Traffic 2000 1: 504–511
Munksgaard International Publishers
Phospholipase A2Antagonists Inhibit Constitutive
Retrograde Membrane Traffic to the Endoplasmic
Paul de Figueiredoa,1, Dan Drecktraha,
Renee S. Polizottoa,2, Nelson B. Coleb,
William J. Browna*
aDepartment of Molecular Biology and Genetics, Cornell
University, Ithaca, NY 14853, USA
bCell Biology and Metabolism Branch, National Institutes of
Child Health and Human Development, National Institutes
of Health, Bethesda, MD 2089, USA
* Corresponding author: W.J. Brown, email@example.com
Eukaryotic cells contain a variety of cytoplasmic Ca2+-
dependent and Ca2+-independent phospholipase A2s
(PLA2s; EC 184.108.40.206.3). However, the physiological roles for
many of these ubiquitously-expressed enzymes is un-
clear or not known. Recently, pharmacological studies
have suggested a role for Ca2+-independent PLA2(iPLA2)
enzymes in governing intracellular membrane trafficking
events in general and regulating brefeldin A (BFA)-stimu-
lated membrane tubulation and Golgi-to-endoplasmic
reticulum (ER) retrograde membrane trafficking, in par-
ticular. Here, we extend these studies to show that
membrane-permeant iPLA2antagonists potently inhibit
the normal, constitutive retrograde membrane traffick-
ing from the trans-Golgi network (TGN), Golgi complex,
and the ERGIC-53-positive ER-Golgi-intermediate com-
partment (ERGIC), which occurs in the absence of BFA.
Taken together, these results suggest that iPLA2en-
zymes play a general role in regulating, or directly medi-
ating, multiple mammalian membrane trafficking events.
Key words: Golgi complex, membrane tubules, phospho-
lipase A2, retrograde transport
Received 19 February 2000, revised and accepted for
publication 21 March 2000
Researchers have employed a variety of biochemical, phar-
macological, and genetic approaches to elucidate the physio-
logical functions of cytoplasmic PLA2s, enzymes that
catalyze the hydrolysis of glycerophospholipids into lysophos-
pholipids and free fatty acids [for reviews, see (1,2)]. The
cytoplasmic PLA2fall into several distinct classes based on
amino acid sequence similarities and include both Ca2+-de-
pendent (cPLA2) and Ca2+-independent (iPLA2) enzymes (3).
Although much is known about the biological functions of
cPLA2in mammals, particularly their roles in arachidonic acid
metabolism (4), much less is known about the physiological
roles of the iPLA2s. These enzymes include the ubiquitously
expressed (Group VI) 80-kDa iPLA2, and two (Group VII and
VIII) iPLA2s specific for platelet-activating factor (PAF) [for
review, see (3)]. Fortunately, pharmacological reagents that
antagonize both intracellular cPLA2and iPLA2s, or are highly
selective for only iPLA2enzymes, have provided investiga-
tors with tools to begin to explore the functions of these
enzymes in living cells [for reviews, see (5,6)].
Recently, we have uncovered additional and unexpected
roles for iPLA2enzyme activities in mammalian cells. Specifi-
cally, we have shown that low concentrations of a broad
spectrum of membrane-permeant PLA2antagonists, includ-
ing bromoenol lactone (BEL), a highly selective mechanism-
based suicide substrate inhibitor of iPLA2enzymes, disrupt
various intracellular membrane trafficking events that appear
to depend on the formation of membrane tubules. These
trafficking events include the membrane tubule-mediated,
step-wise reassembly of Golgi complexes into an intercon-
nected juxtanuclear ribbon (7) and, most relevant to this
work, the tubule-mediated Golgi-to-ER retrograde trafficking
that is induced by the fungal metabolite brefeldin A (BFA) (8).
In mammalian cells, BFA blocks ER-to-Golgi anterograde
membrane trafficking and constitutive protein secretion by
preventing the association of COPI and clathrin coat protein
complexes with Golgi and TGN membranes, respectively (9).
In addition, BFA causes the dramatic formation of long, thin
membrane tubules that extend from the Golgi complex,
TGN, and endosome membranes [for review, see (10)]. In
the case of the Golgi complex, these tubules fuse with the
ER, creating a hybrid Golgi-ER tubulovesicular compartment
(11,12). TGN membranes also tubulate in the presence of
BFA, but these membrane tubules fuse with early endoso-
mal (EE) membranes, creating a hybrid TGN-EE tubulovesicu-
lar network (13,14). Interestingly, BFA does not disrupt the
morphological integrity of the ER-Golgi-intermediate compart-
ment (ERGIC), an organelle through which newly synthesized
secretory proteins travel when trafficking from ER exit sites
to the cis-Golgi proper (15). This last observation demon-
strated that not all membrane-bound organelles in the early
secretory pathway are BFA-sensitive.
A variety of biochemical, morphological, genetic, and phar-
macological studies have now established the existence of
robust recycling pathways from the Golgi complex and TGN
back to the ER in normal cells (16–22). The remaining ques-
tions regarding retrograde trafficking center on identifying
1Present address: Department of Microbiology, University of
Washington, Seattle, Washington 98195-7242.
2Present address: Department of Biological Sciences, Stan-
ford University, Stanford, CA 94305.
PLA2and Golgi Membrane Recycling
31. Knipe DM, Baltimore D, Lodish HF. Maturation of viral proteins in
cells infected with temperature-sensitive mutants of vesicular stom-
atitis virus. J Virol 1977;21: 1149–1158.
Drecktrah D, Brown WL. Phospholipase A(2) antagonists inhibit
nocodazole-induced Golgi ministack formation: evidence of an ER
intermediate and constitutive cycling [In Process Citation]. Mol Biol
Cell 1999;10: 4021–4032.
Tang BL, Low SH, Hauri HP, Hong W. Segregation of ERGIC53 and
the mammalian KDEL receptor upon exit from the 15°C compart-
ment. Eur J Cell Biol 1995;68: 398–410.
Rogalski AA, Singer SJ. Associations of elements of the Golgi
apparatus with microtubules. J Cell Biol 1984;99: 1092–1100.
Thyberg J, Moskalewski S. Microtubules and the organization of the
Golgi complex. Exp Cell Res 1985;159: 1–16.
Hazen SL, Zupan LA, Weiss RH, Getman DP, Gross RW. Suicide
inhibition of canine myocardial cytosolic calcium-independent phos-
pholipase A2. Mechanism-based discrimination between calcium-
dependent and -independent phospholipases A2. J Biol Chem
Street IP, Lin HK, Laliberte F, Ghomashchi F, Wang Z, Perrier H,
Tremblay NM, Huang Z, Weech PK, Gelb MH. Slow- and tight-bind-
ing inhibitors of the 85-kDa human phospholipase A2. Biochemistry
Balsinde J, Dennis EA. Distinct roles in signal transduction for each
of the phospholipase A2enzymes present in P388D1 macrophages.
J Biol Chem 1996;271: 6758–6765.
Morre DJ, Keenan TW. Golgi apparatus buds-vesicles or coated
ends of tubules? Protoplasma 1994;179: 1–4.
Cunningham WP, Morre DJ, Mollenhauer HE. Structure of isolated
plant Golgi apparatus revealed by negative staining. J Cell Biol
Morre DJ, Hamilton RL, Mollenhauer HH, Mahley RW, Cunningham
WP, Cheetham RD, Lequire VS. Isolation of a Golgi apparatus-rich
fraction from rat liver. I. Method and morphology. J Cell Biol
Rambourg A, Clermont Y, Marraud A. Three-dimensional structure
of the osmium-impregnated Golgi apparatus as seen in the high
voltage electron microscope. Am J Anat 1974;140: 27–45.
Rambourg A, Clermont Y, Hermo L. Three-dimensional architecture
of the Golgi apparatus in Sertoli cells of the rat. Am J Anat
44.Rambourg A, Clermont Y. Three-dimensional electron microscopy:
structure of the Golgi apparatus. Eur J Cell Biol 1990;51: 189–200.
Ladinsky MS, Kremer JR, Furcinitti PS, McIntosh JR, Howell KE.
HVEM tomography of the trans-Golgi network: structural insights
and identification of a lace-like vesicle coat. J Cell Biol 1994;127:
Hunziker W, Whitney JA, Mellman I. Selective inhibition of transcyto-
sis by brefeldin A in MDCK cells. Cell 1991;67: 617–627.
Donaldson JG, Lippincott-Schwartz J, Bloom GS, Kreis TE, Klaus-
ner RD. Dissociation of a 110-kDa peripheral membrane protein
from the Golgi apparatus is an early event in brefeldin A action. J
Cell Biol 1990;111: 2295–2306.
Itin C, Schindler R, Hauri HP. Targeting of protein ERGIC-53 to the
ER/ERGIC/cis-Golgi recycling pathway. J Cell Biol 1995;131: 57–
Kappeler F, Itin C, Schindler P, Hauri IP. A dual role for COOH-ter-
minal lysine residues in pre-Golgi retention and endocytosis of
ERGIC-53. J Biol Chem 1994;269: 6279–6281.
Tisdale EJ, Plutner H, Matteson J, Balch WE. p53/58 binds COPI
and is required for selective transport through the early secretory
pathway. J Cell Biol 1997;137: 581–593.
Majoul I, Sohn K, Wieland FT, Pepperkok R, Pizza M, Hillemann J,
Soling HD. KDEL receptor (Erd2p)-mediated retrograde transport of
the cholera toxin A subunit from the Golgi involves COPI, p23, and
the COOH terminus of Erd2p. J Cell Biol 1998;143: 601–612.
Spang A, Schekman R. Reconstitution of retrograde transport from
the Golgi to the ER in vitro. J Cell Biol 1998;143: 589–599.
Cosson P, Letourneur F. Coatomer interaction with di-lysine endo-
plasmic reticulum retention motifs. Science 1994;263: 1629–1631.
Cosson P, Demolliere C, Hennecke S, Duden P, Letourneur F. Delta-
and zeta-COP, two coatomer subunits homologous to clathrin-asso-
ciated proteins, are involved in ER retrieval. EMBO J 1996;15:
Gaynor EC, Ernr SD. COPI-independent anterograde transport:
cargo-selective ER to Golgi protein transport in yeast COPI mutants.
J Cell Biol 1997;136: 789–802.
Gaynor EC, Graham TR, Emr SD. COPI in ER/Golgi and intra-Golgi
transport: do yeast COPI mutants point the way? Biochim Biophys
Acta 1998;1404: 33–51.
Pelharn HR. Getting through the Golgi complex. Trends Cell Biol
Traffic 2000: 1: 504–511