title: ADP-ribosylation factor 1-regulated phospholipase D activity is localized at the plasma membrane and intracellular organelles in HL60 cells.
abstract: ADP-ribosylation factor (ARF), a small GTPase required for vesicle formation, has been identified as an activator of phospholipase D (PLD), thus implying that PLD is localized at intracellular organelles. HL60 cells were prelabelled with [14C]acetate for 72 h and, after disruption, fractionated on a linear sucrose gradient. ARF1-regulated PLD activity in each fraction was assessed by measurement of phosphatidylethanol production. Two peaks of activity were identified, coincident with markers for Golgi/endoplasmic reticulum/granules (endomembranes) and plasma membrane respectively. Analysis of the fractions using exogenous phosphatidylcholine as substrate confirmed the presence of ARF1-dependent PLD activity in endomembranes and plasma membrane, and also identified an additional activity in the cytosol. In formyl-Met-Leu-Phe-stimulated cells, PLD activity as assessed by phosphatidylethanol formation was also associated with both the plasma membrane and endomembranes. Since ARF1-regulated PLD activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), the distributions of inositol lipids and the kinases responsible for lipid phosphorylation were examined. PIP2 was highly enriched at the plasma membrane, whereas phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PI4P), the precursors for PIP2 synthesis, were found predominantly at endomembranes. The distribution of PI 4-kinase and PI4P 5-kinase activities confirmed the plasma membrane as the major site of PIP2 production. However, endomembranes possessed substantial PI 4-kinase activity and some PI4P 5-kinase activity, illustrating the potential for PIP2 synthesis. It is concluded that:(1) ARF1-regulated PLD activity is localized at endomembranes and the plasma membrane, (2) PIP2 is available at both membrane compartments to function as a cofactor for ARF-regulated PLD, and (3) in intact cells, formyl-Met-Leu-Phe stimulates PLD activity at endomembranes as well as plasma membrane.
authors.affiliation: Department of Physiology, University College London, U.K.
authors.forename: J
authors.initials: J
authors.lastname: Whatmore
authors.forename: C P
authors.initials: CP
authors.lastname: Morgan
authors.forename: E
authors.initials: E
authors.lastname: Cunningham
authors.forename: K S
authors.initials: KS
authors.lastname: Collison
authors.forename: K R
authors.initials: KR
authors.lastname: Willison
authors.forename: S
authors.initials: S
authors.lastname: Cockcroft
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000085:Acetates / Q000378:metabolism; D015415:Biomarkers / Q000032:analysis; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020404:Glycerophospholipids; D006056:Golgi Apparatus / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009355:Neomycin / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000032:analysis / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000032:analysis / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010743:Phospholipids / Q000032:analysis / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism
chemical_list: D000085:Acetates; D015415:Biomarkers; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D010743:Phospholipids; C051521:phosphatidylethanol; D009240:N-Formylmethionine Leucyl-Phenylalanine; D006160:Guanosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D019870:1-Phosphatidylinositol 4-Kinase; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D009355:Neomycin