Phospholipase D1 localises to secretory granules and lysosomes and is plasma-membrane translocated on cellular stimulation.
Phospholipase D (PLD) activity has been implicated in the regulation of membrane trafficking [1,2], superoxide generation and cytoskeletal remodelling [3,4]. Several PLD genes have now been identified and it is probable that different isoforms regulate distinct functions. Defining the subcellular localisation of each isoform would facilitate understanding of their roles. Previous PLD localisation studies have been based largely on enzyme activity measurements, which cannot distinguish between isoforms [2,5]. We have cloned the cDNAs encoding human PLD1a and PLD1b from an HL60 cell cDNA library and expressed them as catalytically active fusion proteins with green fluorescent protein (GFP) in COS-1 cells and RBL-2H3 cells, a mast cell model which degranulates upon cross-linking of the high-affinity immunoglobulin E (IgE) receptor. In unstimulated cells, GFP-PLD1b colocalised with secretory granule and lysosomal markers; it was not found at the plasma membrane or nucleus and did not colocalise with markers for the Golgi. Stimulation or RBL-2H3 cells through IgE receptor cross-linking caused plasma membrane recruitment of GFP-PLD1b. Inhibition of IgE-receptor-stimulated, PLD-catalysed phosphatidate formation suppressed secretion of granule and lysosomal contents, but did not affect translocation of GFP-PLD1b. These experiments suggest that PLD1 plays a role in regulated exocytosis rather than endoplasmic reticulum (ER) to Golgi membrane transport.
Brown
F D
FD
Institute for Cancer Studies, Birmingham University Medical School, UK.
Thompson
N
N
Saqib
K M
KM
Clark
J M
JM
Powner
D
D
Thompson
N T
NT
Solari
R
R
Wakelam
M J
MJ
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000201:enzymology*; D006056:Golgi Apparatus / Q000201:enzymology; D049452:Green Fluorescent Proteins; D018922:HL-60 Cells; D006801:Humans; D015471:Leukemia, Basophilic, Acute; D008164:Luminescent Proteins / Q000096:biosynthesis; D008247:Lysosomes / Q000201:enzymology*; D008407:Mast Cells / Q000276:immunology / Q000502:physiology; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017455:Receptors, IgE / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D008164:Luminescent Proteins; D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010739:Phospholipase D; C106801:phospholipase D1