Complex transcriptional and translational regulation of iPLA
2
c
resulting in multiple gene products containing dual competing sites
for mitochondrial or peroxisomal localization
David J. Mancuso
1,2
, Christopher M. Jenkins
1,2
, Harold F. Sims
1,2
, Joshua M. Cohen
1,2
, Jingyue Yang
1,2
and Richard W. Gross
1,2,3,4
1
Division of Bioorganic Chemistry and Molecular Pharmacology, and Departments of
2
Medicine,
3
Chemistry and
4
Molecular Biology
and Pharmacology, Washington University School of Medicine, St. Louis, MO, USA
Membrane-associated calcium-independent phospholipase
A
2
c(iPLA
2
c) contains four potential in-frame methionine
start sites (Mancuso, D.J. Jenkins, C.M. & Gross, R.W.
(2000) J. Biol. Chem. 275, 9937–9945), but the mechanisms
regulating the types, amount and subcellular localization of
iPLA
2
cin cells are incompletely understood. We now:
(a) demonstrate the dramatic transcriptional repression of
mRNA synthesis encoding iPLA
2
cby a nucleotide sequence
nested in the coding sequence itself; (b) localize the site of
transcriptional repression to the most 5¢sequence encoding
the iPLA
2
choloprotein; (c) identify the presence of nuclear
protein constituents which bind to the repressor region by gel
shift analysis; (d) demonstrate the translational regulation of
distinct iPLA
2
cisoforms; (e) identify multiple novel exons,
promoters, and alternative splice variants of human iPLA
2
c;
(f) document the presence of dual-competing subcellular
localization signals in discrete isoforms of iPLA
2
c;and
(g) demonstrate the functional integrity of an N-terminal
mitochondrial localization signal by fluorescence imaging
and the presence of iPLA
2
cin the mitochondrial compart-
ment of rat myocardium. The intricacy of the regulatory
mechanisms of iPLA
2
cbiosynthesis in rat myocardium is
underscored by the identification of seven distinct protein
products that utilize multiple mechanisms (transcription,
translation and proteolysis) to produce discrete iPLA
2
c
polypeptides containing either single or dual subcellular
localization signals. This unanticipated complex interplay
between peroxisomes and mitochondria mediated by com-
petition for uptake of the nascent iPLA
2
cpolypeptide
identifies a new level of phospholipase-mediated metabolic
regulation. Because uncoupling protein function is regulated
by free fatty acids in mitochondria, these results suggest that
iPLA
2
cprocessing contributes to integrating respiration and
thermogenesis in mitochondria.
Keywords: phospholipase; mitochondria; peroxisomes; tran-
scription; translation.
Phospholipases A
2
(PLA
2
s) play critical roles in cellular
growth, lipid homeostasis and lipid second messenger
generation by catalyzing the esterolytic cleavage of the
sn-2 fatty acid of glycerophospholipids [1–5]. The resultant
fatty acids and lysolipids are potent lipid mediators of signal
transduction and alter the biophysical properties of the
membrane bilayer, collectively contributing to the critical
roles that phospholipases play in cellular adaptation,
proliferation and signaling. PLA
2
s constitute a diverse
family of enzymes, which include the intracellular phos-
pholipase families, cytosolic PLA
2
s(cPLA
2
) and calcium-
independent PLA
2
s(iPLA
2
) as well as the secretory PLA
2
s
(sPLA
2
).
More than a decade ago, we identified multiple types of
kinetically distinguishable iPLA
2
activities in the cytosolic,
microsomal and mitochondrial fractions from multiple
species of mammalian myocardium [6–10]. Utilizing the
synergistic power of HPLC in conjunction with MS of
intact phospholipids, initial insights into both the canine
and human mitochondrial lipidomes were made [8,11]. Both
human and canine cardiac mitochondria possess a high
plasmalogen content, and plasmalogens are readily hydo-
lyzed by heart mitochondrial phospholipases [7,8]. Both
cytosolic and membrane-associated iPLA
2
activities are
inhibited by the nucleophilic serine-reactive mechanism-
based inhibitor (E)-6-(bromomethylene)-3-(1-naphthale-
nyl)-2H-tetrahydropyran-2-one (BEL) [12–14]. Recent
studies have shown that BEL has potent effects on
mitochondrial bioenergetics [15] and that fatty acids are a
Correspondence to R. W. Gross, Washington University School of
Medicine, Division of Bioorganic Chemistry and Molecular Phar-
macology, 660 South Euclid Avenue, Campus Box 8020, St. Louis,
MO 63110, USA. Fax: +1 314 362 1402; Tel: +1 314 362 2690;
E-mail: rgross@wustl.edu
Abbreviations: BEL, (E)-6-(bromomethylene)-3-(1-naphthalenyl)-
2H-tetrahydropyran-2-one; cPLA
2
, cytosolic phospholipase A
2
;ECL,
enhanced chemoluminescence; EMSA, electrophoretic mobility shift
analyses; EST, expressed sequence tag; GAPDH, glyceraldehye-
3-phosphate dehydrogenase; iPLA
2
, calcium-independent phosphol-
ipase A
2
; iPLA
2
c, membrane associated calcium-independent phos-
pholipase A
2
(AF263613); MOI, multiplicity of infection; PLA
2
,
phospholipase A
2
; Sf9, Spodoptera frugiperda cells; sPLA
2
, secretory
phospholipase A
2
; TAMRA, 6-carboxytetramethylrhodamine;
UCP, uncoupling protein.
(Received 25 August 2004, revised 10 October 2004,
accepted 13 October 2004)
Eur. J. Biochem. 271, 4709–4724 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04435.x
rate-determining factor in uncoupling protein (UCP) activ-
ity [16]. Thus, the role of mitochondrial iPLA
2
activities in
regulating mitochondrial function is just now beginning to
be understood. Moreover, both fatty acids and lysolipids
alter the physical properties of cell membranes, interact with
specific receptors, and modulate the electrophysiologic
function of many transmembrane ion channels including
K
+
and Ca
2+
channels in many cells and subcellular
contexts [17–20].
In early studies, we purified canine myocardial cytosolic
iPLA
2
activity (iPLA
2
b) to homogeneity [21] identifying a
high specific activity, proteolytically activated form of the
gene whose identity was substantiated by its covalent
radiolabeling with (E)-6-(
3
H)(bromomethylene)-3-(1-napht-
halenyl)-2H-tetrahydropyran-2-one (radiolabeled BEL)
[12]. However, despite our intense efforts at solubilization
and purification, the membrane-associated iPLA
2
activities
we identified in multiple membrane compartments were
resistant to our attempts at their purification. In the
postgenome era it became apparent that multiple different
gene products contributed to the many kinetically diverse
activities of membrane-associated iPLA
2
sinmyocardium
possessing distinct molecular masses and substrate selecti-
vities that resided in multiple discrete subcellular loci [22–27].
Recently, we characterized the genomic organization and
mRNA sequence of a novel iPLA
2
(now termed iPLA
2
c,
GenBank accession number AF263613) located on the long
arm of chromosome 7 at 118 c
M
[26]. Like other members
of the iPLA
2
family–iPLA
2
a(patatin, found in potato
tubers) [28] and iPLA
2
b[23] – iPLA
2
ccontains a consensus
site for nucleotide binding and a lipase consensus motif in its
C-terminal half [26]. Although the intracellular localization
and activity of iPLA
2
bis complex and dynamically
regulated by multiple different cellular perturbations inclu-
ding ATP concentration [7], calcium-activated calmodulin
[29,30], and proteolysis [31,32], the biochemical mechanisms
regulating iPLA
2
cin intact tissues are not known with
certainty. For example, iPLA
2
cis not activated, stabilized
or bound to ATP under any conditions we have examined,
nor does it associate with calmodulin or possess a discern-
able calmodulin-binding consensus sequence [26]. Like
iPLA
2
b,iPLA
2
cis completely inhibited by low micromolar
concentrations (1–5 l
M
) of the mechanism-based inhibitor
BEL [26].
Previously, we demonstrated that iPLA
2
cis synthesized
from a 3.5 kb mRNA containing a putative 2.4 kb coding
region which was most prominent in heart tissue. The
5¢-region of the 2.4 kb coding sequence of iPLA
2
ccontains
four in-frame ATG start sites which can potentially encode
88, 77, 74 and 63 kDa polypeptides [26]. However, in initial
studies in baculoviral and in vitro rabbit reticulocyte lysate
systems, we unexpectedly observed that constructs contain-
ing the full-length 2.4 kb sequence encoding the predicted
88 kDa polypeptide resulted instead in the expression of
only two protein bands of 77 and 63 kDa [26]. Moreover,
the initial characterization of iPLA
2
cin nonrecombinant
cells demonstrated that hepatic iPLA
2
cwas most highly
enriched in the peroxisomal compartment as a 63 kDa
polypeptide [27]. These results raised the intriguing
possibility that iPLA
2
cbiosynthesis was transcriptionally
and/or translationally regulated by as yet unidentified
mechanisms.
To begin to identify the potential modes of the regulation
of iPLA
2
csynthesis at the transcriptional and post-
transcriptional levels, and to identify specific mechanisms
modulating iPLA
2
cexpression and processing in different
cell types, we examined multiple iPLA
2
cconstructs in
different cellular contexts and in intact rat myocardium.
Herein, we demonstrate that iPLA
2
csynthesis is transcrip-
tionally regulated by a transcriptional repressor domain
nested in the 5¢-coding region and translationally regulated
through the differential usage of downstream AUG start
sites. Moreover, this study identifies an N-terminal mito-
chondrial localization signal and demonstrates its functional
integrity by fluorescence colocalization assays. Importantly,
the presence of multiple high molecular mass iPLA
2
c
isoforms in mitochondria from wild-type rat myocardium
was demonstrated. This complex interplay of transcrip-
tional and translational, as well as proteolytic, sculpting of
iPLA
2
cresults in a diverse repertoire of biologic products,
which likely provides the chemical foundations necessary
for iPLA
2
cto fulfill its multiple distinct functional roles in
mammalian tissues.
Experimental procedures
Materials
[
32
P]dCTP[aP] (6000 CiÆmmol
)1
) and enhanced chemolu-
minescence (ECL) detection reagents were purchased from
Amersham Pharmacia Biotech (Piscataway, NJ, USA). A
human heart cDNA library was purchased from Stratagene
(La Jolla, CA, USA). For PCR, a Perkin-Elmer Thermo-
cycler was employed, and all PCR reagents were purchased
from Applied Biosystems (Foster City, CA, USA). The
Luciferase Assay system and TnT Quick coupled Tran-
scription/Translation system were obtained from Promega
(Madison, WI, USA). CV1 cells were generously provided
by D. Kelly (Washington University Medical School).
Vectors pcDNA1.1, pEF1/myc-His and pcDNA 3.1/myc-
His/lacZ were purchased from Invitrogen (Carlsbad, CA,
USA). Vectors pEGFP-N3 and pDsRed-mito were pur-
chased from BD-Biosciences (Palo Alto, CA, USA). Culture
media, CellFECTIN and LipofectAMINE reagents for
transfection, baculovirus vectors and competent DH110Bac
Escherichia coli were purchased from Invitrogen and used
according to the manufacturer’s protocol. QIAfilter plasmid
kits and QIAquick Gel Extraction kits were obtained from
Qiagen (Valencia, CA, USA). Keyhole limpet hemocyanin
was obtained from Pierce (Rockford, IL, USA). BEL was
obtained from Calbiochem (San Diego, CA, USA). Most
other reagents were obtained from Sigma (St. Louis, MO,
USA).
Expression of truncated iPLA
2
c
Constructs encoding the 74- and 63 kDa polypeptides were
prepared as previously described for construction of the full-
length iPLA
2
cconstruct encoding the 88 kDa polypeptide
used for baculoviral expression. In brief, the 74 kDa sense
primer M533 (5¢-TCAAGTCGACATGATTTCACGTTT
AGC-3¢) and the 63 kDa sense primer M530 (5¢-GT
AAGTCGACAATGTCTCAACAAAAGG-3¢)wereeach
paired with reverse primer M458 (5¢-GCATAGCATGCT
4710 D. J. Mancuso et al. (Eur. J. Biochem. 271)FEBS 2004
CACAATTTTGAAAAGAATGGAAGTCC-3¢)forPCR
of 2.0 and 1.7 kb products, respectively, from the full-
length iPLA
2
cpFASTBac1 construct for cloning via SalI/
SphI sites into vector pFASTBac1 (Invitrogen). Subsequent
preparation of bacmids, CellFECTIN-mediated transfec-
tion of Spodoptera frugiperda (Sf9) cells to produce virus,
and the Neutral Red agar overlay method for viral plaque
titering were performed utilizing the Bac-to-Bac Baculovirus
Expression System (Invitrogen) according to the manufac-
turer’s instructions. Sf9 cells were grown and infected for
preparation of recombinant protein extracts as previously
described [26]. In brief, Sf9 cells were cultured in 100-mL
flasks equipped with a magnetic spinner containing supple-
mented Grace’s media [26]. Sf9 cells at a concentration of
1·10
6
cellsÆmL
)1
were prepared in 50 mL of growth media
and incubated at 27 C for 1 h prior to infection with either
wild-type virus or recombinant virus containing human
iPLA
2
ccDNA. After 48 h, cells were pelleted by centrifu-
gation, resuspended in ice-cold NaCl/P
i
and repelleted. The
supernatant was decanted and the cell pellet was resus-
pended in 5 mL of ice-cold homogenization buffer (25 m
M
imidazole, pH 8.0, 1 m
M
EGTA, 1 m
M
dithiothreitol,
0.34
M
sucrose, 20 l
M
trans-epoxysuccinyl-
L
-leucylamido-
(4-guanidino) butane and 2 lgÆmL
)1
leupeptin). Cells were
lysed by sonication (20 ·1 s bursts utilizing a Vibra-cell
sonicator at 30% output) and centrifuged at 100 000 gfor
1hat4C. The supernatant was saved (cytosol) and the
membrane pellet was washed by resuspending with a Teflon
homogenizer in 5 mL of homogenization buffer followed by
a brief sonication step (10 ·1 s bursts) before recentrifu-
gation at 100 000 gfor 1 h at 4 C. After removal of the
supernatant, the membrane pellet was resuspended in 1 mL
of homogenization buffer using a Teflon homogenizer and
then sonicated (5 ·1 s bursts) to prepare a membrane
fraction.
PLA
2
enzymatic assay and immunoblot analysis
Calcium-independent PLA
2
activity was measured by
quantitating the release of radiolabeled fatty acid from
various radiolabeled phospholipid substrates in the presence
of membrane fractions from Sf9 cells infected with wild-type
or recombinant human iPLA
2
cbaculovirus as previously
described [26]. Protein from baculoviral or reticulocyte
lysate samples was separated by SDS/PAGE [33], trans-
ferred to Immobilon-P membranes by electroelution,
probed with anti-iPLA
2
cIg and visualized using ECL as
described previously [26].
Northern blot analysis
Total RNA from Sf9 cells was isolated according to the
protocol for RNeasy (Qiagen). In brief, sample was placed
in tissue lysis buffer containing guanine isothiocarbonate
and disrupted by 20–40 s of pulse homogenation with a
rotor stator homogenizer. Total RNA was then recovered
from a cleared lysate after several washes on an RNeasy
mini spin column and elution with RNase-free water.
Recovery of RNA was determined spectrophotometrically
at 260 nm. RNA (2 lg) was fractionated on a 1.25%
agarose Latitude RNA midi gel (BioWhittaker, Walkers-
ville, ME, USA), blotted onto a nylon membrane,
cross-linked by exposure to a UV light source for 1.5 min
and then baked at 85 C for 60 min. After prehybridization
in ExpressHyb hybridization buffer (BD Biosciences) for
30 min, the blot was hybridized 1 h at 68 C with radio-
labeled iPLA
2
cprobe prepared as previously described [26]
in hybridization buffer and then washed with 2·NaCl/Cit
containing 0.1% (w/v) SDS twice for 30 min each, followed
by two washes with 0.1·NaCl/Cit containing 0.1% (w/v)
SDSfor40mineachat50C, as described in the
manufacturer’s instructions. Hybridized sequences were
identified by autoradiography for 16 h.
RNA stability assay
Spinner flasks (100 mL) were infected with equivalent
volumes of each truncated viral iPLA
2
cconstruct [multi-
plicity of infection (MOI) ¼1] and 48 h later, actino-
mycin D was added to a concentration of 10 lgÆmL
)1
.At0,
15, 30, 60, 120 and 240 min following actinomycin D
addition, 2-mL aliquots were removed, centrifuged to
collect pellets and quick-frozen in liquid N
2
.RNAwas
then prepared following the RNeasy (Qiagen) protocol.
RNA samples (2 lg) were fractionated on a latitude RNA
midi-gel for northern analysis as described above.
Quantitative PCR
RNA was prepared from Sf9 cell pellets following the
RNeasy protocol supplemented with on-column RNase-
free DNase treatment to remove baculoviral DNA as
described by the manufacturer. Completeness of removal of
baculoviral DNA was monitored by including control
samples spiked with plasmid DNA (either cell pellets from
uninfected Sf9 cells or water blanks). Quantitative PCR of
DNase-treated control samples routinely did not generate
detectable signal. For analysis of actinomycin D-treated test
samples, 0.2–1 lg of the total RNA was reverse tran-
scribed using MultiScribe reverse transcriptase in a TaqMan
Gold RT-PCR kit (Applied Biosystems) by incubation for
10 min at 25 C followed by 30 min at 48 C and a final
step of 5 min at 95 C and 20 ng of cDNA was used per
reaction in quantitative PCR. Specific iPLA
2
cprimer pairs
and probe were designed using
PRIMER EXPRESS
software
from PE Biosystems. Forward and reverse primers, respect-
ively (5¢-AGCTCTTTGATTACATTTGTGGTGTAA-3¢
and 5¢-CACATTCATCCAAGGGCATATG-3¢)wereused
for amplification of an 100 nucleotide product flanking
the boundary between exons 5 and 6 of the iPLA
2
cgene. A
30-mer hybridization probe (5¢-CCCAACATGAAAGC
TAATATGGCACCTGTG-3¢) was designed to anneal
between the PCR primers, at the exon 5/6 boundary,
5¢-labeled with reporter dye 6-FAM and 3¢-labeled
with quenching dye, 6-carboxytetramethylrhodamine
(TAMRA). PCRs were carried out using TaqMan PCR
reagents (Applied Biosystems) as recommended by the
manufacturer. Each PCR amplification was performed in
triplicate, using the following conditions: 2 min at 50 C
and 10 min at 95 C, followed by a total of 40 two-
temperature cycles (15 s at 95 C and 1 min at 60 C). For
the generation of standard curves, serial dilutions of a
cDNA sample were used and mRNA levels were compared
for various time points after correction using concurrent
FEBS 2004 Regulation of iPLA
2
cbiosynthesis (Eur. J. Biochem. 271) 4711
glyceraldehye-3-phosphate dehydrogenase (GAPDH) mes-
sage amplification with GAPDH primers and probe as an
internal standard. Results were plotted as relative mRNA
level vs. time (hours) and the slopes of exponential
trendlines for each construct were compared.
Luciferase assay
PCR primers in Table 1 were used to amplify segments
containing 124 nucleotides of sequence upstream of the
iPLA
2
c63 kDa start site. All 3¢PCR primers in Table 1
were designed to generate identical Kozak (GCCACC)
sequences [34,35] upstream of the ATG start. In each case,
the sequence around the ATG start is ÔGCCAX
CATGÕ(where ÔXÕis a ÔCÕnucleotide in all constructs
except 83 which contains an ÔAÕnucleotide). In each case,
PCR products were cloned into HindIII/NcoIrestriction
sites within the polylinker region of pGL3-Promoter vector
(pGL3P). Also, because of the presence of a naturally
occurring NcoI site within the 83 construct, an AflIII
restriction site was utilized at the 3¢-end of this construct
(instead of NcoI) to generate a compatible cohesive end for
cloning into the NcoI restriction site of pGL3-Promoter
vector (pGL3P). Transient transfection of CV1 cells with
each of the inhibitory constructs was performed using
LipofectAMINE Plus (Invitrogen). For each transfection,
1–2 lg of luciferase reporter plasmid was cotransfected with
100 ng of pcDNA 3.1/myc-His/lacZ vector and b-galac-
tosidase activity was measured utilizing the b-galactosidase
enzyme assay system (Promega) for normalization of
results. Background measurements were uniformly low
and cell survival was indistinguishable in all transfections
performed. The cells were harvested 24 h later and luciferase
activity was assayed using the luciferase assay system
(Promega) following the manufacturer’s protocol. Relative
luminescence values were measured in a Beckman Scintil-
lation counter with a wide-open window.
Subcellular fractionation of rat heart
Subcellular fractionation of rat heart by differential centri-
fugation was performed essentially as described previously
for rat liver [27]. In brief, rat heart was minced on ice and
then homogenized in 3 vol. (w/v) of ice-cold homogeniza-
tion buffer [0.25
M
sucrose, 5 m
M
Mops,pH7.4,1m
M
EDTA and 0.1% (v/v) ethanol, 0.2 m
M
dithiothreitol
containing protease inhibitors (0.2 m
M
phenylmethylsulfo-
nyl fluoride, 1 lgÆmL
)1
leupeptin, 1 lgÆmL
)1
aprotinin and
15 lgÆmL
)1
phosphoramidon)] using a Potter-Elvehjem
homogenizer at 1000 r.p.m. with 8–10 strokes. The homo-
genate was first centrifuged at 100 gfor 10 min to remove
cellular debris and then at 1000 gto obtain a nuclear pellet
(nuclear fraction) and a supernatant fraction. The 1000 g
supernatant fraction was further centrifuged at 3000 gfor
20 min to collect a heavy mitochondrial pellet (heavy
mitochondrial fraction). The 3000 gsupernatant was then
centrifuged at 23 500 gfor 20 min to collect the light
mitochondrial fraction pellet 23 500 g(light mitochondrial
fraction). The 23 500 gsupernatant was then centrifuged at
70 000 gfor 20 min to collect a second light mitochondrial
pellet (70 000 glight mitochondrial fraction). Utilizing the
above subcellular fractionation technique, the majority of
mitochondria were present in the 3000 and 23 500 gpellets,
whereas the large majority of peroxisomal marker PMP70
was present in the supernatant.
Promoter analysis
iPLA
2
csequences were examined for the presence of
putative promoter elements utilizing the internet-based
program
TFSEARCH
(http://150.82.196.184/research/db/
TFSEARCH.html). Promoter activity of iPLA
2
csequences
was analyzed by cloning sequences upstream of the
luciferase reporter gene in promoterless vector pGL3-
Enhancer (Promega). The following primers were utilized
to amplify PCR products containing iPLA
2
csequence:
P1, 5¢-TCAAGGTACCATGATTTCCTGAAGG-3¢;P2,
5¢-CTGAAGATCTAGCCTTTACTTTCA-3¢;P3,5¢-GC
TAGGTACCAATACAGTAATATATG-3¢;P4,5¢-TGC
TAGATCTCCACCCACTCA-3¢;P5,5¢-TTATGGTACC
TGAAAGGGAATAGCGGC-3¢;P6,5¢-GGCTGGTAC
CCTTGCGCTCCGTC-3¢;P7,5¢-GGAGAGATCTGCG
GGAAGCCGCGACAGA-3¢;p8,5¢-TTCCAGATCTG
CAGAGATAAGCCTCCC-3¢;p9,5¢-GCGTGAGATCT
CTGGTTGGTTGC-3¢;P10,5¢-ACCAGGTACCGCA
CAGCACGCCCC-3¢; and P11, 5¢-GTCCGGTACCGG
AAGGCAAAACGA-3¢. Primers P1 and P2 were utilized
to amplify a 584-nucleotide product containing sequence
Table 1. PCR primer pairs for localization of transcriptional regulatory elements in the 5¢-coding region of iPLA
2
c.Underlined residues indicate the
locations of HindIII (AAGCTT), NcoI (CCATGG), or Af l III (ACATGT) restriction sites utilized for cloning PCR products.
Construct PCR primer pairs 5¢-to3¢-sequence
88 88F GTTGAAGCTTGTGTCTATTAATCTGACTGTA
88R TAGACCATGGTGGCTTATCCTCCAGTAATGC
87 87F GTGTAAGCTTGAAGCAGAGAAGCAAGCAACTG
87R ACTGCCATGGTGGCCTTCACTTTTGGTCCATTTAC
85 85F TGGAAAGCTTGCCACATCAGTCTACAAAG
85R TGCTCCATGGTGGCATCCCAATATGTAAACCA
83 83F GAACCAAGCTTGAAGCACATTCTTGCAGTAAGCA
83R CAAAACATGTTGGCTACGGGACATACAAATGTTCA
80 80F GTTGAAGCTTTTTGAAACTTAGCACTTCTGC
80R ATTCCATGGTGGCTGAAATCATTTCATTTTGATTGCC
74 74F TCAAAAGCTTATGATTTCACGTTTAGCTC
74R CTTTCCATGGTGGCTGTCACTATATTTTTTCA
4712 D. J. Mancuso et al. (Eur. J. Biochem. 271)FEBS 2004
upstream from iPLA
2
cexon 1. For construct I, primers P3
and P4 were utilized to amplify a 584 nucleotide product
containing sequence upstream from iPLA
2
cexon 2. PCR
products for constructs II–IX were prepared as follows:
primers P5 and P4 were paired to amplify a 390-nucleotide
product for construct II; primers P6 and P4 were utilized to
amplify a 197-nucleotide product for construct III; primers
P5 and P8 were employed to amplify a 215-nucleotide
product for construct IV; primers P3 and P8 were utilized to
amplify a 216-nucleotide product for construct V; primers
P3 and P7 were paired to amplify a 409-nucleotide product
for construct VI; primers P5 and P9 were utilized to amplify
a 131-nucleotide product for construct VII; primers P10 and
P9 were paired to amplify a 106-nucleotide product for
construct VIII; and primers P11 and P7 were employed to
amplify a 155-nucleotide product for construct IX. PCR
products were subsequently cloned via KpnI/BglII restric-
tion sites into the promoterless vector pGL3-Enhancer
(Promega) and then utilized for LipofectAMINE Plus-
mediated transient transfection of CV1 cells followed 24 h
later by analysis of luciferase activity utilizing the Luciferase
Assay System of Promega. Empty pGL3-Enhancer vector
and the SV40-containing promoter vector pGL3-Promoter
were used as controls. MyoD vector used for cotransfection
of CV1 cells with the pre-exon 1 iPLA
2
cconstruct was
obtained from M. Chin (Harvard Medical School) [36].
Results were normalized to b-gal resulting from cotransfec-
tion with a LacZ vector.
5¢-Rapid amplification of cDNA ends (RACE)
5¢-RACE was performed as previously described employing
human heart Marathon-Ready cDNA (BD Biosciences)
and primers AP1 and M460 [26]. PCR products were gel
purified with a QIAquick gel extraction kit, subcloned into
pGEM-T vector (Promega), sequenced and analyzed by
alignment with iPLA
2
csequences.
Electrophoretic mobility shift analyses
Electrophoretic mobility shift analyses (EMSA) were per-
formed with the Promega gel shift assay system according to
the manufacturer’s specifications by using 2 lg of nuclear
protein for each gel shift reaction. For analysis of the
5¢-transcription inhibitory region of iPLA
2
c, double-stran-
ded oligonucleotides containing 5¢-iPLA
2
c, sequence were
end-labeled with [
32
P]ATP using T
4
polynucleotide kinase,
as instructed by the manufacturer (Promega). Competition
studies were performed by adding a 100-fold molar excess of
unlabeled oligonucleotide or nonspecific control oligo-
nucleotide to the reaction mixture prior to the addition of
radiolabeled probe. Reaction mixtures were analyzed on
Novex 6% DNA retardation polyacrylamide gels in 0.5·
TBE (89 m
M
Tris/HCl, pH 8.0; 89 m
M
boric acid; 2 m
M
EDTA) as the running buffer. Electrophoresis was per-
formed at 298 V for 20 min, at 4 C followed by drying of
the gel at 80 C under vacuum and visualization of DNA–
protein complexes by autoradiography for 12–18 h. Sense
and reverse complement oligonucleotide sequences corres-
ponding to the following sequences were synthesized and
annealed: g50 (5¢-TATTAATCTGACTGTAGATATAT
ATATATTACCTCCTTAGTAATGC-3¢) and random-
ized control g50c (5¢-TTGATAGTTATCTATTACAG
TCTTCTTAGATTGAAACAA-3¢), g177 (5¢-CATACAA
ACATAATAAGATGTAAATGG-3¢) and control g177c
(5¢-TCATCTAAGTACAATAGATAGAAGAAA-3¢),
g230 (5¢-TGTTACTCTCCAAGCAACCA-3¢) and control
g230c (5¢-GACACTTGTCATCACACTCA-3¢). For ana-
lysis of the pre-exon 1 region, myo2 double-stranded DNA
having the sequence 5¢-GAAGTACAGGTGTGGCTGG-
3¢was utilized along with control myo2ctl (5¢-GATCG
TTGTGAAGAGGGCG-3¢). For analysis of the pre-
exon 2 promoter region, Inr double-stranded DNA having
the sequence 5¢-GCGTCACTTCCGCTGGGGGCGG-3¢
was utilized along with randomized control Inrc (5¢-GTG
GCCGGGTGGTCCACCTCGG-3¢).
Mitochondrial target prediction, iPLA
2
c–GFP constructs
and confocal microscopy
The internet-based
MITOPROT
computer program (http://
www.mips.gsf.de/cgi-bin/proj/medgen/mitofilter) [37] was
utilized for prediction of mitochondrial targeting sequences
in iPLA
2
c. To prepare the 74-GFP construct, complement-
ary 5¢-phosphorylated primers (5¢-TCGAGCCACCAT
GATTTCACGTTTAGCTCAATTTAAGCCAAGTTCC
CAAATTTTAAGAAAAGTAG-3¢and 5¢-TCGACTACT
TTTCTTAAAATTTGGGAACTTGGCTTAAATAAA
CGTGAAATCATGGTGGC-3¢) were annealed by heat-
ing a 4-l
M
mixture of primers to 95 C for 3 min followed
by cooling to 22 C prior to cloning into the Xho1/Sal1
sites of vector pEGFP-N3. Integrity and orientation of the
N-terminal fusion products were verified by sequencing.
Vector pDsRed2-Mito (BD Biosciences), which encodes
a mitochondrial-targeting sequence of human cyto-
chrome coxidase fused to red fluorescent protein, was
utilized as a mitochondrial marker. HeLa cells were grown
on two-well Laboratory Tek chamber slides to 60–80%
confluency prior to LipofectAMINE Plus (Invitrogen)
mediated single or cotransfection according to the manu-
facturer’s suggested protocol. After 48 h, cells were
washed in NaCl/P
i
, fixed with 4% (v/v) paraformaldehyde,
coverslipped and fluorescence was analyzed utilizing a
Zeiss Axiovert 200 (Carl Zeiss Inc., Thornwood, NY,
USA) equipped with Zeiss LSM-510 confocal system with
a63·oil immersion objective and excitation wavelengths
of 488 and 633 nm. Single transfections with either
pDsRed2-Mito or 74-GFP construct were utilized to
optimize immunofluorescence conditions and eliminate
bleed-through. Filters were optimized for double-label
experiments to minimize bleed-through and fluorescence
images were collected by utilizing Zeiss
LSM
software.
Results
Identification of transcriptional regulatory elements
nested in the 5¢-coding region of iPLA
2
c
In previous work, we demonstrated that expression of a
baculoviral construct encoding the full-length 88 kDa
coding sequence of iPLA
2
cin Sf9 cells resulted instead
in the production of downstream polypeptides of 77 and
63 kDa in nearly equal amounts [26]. This was remarkable
because translation initiation almost always occurs at the
FEBS 2004 Regulation of iPLA
2
cbiosynthesis (Eur. J. Biochem. 271) 4713