Localization of a highly active pool of type II phosphatidylinositol 4-kinase in a p97/valosin-containing-protein-rich fraction of the endoplasmic reticulum.
Different phosphoinositides are synthesized in cell membranes in order to perform a variety of functions. One of the most abundant of these lipids is phosphatidylinositol (PI) 4-phosphate (PI4P), which is formed in human eukaryotes by type II and type III phosphatidylinositol 4-kinase (PI4K II and III) activities. PI4K II activity occurs in many different subcellular membranes, although no detailed analysis of the distribution of this activity has been reported. Using density gradient ultracentrifugation, we have previously found that in A431 cells the predominant PI4K activity arises from a type II alpha enzyme that is localized to a buoyant membrane fraction of unknown origin [Waugh, Lawson, Tan and Hsuan (1998) J. Biol. Chem. 273, 17115-17121]. We show here that these buoyant membranes contain an activated form of PI4K II alpha that can be separated from the bulk of the PI4K II alpha protein in A431 and COS-7 cells. Proteomic analysis revealed that the buoyant membrane fraction contains numerous endoplasmic reticulum (ER)-marker proteins, although it was separated from the bulk of the ER, ER-Golgi intermediate compartment, transitional ER, Golgi and other major subcellular membranes. Furthermore, the majority of the cytoplasmic valosin-containing protein (VCP), an AAA+ATPase implicated in various ER, transitional ER, Golgi and nuclear functions, was almost completely localized to the same buoyant membrane fraction. Co-localization of VCP and PI4K activity was confirmed by co-immunoprecipitation. These results suggest the previously unsuspected existence of an ER-related domain in which the bulk of the cellular PI4P synthesis and VCP are localized.
Waugh
Mark G
MG
Centre for Molecular Cell Biology, Department of Medicine, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, UK.
Minogue
Shane
S
Anderson
J Simon
JS
Balinger
Adam
A
Blumenkrantz
Deena
D
Calnan
Denis P
DP
Cramer
Rainer
R
Hsuan
J Justin
JJ
D019870:1-Phosphatidylinositol 4-Kinase / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002458:Cell Fractionation / Q000379:methods; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006801:Humans; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; C046951:valosin; D019870:1-Phosphatidylinositol 4-Kinase