Journal
BIOCHEMICAL JOURNAL
Volume 373, Issue -, Pages 57-63Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20030089
Keywords
endoplasmic reticulum; phosphatidylinositol kinase; phosphoinositide signalling; subcellular fractionation; valosin-containing protein
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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 11 and type III phosphatidylinositol 4-kinase (P14K II and III) activities. P14K 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 P14K activity arises from a type IIalpha 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 P14K IIalpha that can be separated from the bulk of the P14K IIalpha 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 P14K activity was confirmed by coimmunoprecipitation. These results suggest the previously unsuspected existence of an ER-related domain in which the bulk of the cellular P14P synthesis and VCP are localized.
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