34 expression reduced the cellular growth rate and caused a striking accumulation of large acidic phase-lucent vacuoles that contain lysosomal membrane proteins LAMP1 and LGP85. Analysis of these structures by electron microscopy suggests that they represent swollen late endosomes that have lost the capacity for inward vesiculation but retain the capacity to fuse with lysosomes. Morphological perturbation of the late endosome compartment was accompanied by a reduced rate of processing of the endosomal intermediate form of cathepsin D to the mature lysosomal form. There was also a reduction in the rate of epidermal growth factor receptor (EGFR) dephosphorylation and degradation following ligand stimulation, consistent with the retention of the EGFR on the limiting membranes of the enlarged late endosomes. By contrast, the suppression of hVps34 expression did not block trafficking of cathepsin D between the TGN and late endosomes, or endocytic uptake of fluid-phase markers, or association of a PtdIns(3)P-binding protein, EEA1, with early endosomes. LAMP1-positive vacuoles were depleted of PtdIns(3)P in the hVps34-knockdown cells, as judged by their inability to bind the PtdIns(3)P probe GFP-2xFYVE. By contrast, LAMP1-negative vesicles continued to bind GFP-2xFYVE in the knockdown cells. Overall, these findings indicate that hVps34 plays a major role in generating PtdIns(3)P for internal vesicle formation in multivesicular/late endosomes. The findings also unexpectedly suggest that other wortmannin-sensitive kinases and/or polyphosphoinositide phosphatases may be able to compensate for the loss of hVps34 and maintain PtdIns(3)P levels required for vesicular trafficking in the early endocytic pathway or the TGN.