Abstract
Polyphosphoinositides regulate numerous steps in membrane transport. The levels of individual phosphatidylinositols are controlled by specific lipid kinases, whose activities and localization are in turn regulated by a variety of effectors. Here we have examined the effect of overexpression of frequenin, a modulator of phosphatidylinositol 4-kinase activity, on biosynthetic and postendocytic traffic in polarized Madin-Darby canine kidney cells. Endogenous frequenin was identified in these cells by polymerase chain reaction, Western blotting, and indirect immunofluorescence. Adenoviral-mediated overexpression of frequenin had no effect on early Golgi transport of membrane proteins, as assessed by acquisition of resistance to endoglycosidase H. However, delivery of newly synthesized influenza hemagglutinin from the trans-Golgi network to the apical cell surface was severely inhibited in cells overexpressing frequenin, whereas basolateral delivery of the polymeric immunoglobulin receptor was unaffected. Overexpression of frequenin did not affect postendocytic trafficking steps including apical and basolateral recycling and basal-to-apical transcytosis. We conclude that frequenin, and by inference, phosphatidylinositol 4-kinase, plays an important and selective role in apical delivery in polarized cells.
Highlights
We have been interested in the mechanisms that regulate cargo sorting and vesicle formation along the biosynthetic and postendocytic pathways
Digital deconvolution of uninfected Madin-Darby canine kidney (MDCK) cells processed for indirect immunofluorescence using rabbit polyclonal anti-frequenin antibody revealed punctate staining that was concentrated throughout the cytoplasm (Fig. 4, panels A-H), consistent with frequenin localization on membranous organelles
We have demonstrated that frequenin is endogenously expressed in MDCK cells, and that overexpressed frequenin coimmunoprecipitates with the wortmannin-sensitive  isoform of phosphatidylinositol 4-OH kinase (PI4K)
Summary
We have been interested in the mechanisms that regulate cargo sorting and vesicle formation along the biosynthetic and postendocytic pathways. Rapidly accumulating evidence has implicated polyphosphoinositides as key players in membrane trafficking (reviewed in Ref. 1). In addition to their function as second-messenger precursors, these lipids are thought to participate physically in the formation and release of vesicles from various compartments along the secretory pathway. Frequenin was demonstrated to regulate the activity of the yeast phosphatidylinositol 4-OH kinase (PI4K) Pik1 [5], which is homologous to the mammalian PI4K  isoform (PI4K [6]). We tested whether mammalian frequenin interacts with PI4K, and investigated the effect of frequenin overexpression on biosynthetic and postendocytic protein traffic in this polarized epithelial cell line
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