Abstract
Phosphatidylinositol 4-phosphate (PI4P) regulates biosynthetic membrane traffic at multiple steps and differentially affects the surface delivery of apically and basolaterally destined proteins in polarized cells. Two phosphatidylinositol 4-kinases (PI4Ks) have been localized to the Golgi complex in mammalian cells, type III PI4Kbeta (PI4KIIIbeta) and type II PI4Kalpha (PI4KIIalpha). Here we report that PI4KIIIbeta and PI4KIIalpha localize to discrete subcompartments of the Golgi complex in Madin-Darby canine kidney (MDCK) cells. PI4KIIIbeta was enriched in early Golgi compartments, whereas PI4KIIalpha colocalized with markers of the trans-Golgi network (TGN). To understand the temporal and spatial control of PI4P generation across the Golgi complex, we quantitated the steady state distribution of a fluorescent PI4P-binding domain relative to cis/medial Golgi and TGN markers in transiently transfected MDCK cells. The density of the signal from this PI4P reporter was roughly 2-fold greater in the early Golgi compartments compared with that of the TGN. Furthermore, this ratio could be modulated in vivo by overexpression of catalytically inactive PI4KIIIbeta and PI4KIIalpha or in vitro by the PI4KIIIbeta inhibitor wortmannin. Our data suggest that both PI4KIIIbeta and PI4KIIalpha contribute to the compartmental regulation of PI4P synthesis within the Golgi complex. We discuss our results with respect to the kinetic effects of modulating PI4K activity on polarized biosynthetic traffic in MDCK cells.
Highlights
The synthesis and metabolism of phosphatidylinositols (PIs)1 provide a mechanism to regulate myriad cellular processes
These data suggest that PI4KIII and PI4KII␣ function independently to generate Phosphatidylinositol 4-phosphate (PI4P)-rich domains in the cis/medial Golgi and trans-Golgi network (TGN) of Madin-Darby canine kidney (MDCK) cells
No residual Golgi staining was observed (Fig. 4C, arrows). These data suggest that PI4KIII is largely responsible for maintaining the steady state pool of PI4P in the Golgi complex at steady state in MDCK cells
Summary
The synthesis and metabolism of phosphatidylinositols (PIs) provide a mechanism to regulate myriad cellular processes. Cellular signaling and intracellular trafficking events are activated independently, which requires spatial and temporal control of PI metabolism This is mediated in part by the subcellular distribution of phosphatidylinositol kinases, which phosphorylate PIs in specific compartments of the cell, including the plasma membrane, endosomes, secretory granules, and the Golgi complex [3,4,5,6]. PI4KII␣ appears to have a broader distribution in the cell and has been shown to be associated with several membrane bound organelles, including endosomes, a subcompartment of the endoplasmic reticulum, synaptic vesicles, and the Golgi complex [12, 15,16,17] These two proteins function in the same organelle to generate PI4P, the suborganellar distribution of these kinases and the locations of the pools of PI4P they generate are unknown. Overexpression of wild-type PI4KIII had no effect on early Golgi transport but inhibited TGN-to-apical membrane delivery [9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
