370 C. Donly et al. Insect Biochemistry and Molecular Biology 30 2000 369–376
In insects, a glutamate transporter cDNA isolated and cloned from the head of the caterpillar Trichoplusia ni,
TrnEAAT1, was also shown to accept both L- and D- enantiomers of aspartate as transport substrates Donly
et al., 1997, while a glutamate transporter isolated from Drosophila
embryos, dEAAT1, discriminates only weakly between these two substrates Seal et al., 1998.
The situation, however, is quite different for a trans- porter cDNA isolated from the brain of the cockroach,
Diploptera punctata . We report here that functional
characterization of
this cockroach
transporter, DipEAAT1, in a baculovirus-cell expression system
revealed that this transporter is strongly stereoselective. [
3
H]D-aspartate is not a high affinity transport substrate for this transporter, nor are D-aspartate or threo-3-hyd-
roxy-D-aspartate potent blockers of L-glutamate uptake.
Fig. 1. Amino acid sequence comparison of glutamate transporters cloned from insects. Residues identical in all 3 sequences are shaded in black,
while positions having 2 out of 3 matches are shaded grey. Bars are drawn over regions of sequence hydrophobicity that may correspond to transmembrane structures. Two histidine residues in the DipEAAT1 sequence analogous to a putative Zn
++
binding site are boxed. The sequence of TrnEAAT1 is from Donly et al. 1997 and the sequence of DrmEAAT1 is from Seal et al. 1998. The sequence of the DipEAAT1 cDNA
reported here has been deposited in the GenBankEMBL database under the accession AF208521.
2. Methods
2.1. Isolation of a cockroach glutamate transporter cDNA
A clone encoding a glutamate transporter was isolated from a cockroach brain cDNA library essentially as
described by Donly et al. 1997 for the isolation of a lepidopteran transporter. Briefly, the cDNA fragment
used for library screening was generated by PCR from cockroach brain cDNA using degenerate oligonucleot-
ides designed to regions of high homology among mam- malian glutamate transporters Donly et al., 1997. The
250 bp fragment produced was labeled with
32
P and used to screen 1,000,000 plaques from a cDNA library rep-
resenting RNA isolated from brains of mated female D.
371 C. Donly et al. Insect Biochemistry and Molecular Biology 30 2000 369–376
Fig. 2. Expression of glutamate transporter mRNAs. RNAs extracted
from the brains of the cockroach D. punctata-left and the caterpillar T. ni-right were separated by denaturing agarose gel electrophoresis
and transferred to nylon membranes.
32
P-Labeled fragments of the DipEAAT1 and TrnEAAT1 cDNAs were hybridized with the corre-
sponding RNAs and then the washed blots exposed to X-ray film. RNA products of approximately 5.0 kb cockroach and 3.6 kb caterpillar
were observed to be present in the insect brains.
punctata Donly et al., 1996. Eleven positive clones
were identified and the corresponding phagemids sub- sequently excised Stratagene. A single clone was selec-
ted and subjected to complete automated dideoxynucleo- tide chain termination sequencing Mobix.
2.2. RNA isolation and Northern blot analysis RNA was isolated from brains of mated female cock-
roaches using the Pharmacia QuickPrep mRNA purifi- cation system Donly et al., 1996. The isolated RNA
was used for cDNA library construction Stratagene, cDNA synthesis using the First-Strand cDNA Synthesis
Kit Pharmacia Biotech, or Northern blotting. Total RNA was isolated from brains of Trichoplusia ni cater-
pillars by homogenization in Trizol Reagent Life Technologies, and used directly for Northern blotting.
4 µ
g of polyA
+
-enriched cockroach brain RNA, or 10 µ
g of total caterpillar brain RNA were separated on denaturing agarose gels and transferred to Hybond N
+
nylon Amersham, where they were fixed in a CL-1000 Ultraviolet Crosslinker UVP Inc.. Hybridizations were
performed at 65 °
C in QuikHyb solution Stratagene, using
32
P-labeled fragments of each transporter. For the cockroach RNA, the 250 bp fragment of the D. punctata
transporter cDNA generated by PCR above was used as the probe, while for the caterpillar RNA, an 800 bp
fragment comprising the 59 segment of the TrnEAAT1 cDNA was used. Washes and exposure to X-ray film
were
performed according
to standard
protocols Sambrook et al., 1989.
2.3. Baculovirus expression The
cockroach glutamate
transporter cDNA
DipEAAT1 was ligated into the transfer vector pFastBac1 for expression using the Bac-to-Bac baculo-
virus expression system Life Technologies. Two differ- ent versions of the cDNA were used for expressions.
One version included the entire cloned cDNA, while the other included only the open reading frame ORF. The
shortened version was created using oligonucleotide pri- mers designed from the ends of the peptide ORF with
restriction endonuclease recognition sequences at their 59 ends. The PCR amplification was performed using
Expand polymerase Roche to minimize the occurrence of errors in the amplified products. Both versions pro-
duced similar expression levels. The caterpillar cDNA TrnEAAT1 was previously cloned in the Bac-to-Bac
expression system as described in Donly et al. 1997. The constructs were transposed to bacmid as directed by
the supplier Life Technologies and used to transfect Sf9 cells grown in TC-100 medium supplemented with
10 FBS Life Technologies. Viral stocks were ampli- fied in BTI-TN-5B1-4 High Five cells Invitrogen.
High Five cells were maintained in monolayer culture at 27
° C in EX-CELL 405 serum-free medium JRH
Biosciences. For expression of transporters, cells were infected at a multiplicity of infection of 0.35–0.95 in 12
well microtitre plates using 200,000–300,000 cells per well. Cells were assayed for activity after 48 h.
2.4. Transport assays Functional assays were performed 48 h after infection
with recombinant baculovirus, as described by Donly et al. 1997. The transport activity of virus-infected cells
expressing DipEAAT1 was compared with that of infected cells expressing the lepidopteran glutamate
transporter TrnEAAT1 Donly et al., 1997. Cells grown
372 C. Donly et al. Insect Biochemistry and Molecular Biology 30 2000 369–376
in 12 well plates were washed with saline containing 66 mOsm Na
+
composition listed in Donly et al., 1997 and then exposed to 500
µ l saline containing 10
µ l [
3
H]L- glutamate specific activity 46–63 Cimmol or [
3
H]D- aspartate specific activity 20 Cimmol Amersham for
5 min at 27 °
C. Uptake was terminated by washing the cells in Na
+
-free saline. The cells were air dried and the accumulated radiolabel extracted in 500
µ l 70 ethanol.
A 200 µ
l aliquot was added to scintillation fluid and its radioactivity measured.
For kinetic studies, the corresponding unlabeled amino acid was added to the saline to give final L-gluta-
mate or D-aspartate concentrations between 1 µ
M and 500
µ M. Only the Na
+
-dependent accumulation of radi- olabel by the cells greater than 95 of total uptake at
non-saturating concentrations of amino acid was used to determine transporter kinetics. [
3
H]L-glutamate was used for the competitive inhibition studies. In these
experiments, unlabeled inhibitor was added to the medium over the range 1
µ M to 500
µ M.
Amino acids were obtained from Sigma L-glutamate, D-glutamate, L-aspartate, D-aspartate, L-cysteate or
from Tocris Cookson D-cysteate, threo-3-hydroxy-L- aspartate and threo-3-hydroxy-D-aspartate.
3. Results and discussion
