Immunolocalisation of phospholipase D1 on tubular vesicular membranes of endocytic and secretory origin

We have examined the localisation of overexpressed phospholipase DI (PLD1) using antibodies against its amino- and carboxyl-terminal domains. PLD1 ove rexpressed in COS-7 cells showed variable distribution by immunofluorescenc e but was mainly in punctate structures in the perinuclear region and at th e plasma membrane. Downregulation by an anti-sense plasmid resulted in almo st exclusively perinuclear distribution in punctate structures that contain ed immunoreactivity for the endogenous KDEL receptor and the early endosoma l antigen EEA1 protein. Influenza haemagglutinin (HA) and HA-derived mutant s designed to locate primarily to secretory or endocytic membranes were pre sent in PLD1-positive membranes. Immunofluorescence analysis in permanent C HO cell lines that express PLD1 inducibly confirmed the presence of PLD1 on both endocytic and secretory membranes. Analysis of PLDI distribution by i mmunocytochemistry and electron microscopy of intact CHO cells and of isola ted membranes revealed that PLD1 was present in tubulovesicular elements an d multivesicular bodies. Some of these were close to the Golgi region where as others stained positive for endocytic cargo proteins. Morphometric analy sis assigned the majority of PLDI immunoreactivity on endosomal membranes a nd a smaller amount on membranes of secretory origin. PLDI, via signals tha t are currently not understood, is capable of localising in tubulovesicular membranes of both endocytic and secretory origin.