Stimulation of Golgi membrane tubulation and retrograde trafficking to the ER by phospholipase A(2) activating protein (PLAP) peptide.
Recent pharmacological studies using specific antagonists of phospholipase A(2) (PLA(2)) activity have suggested that the formation of Golgi membrane tubules, 60-80 nm in diameter and up to several microns long, both in vivo and in a cell-free cytosol-dependent reconstitution system, requires the activity of a cytoplasmic Ca(2+)-independent PLA(2). We confirm and extend these studies by demonstrating that the stimulators of PLA(2), melittin and PLA(2) activating protein peptide (PLAPp), enhance cytosol-dependent Golgi membrane tubulation. Starting with preparations of bovine brain cytosol (BBC), or a fraction of BBC that is highly enriched in tubulation activity, called the gel filtration (GF) fraction, that are at subsaturating concentrations for inducing tubulation in vitro, we found that increasing concentrations of melittin or PLAPp produced a linear and saturable stimulation of Golgi membrane tubulation. This stimulation was inhibited by cytosolic PLA(2) antagonists, including the Ca(2+)-independent PLA(2)-specific antagonist, bromoenol lactone. The stimulatory effect of PLAPp, and its inhibition by PLA(2) antagonists, was reproduced using a permeabilized cell system, which reconstitutes both cytosol-dependent Golgi membrane tubulation and retrograde trafficking to the endoplasmic reticulum (ER). Taken together, these results are consistent with the idea that cytosolic PLA(2) activity is involved in the formation of Golgi membrane tubules, which can serve as trafficking intermediates in Golgi-to-ER retrograde movement.
Polizotto
R S
RS
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
de Figueiredo
P
P
Brown
W J
WJ
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D001921:Brain / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D002417:Cattle; D002463:Cell Membrane Permeability; D002999:Clone Cells; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000201:enzymology* / Q000648:ultrastructure; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006056:Golgi Apparatus / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure*; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D008555:Melitten / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats
D004791:Enzyme Inhibitors; D011506:Proteins; C055324:phospholipase A2-activating protein; D008555:Melitten; D010741:Phospholipases A