PIKfyve controls fluid phase endocytosis but not recycling/degradation of endocytosed receptors or sorting of procathepsin D by regulating multivesicular body morphogenesis
PIKfyve controls fluid phase endocytosis but not recycling/degradation of endocytosed receptors or sorting of procathepsin D by regulating multivesicular body morphogenesis
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DOI：
10.1091/mbc.e03-04-0222
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发表时间：
2003-11-01
影响因子：
3.3
通讯作者：
Shisheva, A
中科院分区：
文献类型：
--
作者：
Ikonomov, OC;Sbrissa, D;Shisheva, A
哺乳动物磷脂酰肌醇（PtdIns）5-P/PtdIns 3，5-P-2-产生激酶PIKfyve已涉及维持哺乳动物细胞内膜稳态。为了解决PIKfyve在运输过程中的作用，我们检查了诱导表达野生型或激酶缺陷显性阴性形式的稳定人胚肾293细胞系中生物合成，内吞和再循环途径的功能。PIKfyve（WT）或PIKfyve（K1831 E）表达不影响新合成的组织蛋白酶原D的加工和溶酶体靶向。同样，转铁蛋白摄取/回收或表皮生长因子受体降解的速率在任一蛋白质表达后均未改变。相比之下，PIKfyve（K1831 E）而不是PIKfyve（WT）表达显著损害了液相标记辣根过氧化物酶的晚期摄取。在瞬时表达PIKfyve（K1831 E）的COS细胞中，通过具有特异性标记物的共聚焦显微镜检查细胞器形态显示，尽管PIKfyve（K1831 E）诱导的内膜空泡化显著，但高尔基体、末端溶酶体和再循环隔室与未转染的细胞不可区分。与此相反，我们观察到一个显着的影响，后期内吞隔室，标志着破坏葡聚糖标记的核周内体隔室和分散的扩大囊泡的形成。电子显微镜鉴定了表达PlKfyve（K1831 E）的人胚肾293细胞中的细胞质空泡，其为扩大的多泡体样结构，内部囊泡和膜涡的数量显著减少。总之，这些数据表明，PIKfyve通过控制多泡体的形态发生和功能，选择性地调节含有溶酶体定向液相货物的外周内体的分选和运输。
The mammalian phosphatidylinositol (PtdIns) 5-P/PtdIns 3,5-P-2-producing kinase PIKfyve has been implicated in maintaining endomembrane homeostasis in mammalian cells. To address the role of PIKfyve in trafficking processes, we examined the functioning of the biosynthetic, endocytic, and recycling pathways in stable human embryonic kidney 293 cell lines inducibly expressing the wild-type or kinase-defective dominant-negative form. PIKfyve(WT) or PIKfyve(K1831E) expression did not affect the processing and lysosomal targeting of newly synthesized procathepsin D. Likewise the rates of transferrin uptake/recycling or epidermal growth factor receptor degradation were not altered upon expression of either protein. In contrast, PIKfyve(K1831E) but not PIKfyve(WT) expression markedly impaired the late uptake of fluid phase marker horseradish peroxidase. Inspection of the organelle morphology by confocal microscopy with specific markers in COS cells transiently expressing PIKfyve(K1831E) Showed the Golgi apparatus, end lysosomes, and the recycling compartment indistinguishable from nontransfected cells, despite the dramatic PIKfyve(K1831E)-induced endomembrane vacuolation. In contrast, we observed a striking effect on the late endocytic compartment, marked by disruption of the dextran-labeled perinuclear endosomal compartment and formation of dispersed enlarged vesicles. Electron microscopy identified the cytoplasmic vacuoles in the PlKfyve(K1831E)-expressing human embryonic kidney 293 cells as enlarged multivesicular body-like structures with substantially lower number of internal vesicles and membrane whorls. Together, these data indicate that PIKfyve selectively regulates the sorting and traffic of peripheral endosomes containing lysosomaly directed fluid phase cargo through controlling the morphogenesis and function of multivesicular bodies.