Fd. Brown et al., PHOSPHOLIPASE D1 LOCALIZES TO SECRETORY GRANULES AND LYSOSOMES AND ISPLASMA-MEMBRANE TRANSLOCATED ON CELLULAR-STIMULATION, Current biology, 8(14), 1998, pp. 835-838
Phospholipase D (PLD) activity has been implicated in the regulation o
f membrane trafficking [1,2], superoxide generation and cytoskeletal r
emodelling [3,4], Several PLD genes have now been identified and it is
probable that different isoforms regulate distinct functions. Definin
g the subcellular localisation of each isoform would facilitate unders
tanding of their roles. Previous PLD localisation studies have been ba
sed 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 catalyt
ically active fusion proteins with green fluorescent protein (GFP) in
COS-1 cells and RBL-2H3 cells, a mast cell model which degranulates up
on crosslinking of the high-affinity immunoglobulin E (IgE) receptor.
In unstimulated cells, GFP-PLD1b colocalised with secretory granule an
d lysosomal markers; it was not found at the plasma membrane or nucleu
s and did not colocalise with markers for the Golgi, Stimulation of RB
L-2H3 cells through IgE receptor cross-linking caused plasma membrane
recruitment of GFP-PLD1b, Inhibition of IgE receptor-stimulated, PLD-c
atalysed phosphatidate formation suppressed secretion of granule and l
ysosomal contents, but did not affect translocation of GFP-PLD1b, Thes
e experiments suggest that PLD1 plays a role in regulated exocytosis r
ather than endoplasmic reticulum (ER) to Golgi membrane transport.