Du Guangwei, Altshuller Yelena M, Vitale Nicolas, Huang Ping, Chasserot-Golaz Sylvette, Morris Andrew J, Bader Marie-France, Frohman Michael A
Department of Pharmacology, University Medical Center, State University of New York at Stony Brook, Stony Brook, NY 11794, USA.
J Cell Biol. 2003 Jul 21;162(2):305-15. doi: 10.1083/jcb.200302033.
The signaling enzyme phospholipase D1 (PLD1) facilitates membrane vesicle trafficking. Here, we explore how PLD1 subcellular localization is regulated via Phox homology (PX) and pleckstrin homology (PH) domains and a PI4,5P2-binding site critical for its activation. PLD1 localized to perinuclear endosomes and Golgi in COS-7 cells, but on cellular stimulation, translocated to the plasma membrane in an activity-facilitated manner and then returned to the endosomes. The PI4,5P2-interacting site sufficed to mediate outward translocation and association with the plasma membrane. However, in the absence of PX and PH domains, PLD1 was unable to return efficiently to the endosomes. The PX and PH domains appear to facilitate internalization at different steps. The PH domain drives PLD1 entry into lipid rafts, which we show to be a step critical for internalization. In contrast, the PX domain appears to mediate binding to PI5P, a lipid newly recognized to accumulate in endocytosing vesicles. Finally, we show that the PH domain-dependent translocation step, but not the PX domain, is required for PLD1 to function in regulated exocytosis in PC12 cells. We propose that PLD1 localization and function involves regulated and continual cycling through a succession of subcellular sites, mediated by successive combinations of membrane association interactions.
信号酶磷脂酶D1（PLD1）促进膜泡运输。在此，我们探究PLD1的亚细胞定位是如何通过Phox同源（PX）结构域、普列克底物蛋白同源（PH）结构域以及对其激活至关重要的PI4,5P2结合位点来调控的。在COS-7细胞中，PLD1定位于核周内体和高尔基体，但在细胞受到刺激时，它会以一种促进活性的方式转运至质膜，然后再回到内体。PI4,5P2相互作用位点足以介导向外转运并与质膜结合。然而，在缺乏PX和PH结构域的情况下，PLD1无法有效地回到内体。PX和PH结构域似乎在不同步骤促进内化作用。PH结构域驱动PLD1进入脂筏，我们发现这是内化作用的关键步骤。相比之下，PX结构域似乎介导与PI5P的结合，PI5P是一种新发现的在内吞小泡中积累的脂质。最后，我们表明在PC12细胞中，PLD1在调节性胞吐作用中发挥功能需要依赖PH结构域的转运步骤，而不是PX结构域。我们提出，PLD1的定位和功能涉及通过一系列亚细胞位点进行的受调控的连续循环，这是由膜结合相互作用的连续组合介导的。
