The opt1 gene of
Drosophila melanogaster encodes a
proton-dependent dipeptide transporter
Gregg
Roman,
Victoria
Meller,
Kwok Hang
Wu, and
Ronald L.
Davis
Department of Cell Biology, Baylor College of Medicine, Houston
Texas 77030
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ABSTRACT |
We have cloned and
characterized the opt1 gene of
Drosophila melanogaster. This gene
encodes a protein with significant similarity to the PTR family of
oligopeptide transporters. The OPT1 protein is localized to the apical
epithelial membrane domains of the midgut, rectum, and female
reproductive tract. The opt1 message is maternally loaded into developing oocytes, and OPT1 is found in the
-yolk spheres of the developing embryo. It is also found throughout
the neuropil of the central nervous system, with elevated expression
within the
- and
-lobes of the mushroom bodies. Transport activity was examined in HeLa cells transiently expressing OPT1. This
protein is a high-affinity transporter of alanylalanine; the
approximate Km
constant is 48.8 µM for this substrate. OPT1 dipeptide transport
activity is proton dependent. The ability of selected
-lactams to
inhibit alanylalanine transport suggests that OPT1 has a broad
specificity in amino acid side chains and has a substrate requirement
for an
-amino group. Together these data suggest an important role
for OPT1 in regulating amino acid availability.
PTR transport proteins; oligopeptide transport; protein metabolism; yolk; nutrient uptake
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INTRODUCTION |
THE CELLULAR UPTAKE of small peptides is fundamental to
nutrition and the economy of amino acids in many organisms. This
process occurs primarily through saturable carrier proteins. Many
bacteria and yeast actively take up short peptides directly from the
environment and are capable of using these peptides as their sole
source of nitrogen (61, 63). Carrier proteins within the roots of
Arabidopsis thaliana can also
transport di- and tripeptides from the growth media, thereby providing
an additional source of fixed nitrogen for this plant (79). In mammals,
as much as 60% of digested proteins is absorbed into the intestinal
epithelium as di- or tripeptides (14, 57). Furthermore, the loss of
small peptides from the mammalian glomerular filtrate is minimized by a
saturable oligopeptide transport system found in the renal proximal
tubule (28).
Small peptide transport may have additional importance in the early
development of many organisms. Many plants and animals stockpile
proteins in their oocytes or associated maternal tissue as a supply of
amino acids for developing embryos. In
Arabidopsis, di- and tripeptides are
transported from the protein stores of the endosperm into the
cotyledons of the developing embryo (77). In most invertebrates and
many vertebrates, yolk proteins are stored in membrane-limited
compartments within the oocyte. These protein stores are digested,
primarily by a cathepsin B-like peptidyl dipeptidase, and used as a
source of amino acids by the developing embryo. The mechanism by which
these amino acids are released from yolk vesicles remains largely
unexamined.
The transport and metabolism of small peptides may have a particularly
profound role in the nutrition and physiology of insects. In
Drosophila melanogaster, di- and
tripeptides are found in body fluids at unusually high concentrations,
constituting up to 30% of the total amino acids in adults (15, 18,
54). It has been proposed that these peptides may function in
osmoregulation, as has been shown in some marine invertebrates (15,
18). Most dietary protein digestion in
Drosophila occurs within the midgut; the end products of this proteolytic digestion are thought to be
quickly absorbed into the epithelia (40, 45, 75). The formation of
primary urine in insects occurs within the Malpighian tubules (48, 64).
The secretions of this organ are deposited into the hindgut, where
amino acids are reabsorbed into the rectal epithelia (64). Despite the
abundance of peptides within
Drosophila, virtually nothing is known
of their fate within the digestive or excretory systems.
Recently, several genes encoding a family of peptide transporters have
been cloned from mammals, yeast, plants, and bacteria (PTR family;
Refs. 59, 78). The characterized PTR proteins transport di- and
tripeptides with little specificity for amino acid composition (10, 25,
46, 47, 63, 69). Activity has been shown to be coupled to proton
symport for several of the family members (9, 25, 34, 47). The
identified mammalian PTR proteins are subdivided into two types: pepT1
and pepT2. This classification derives from the sequence similarity,
biochemical activity, and expression patterns of these proteins (9, 25, 47). The pepT1 proteins from human, rabbit, and rat are high-capacity, low-affinity transporters. These proteins are expressed abundantly in
the small intestine and at lower levels in the kidney (25, 46, 69). The
human, rabbit, and rat pepT2 proteins are low-capacity, high-affinity
transporters. The pepT2 proteins are expressed predominantly in the
kidney proximal tubules without detectable expression in the intestines
(9, 47, 53).
Here we report the characterization of
opt1, a
Drosophila member of the PTR family of
transporters. This gene was originally identified as a transcript
expressed preferentially in females, located adjacent to the
roX1 untranslated nuclear RNA (3, 52). We show that the opt1 locus encodes a
high-affinity di- and tripeptide transporter. Furthermore, our
experiments suggest that the OPT1 transport activity is proton
dependent. Opt1 mRNA is expressed in
germinal and somatic tissue of both genders but is most highly expressed in the nurse cells of the female ovary. OPT1 protein is found
on distinct membrane domains in neurons and epithelial cells of the
midgut, rectum, and female reproductive tract. The biochemical activity
and the sites of OPT1 expression suggest an integral role for this
protein in governing amino acid availability in
Drosophila.
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METHODS AND MATERIALS |
Strains.
Fly stocks were raised on cornmeal agar food at 22-25°C.
Wild-type control strains Canton-S or
ry506 were used.
The isolation of the MB710 line has previously been described (36). The
relevant genotypes of the three sex determination lines are as follows:
1) w
SxlM1,f3
sn/C(1)DX
y/Y;
2) y cm
Sxlf7,M1
ct6
v/C(1)DX
y,/BsY;
and 3)
X/BsY;th
st tra1 cp ri
pp/TM3.
Molecular biology.
The isolation of the
opt1/roX1
genomic clones has previously been described (52). The
opt1 transcribed region was identified by hybridizing isolated genomic clones to Northern blots containing 5 µg poly(A)+ RNA isolated from
whole flies. Selected genomic fragments were then used to isolated cDNA
clones from head-specific libraries kindly supplied by P. Salvaterra
(City of Hope, Duarte, CA), T. Schwarz (Stanford, CA), and C. Hall
(Baylor College of Medicine, Houston, TX). Two partial cDNAs and one
full-length cDNA were sequenced entirely. For the preparation of
sex-specific RNAs, ~28,000
ry506 flies
between 0 and 4 days old were harvested and sexed by hand. Heads and
bodies were separated, and RNA was isolated using Trizol reagent
[Bethesda Research Laboratories (BRL), Rockville, MD]. Both
formaldehyde and glyoxal-DMSO methods were used in the Northern analysis (70).
For the developmental RT-PCR analysis, ~200 mg of animals from each
stage were isolated by hand. The animals were homogenized in a 1.5-ml
Eppendorf tube, and RNA was extracted with Trizol reagent according to
manufacturer's protocols. Total RNA (2 µg) from each stage was
directly reverse transcribed with Superscript II RT (BRL). Control
reactions were digested with 10 µg DNase-free RNase for 1 h at
37°C before reverse transcription. PCR reactions were performed
with 1% of total reverse transcription reaction. PCR amplicons for
opt1 long and short shared the same
antisense primer and utilized exon-specific sense primers (sequence
available on request). PCR conditions were 94°C for 20 s, 64°C
for 20 s, and 72°C for 40 s, with 30 cycles for
opt1 long and 40 cycles opt1 short.
Computer analysis.
DNA and protein sequence analyses were performed with the GCG suite of
programs (23) and DNA Strider (version 1.2). Additional opt1 splice variants were sought in
the genomic DNA sequence with the GeneFinder program (76). The MAR
Finder algorithm was used to identify regions with high probability of
forming matrix attachment sites; for this analysis, all six rules were
utilized (71). The TOPPREDICT and MEMSAT programs were used for
membrane topology prediction (38, 72). The FASTA (62) and BLAST (1)
algorithms were used to identify homologous sequences in the
GenBank and EMBL databases. Protein alignments were
performed with a PAM250 matrix specific for integral membrane
proteins (39). The phylogenetic tree was generated by the TreeGen web
server (http://cbrg.inf.ethz.ch/subsection3_1_6.html; Ref.
32). The accession numbers for the proteins used in sequence comparisons are as follows: rabbit pepT2 (U32507; Ref. 9), human pepT2
(S78203; Ref. 47), rat pepT2 (D63149; Ref. 69), rabbit pepT1 (U06467;
Ref. 25), human pepT1 (U21936; Ref. 47), rat pepT1 (D50664; Ref. 53),
Caenorhabditis elegans ORF2 (g1246435;
Ref. 87), C. elegans ORF1 (1049410;
Ref. 87), cucumber chloroplast (Z69370),
Arabidopsis Chl1 (L10357; Ref. 85),
AtPtr2B (L39082; Ref. 77), Candida
Ptr2 (U09781; Ref. 8), AtPtr2A (U01171; Ref. 79),
Saccharomyces Ptr2 (L11994; Ref. 63),
Escherichia coli YHIP (1789911; Ref.
53), Lactococcus lactis DtpT1 (U05215;
Ref. 34), and E. coli YJDL (1786927).
Antibodies.
The Histag-outer loop fusion protein was generated by inserting the
0.65-kb Bgl
II-Pst I fragment from the
opt1 cDNA into the pRSETA vector
(Invitrogen, Carlsbad, CA). The resulting
Histag-OPT1(I417-A632)
fusion protein was induced with 0.4 mM
isopropyl-
-D-thiogalactopyranoside (IPTG) in BL21(DE3) pLysS and purified over a nickel-agarose column (Qiagen, Santa Clara, CA). The anti-outer loop (
-OL) antibody was
raised against this purified fusion protein. The glutathione S-transferase fusion to the
OPT1(L677-A743)
COOH-terminal peptide was generated by inserting the 0.3-kb Nhe I fragment from the
opt1 cDNA into the
Xba I site of the pGEX-KG vector.
Production of the fusion protein was induced with 0.4 mM IPTG in XL-1
Blue and purifed over glutathione-agarose according to the
manufacturer's recommendations (Pharmacia, Uppsala, Sweden). The
anti-COOH-terminal (
-Cterm) antibody was raised against this fusion
protein. Two New Zealand White female rabbits were injected for each
fusion protein. Fusion proteins were coupled to Reacti-Gel 6X
CDI-agarose according to manufacturer's recommendations (Pierce, Rockford, IL). The methods of Smith and Fisher (74) were used to purify
the antibodies from the fusion protein-agarose columns.
Standard procedures were used for Western blotting experiments (70).
Drosophila proteins were isolated by
using Trizol. Proteins from transfected HeLa cells were extracted in
1% SDS. The affinity-purified 2141
-Cterm antibody was used at
1:100 dilution. We used a goat anti-rabbit horseradish
peroxidase-conjugated secondary antibody from Vector Laboratories
(Burlingame, CA) at a 1:10,000 dilution.
Transport assays.
HeLa cells were seeded at 2 × 105 cells per 35-mm well and
incubated for 24 h before transfections. For each transfection, either
1 µg of pCMVOPT1 or 1 µg of pCMV2 was mixed with 6 µl of lipofectamine (BRL) according to manufacturer's recommendations. Transfection proceeded for 6 h, after which cells were cultured in DMEM
(BRL) for an additional 18 h. Transport assays were performed directly
in the 35-mm wells. All data points represent three independent transfections. For these transport assays, transfected cells were rinsed twice with transport buffer [25 mM MES-Tris (pH 6.0), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2,
0.8 mM MgSO4, and 5 mM
glucose] and then incubated with assay buffer at 22°C; this
is also the incubation temperature at which we raise
Drosophila. Assay buffer consisted of
1 ml of
L-[3H]alanylalanine
(Moravek Biochemicals, Brea, CA) diluted at the specified concentration
in transport buffer. Specific activity of
L-[3H]alanylalanine
was 1 Ci/mM, with the exception of the 400 µM alanylalanine transport
assays, which were at 0.5 Ci/mM. Transport was stopped by the addition
of 5 ml of ice-cold 1× PBS (pH 7.5), followed immediately by a
second rinse in the same buffer. Cells were lysed in 1 ml of 1% SDS,
and
L-[3H]alanylalanine
was measured by scintillation counting. In the time course experiment,
assay buffer contained 400 µM alanylalanine. In most of the
inhibition experiments presented in Table
1, 10 µM alanylalanine was incubated in
the presence of 10 mM peptide or peptidomimetics competitors (Sigma,
St. Louis, MO). The protonophore carbonyl cyanide
p-trifluoromethoxyphenylhydrazone (FCCP)
(Sigma) was applied at 25 µM simultaneously with 10 µM
alanylalanine. Uptake was measured at 2 min in both dose-response and
inhibition experiments.
Histology.
Cryosections (10 µm) for LacZ staining, in situ hybridizations, and
immunohistochemistry were as described by Han et al. (35). Paraffin
sections (5 µm) of female and male abdomens were processed as
described by Skoulakis and Davis (73). The collection, dechorionation, and fixation of embryos were as described (82). RNA in situ hybridization to sectioned material was essentially as described by
Skoulakis and Davis (73). Riboprobes were generated by in vitro
transcription of portions of the opt1
coding region from the c5 cDNA using digoxigenin-UTP (Boehringer
Mannheim, Indianapolis, IN). In situ hybridizations to embryos were as
previously described (52). OPT1 digoxigenin-labeled DNA probes for
these experiments were generated by random prime labeling of cDNA
fragments.
Immunohistochemistry procedures were as previously described (73). The
1476
-OL affinity-purified antibody was used at a 1:800 dilution for
immunohistochemistry of frontal head cryosections. Immunohistochemistry
on the paraffin abdominal sections was performed with either a 1:100
dilution of 2141
-Cterm affinity-purified antibody or a 1:10
dilution of 1476
-OL affinity-purifed antibody. A 1:2,000 dilution
of 2141
-Cterm affinity-purified antibody was used in
immunohistochemistry of embryos. The Vectastain ABC kit (Vector) was
used for signal detection in all immunohistochemistry experiments. Yolk
spheres from stage 10 and
14 oocytes were measured at ×100
magnification under oil immersion with an ocular reticle calibrated
with a stage micrometer. Ten neighboring spheres from two oocytes were
measured for each stage.
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RESULTS |
Identification and primary structure of opt1.
The MB710 enhancer detector line was identified in a screen for genes
preferentially expressed in the mushroom bodies of the female
Drosophila brain (36). The P element
in MB710 was inserted at cytological position 3F (36, 52). Two genes
were identified at this locus by Northern analysis:
roX1 and
opt1 (Fig.
1A)
(52). No additional RNAs within 15 kb on either side of the P element were detected by hybridization to
poly(A)+ RNA isolated from whole
flies (data not shown). The MB710 element interrupts and greatly
reduces roX1 expression (52).
RoX1 expression in wild-type adult
flies is principally limited to the male central nervous system (3,
52). Therefore, the LacZ activity in MB710 does not reflect this
gene's expression pattern. The opt1
gene was examined further as a potential gene expressed in mushroom bodies.

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Fig. 1.
Structure of
opt1/roX1
locus. A: transcript maps for
opt1 and
roX1 are shown below a genomic
restriction map for this locus. Shaded region within
opt1 transcript indicates 2 long open
reading frames produced by alternative 5'-exons. 5'- and
3'-ends of transcripts are indicated above respective maps.
Position of P[lArB] element within locus is indicated by
triangle below genomic map. Arrow within this triangle denotes
direction of LacZ transcription within P[lArB] element.
Vertical arrows indicate location of potential nuclear matrix
attachment sites. b, Xba I; B,
Bgl II; K,
Kpn I; S,
Sac I; X,
Xho I. B: model for topology of OPT1 is
shown. MEMSAT algorithm (38) was used to predict positions of
transmembrane helices in opt1 gene
product and orientation of this protein within membrane. Consensus
cAMP-dependent kinase phosphorylation site is denoted by an encircled P
in 4th cytoplasmic domain.
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Eight independent opt1 cDNAs were
isolated from several Drosophila
head-specific libraries. We estimate the abundance of
opt1 within the head to be ~1 in
23,000 transcripts as determined by the hybridization to an unamplified
cDNA library. Selected cDNA and genomic clones were completely
sequenced and compared. The longest cDNA was 2.85 kb; this cDNA starts
18 bp 3' to a consensus transcription start site and ends in a
poly(A) tail. Both opt1 and
roX1 are transcribed in the same
direction, with just 529 bp separating the two genes. The
opt1 gene is divided by three introns
(Fig. 1A). We identified two
alternative splice forms of opt1. Four
of the isolated cDNAs contained the 5'-most exon shown in Fig.
2, and the rest were truncated before this
splice juncture. Transcripts containing this exon are referred to as opt1 long. The GeneFinder algorithm,
however, predicts the presence of a novel 5'-exon within the
first intron of opt1 (76). This exon
was verified by Northern blots and RT-PCR and was also found in a cDNA
identified by Amrein and Axel (3). Transcripts containing this
alternative exon are referred to as
opt1 short. Hybridization of probes
specific for the two alternative exons to Northern blots demonstrate
that opt1 long transcripts represent
most opt1 messages (data not shown).
Two Drosophila-expressed sequence tags
with identity to opt1 were also
identified: LD02644 begins in the 5'-most exon of
opt1 long, and HL05718 shares identity
to the 3'-end of this gene. Additionally, two regions with strong
potentials for nuclear matrix attachment sites were identified at the
opt1/roX1 locus by the MAR Finder
algorithm [avg strength 0.72 (Ref. 71) and 0.74 (Ref. 42),
respectively]. These two sites flank the P-element insert in
MB710 (Fig. 1A).

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Fig. 2.
OPT1 is a member of proton-dependent oligopeptide transporter family.
Phylogenetic tree representing family of proton-coupled oligopeptide
transporters was generated by a least-squares dynamic programming
algorithm (32). Numbers are calculated PAM distances, and small circle
represents weighted centroid of tree.
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The longest opt1 cDNA contains a
single long open reading frame. Conceptual translation of this open
reading frame reveals a predicted polypeptide of 743 amino acids with a
molecular mass of 82.2 kDa. The opt1
short open reading frame encodes a protein of 737 amino acids (3). Both
OPT1 proteins possess regions of extreme hydrophobicity; the TOPPREDICT
and MEMSAT topology modeling algorithms predict 12 transmembrane helices in both OPT1 proteins (Fig.
1B) (38, 43, 72). One striking
feature of this topology model is the large 200-amino acid fifth outer
loop. The OPT1 protein also contains a single consensus protein kinase A phosphorylation site
(RRHS259-262) in the fourth
cytoplasmic domain.
OPT1 is a member of the peptide transporter family of proteins.
The conceptual translation of opt1
revealed a significant degree of similarity to a family of membrane
carrier proteins that includes the PTR transport proteins (59, 78).
These proteins transport di- and tripeptides across membranes energized
by a proton motive force (18, 25, 34, 47).
Opt1 was found to be most similar to
the human pepT2 oligopeptide transporter, with 40% identity and 62%
similarity (47). Significant similarity was also found to several other
proteins that are yeast and
Arabidopsis peptide transporters (63,
77, 79), nitrate transporters (85), or uncharacterized ORFs (53, 87).
We generated a phylogenetic tree to examine the possible relationships
among these proteins (Fig. 2). There are four major branches in this
family; OPT1 is found within a branch exclusively containing members of
the animal kingdom. The pepT1/pepT2 split within this subfamily occurs
after the divergence with OPT1 (Fig. 2).
We have optimally aligned the sequences of the PTR animal subfamily
(Fig. 3). The regions of identity and
similarity within this subfamily are found throughout the lengths of
these proteins. OPT1 is only slightly more like the renal pepT2
proteins than the intestinal pepT1 proteins and contains several
regions of identity that are specific to either all pepT1 or pepT2
proteins (Fig. 3). A conserved histidine residue that is obligatory for the transport function of both human pepT1 and pepT2 is also conserved in OPT1 (Fig. 3, position 88) (26). This histidine probably functions
in the binding and translocation of protons in the pepT1 and pepT2
proteins (26). The size, but not the sequence, of the large fifth outer
loop of OPT1 is well conserved in this branch of the family tree, but
the length of this domain is poorly conserved in the nonanimal family
members (data not shown). The strong sequence identity and similar
topology among these animal transporters present cogent evidence that
they arose from a common ancestor (49).

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Fig. 3.
Amino acid sequence comparison of animal proton-dependent
oligopeptide transporter subfamily. Caenorhabditis
elegans ORF1, C. elegans ORF2, human pepT2, rabbit pepT2, rat pepT2,
human pepT1, rat pepT1, rabbit pepT1, and OPT1 proteins are optimally
aligned. Conserved 28 amino acid
NH2-terminus of
C. elegans ORF1 sequence was generated
by splicing C. elegans cosmid K04E7
+6126 sequence (accession no. U39666) to splice acceptor sequence at
position +6553 in 2nd exon. Identities in all family members are
indicated by white lettering within black boxes. Amino acids that are
conserved in 6 or more family members are shaded in gray. Positions of
putative opt1 transmembrane domains
are underlined.
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OPT1 has proton-dependent oligopeptide transport activity.
Because OPT1 shares significant sequence similarity to nitrate as well
as oligopeptide transporters, we investigated the transport properties
of this protein in transfected HeLa cells. The kinetic parameters were
examined by measuring
[3H]alanylalanine
influx as a function of time and substrate concentration (Fig.
4). OPT1-transfected HeLa cells
demonstrated significant alanylalanine uptake; this activity was found
to be linear at 2 min for substrate concentrations ranging from 5 to
400 µM (Fig. 4A, data not shown).
Additionally, after 2 min, we failed to detect any degradation of
[3H]alanylalanine
within the Hela cell extracts by TLC (data not shown). These data
indicate that the substrate was not signifcantly processed within the
first 2 min of the assay and that we could measure the rate of
alanylalanine uptake.

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Fig. 4.
OPT1 facilitates cellular uptake of alanylalanine.
A: alanylalanine uptake was measured
in pCMVOPT-transfected HeLa cells or control cells transfected with
empty vector as a function of time. Transfected HeLa cells were
incubated in presence of 50 µM
[3H]alanylalanine (1 Ci/mmol) for indicated times. Assay buffer was pH 6. SE bars are shown.
B:
[3H]alanylalanine
uptake was examined at substrate concentrations ranging from 5 to 400 µM. Assays were conducted at pH 6 for 2 min.
Inset: Eadie-Hofstee plot. From this
plot, Km and
Vmax were
determined to be 48.8 µM and 384 pmol/2 min for
106 cells, respectively. SE bars
are shown.
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The OPT1-dependent alanylalanine transport activity displayed
Michaelis-Menten saturation kinetics with an apparent
Km and Vmax of 48.8 µM
and 384 pmol/2 min for 106 cells,
respectively (Fig. 4B). We further
investigated whether this transport process is driven by a proton
motive force by either increasing the pH of the transport buffer or by
removing the proton gradient. Alanylalanine transport is reduced
~10-fold at pH 7 and 20-fold at pH 8 (Table 1). When the protonophore
FCCP (25 µM) was used to collapse the proton gradient, alanylalanine
uptake was almost eliminated (Table 1). Together, these data strongly suggest a proton dependence for OPT1-driven dipeptide transport.
The potential substrate specificity of OPT1 was examined by measuring
uptake of 10 µM
[3H]alanylalanine in
the presence of 10 mM competitor (Table 1). The capability of OPT1 to
transport peptides of different lengths was examined with a series of
alanine peptides. Alanine and tetraalanine did not significantly
inhibit uptake (P > 0.05, Student's
t-test), whereas di- and trialanine
peptides practically eliminate measurable uptake (Table 1). The
D-enantiomer of alanylalanine
failed to significantly inhibit uptake, indicating that OPT1 is also
stereoselective in substrate recognition (Table 1). Glutathione is one
of the most abundant peptides in
Drosophila (58). Nevertheless, this
-glutamyl-linked tripeptide was a poor inhibitor of alanylalanine uptake, suggesting that it is an unlikely substrate for OPT1 transport in vivo (Table 1). We also examined the ability of several
peptidomimetic drugs to inhibit alanylalanine transport. For this
comparison, we used the closely related
-lactams, ampicillin,
carbenicillin, and benzylpenicillin. These drugs have identical
backbones but differ at the
-substituent; ampicillin has an
-amino group, whereas carbenicillin has a carboxyl moiety and
benzylpenicillin has a hydrogen. Of these three, only ampicillin
significantly inhibited transport, suggesting that the
-amino group
may be essential for substrate recognition (Table 1). Consistent with this hypothesis, the peptidomimetic angiotensin-converting enzyme inhibitor captopril, which has an
-sulfhydryl group, was a poor inhibitor, and the aminocephalosporin cefadroxil was an effective inhibitor of transport (Table 1).
Distribution of OPT1.
We examined the distribution and timing of
opt1 expression by Northern analysis
and RT-PCR (Fig. 5). The full-length
opt1 cDNA hybridized to a single
3.0-kb message in the heads and bodies of both adult male and females,
with most of the signal located in the female body (Fig.
5A). Additionally, both
opt1 long and short splice variants
are present throughout development (Fig. 5B). The short splice form, however,
was barely detectably during larval stages.

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Fig. 5.
Analysis of opt1 transcripts.
A: Northern blot containing 5 µg of
poly(A)+ RNA from heads and bodies
of males (M) and females (F) was hybridized with a full-length
Opt1 cDNA. Alcohol dehydrogenase (ADH)
was hybridized as a loading control. Because chorion genes are only
expressed in female bodies, they are used as a control for sex
specificity of RNAs. B: RT-PCR
amplification of opt1 long and short
transcripts from different developmental stages demonstrating presence
of both messages throughout development. Sense primers for each
amplicon were specific to indicated exon; same antisense primer from
exon 3 was used in both reactions.
Opt1 long was amplified for 30 cycles
and opt1 short for 40 cycles.
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We further identified the foci of opt1
expression with both in situ hybridization and immunohistochemistry. To
ensure that we detected authentic OPT1 immunoreactivity, we used four
independent affinity-purified antibodies raised against either the
COOH-terminus or a polypeptide containing most of the large fifth outer
loop (see METHODS AND MATERIALS). On
Western blots, one of the antibodies raised against the COOH-terminus
could specifically recognize OPT1 expressed in both HeLa cells and
Drosophila (Fig.
6). The other three antibodies failed to
reproducibly recognize OPT1 from Drosophila extracts but specifically
recognized OPT1 expressed in HeLa cells (data not shown). Nevertheless,
we had an excellent correlation between the patterns of expression
detected by in situ hybridization and all four antibodies.

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Fig. 6.
OPT1 is detected in embryos and adults by Western analysis. An 81.6-kDa
protein is detected by affinity-purified anti-COOH-terminal ( -Cterm)
antibody in 10 µg of protein extract from HeLa cells expressing
opt1 gene but not in controls cells
transfected with empty vector. OPT1 is also detected in 100 µg of
embryo and 200 µg of adult male protein extracts. Molecular masses
(in kDa) are indicated.
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Opt1 is expressed in both somatic and
germinal tissues. The opt1 message and
protein are detected within the epithelia of the midgut, rectum, and
the oviducts, seminal receptacles, and spermathecal ducts of the female
reproductive tract (Fig. 7). OPT1
immunoreactivity is clearly limited to the apical membranes of the
midgut and rectal epithelium (Fig. 7,
A and
B). OPT1 is detected along the
entire length of the midgut, beginning at the cardia and ending at the
junction with the anterior intestine. We also detected OPT1
immunoreactivity within the basal portions of all four rectal papilla,
but no signal is found in the more apical regions, suggesting the
presence of specific domains within this tissue (Fig.
7C). The oviducts, seminal
receptacles, and spermathecal ducts of the female reproductive tract
also stain specifically with anti-OPT1 antibodies, and a low amount of
OPT1 is detected within the uterus (Fig.
7C). OPT1 may be expressed at very
low levels within almost all neuropil regions of the central nervous
system (Fig. 7D). An increase in
signal is seen in the
- and
-lobes of the mushroom bodies,
consistent with a modest preferential expression in this area. There
was no increase in protein levels detected in the
-lobes, suggesting
that
- and
-lobe mushroom body neurons may require or benefit
from more peptide transport activity than the
-lobes. OPT1 is also
detected in the antennal nerve (data not shown). Nevertheless, no
sexually dimorphic staining patterns are found in the central nervous
system. In contrast to the weak expression in the brain, a strong
signal is seen within the fat bodies surrounding the central nervous system (Fig. 7D). It is therefore
probable that opt1 expression in the
fat bodies accounts for the majority of transcripts present the head
RNA population (Fig. 5A).

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Fig. 7.
OPT1 expression in somatic membrane domains.
A: immunohistochemical staining of a
sagittal section of boundary between thorax and 1st abdominal segment.
-Cterm antibody was used to detect OPT1 in sectioned tissue. OPT1 is
found within apical membranes of midgut. Magnification: ×40.
B: immunohistochemical staining of a
sagittal section of posterior abdomen of a wild-type female. OPT1 was
detected within apical membranes of rectal epithelium with -Cterm
antibody. Magnification: ×40. C:
immunohistochemical staining of a sagittal section of posterior abdomen
of a wild-type female. -Cterm antibody was used to detect OPT1. OPT1
immunoreactivity is detected within rectal papilla (rp), oviducts (od),
seminal receptacle (sr), spermathecal ducts (sd), and oocytes (oo).
Magnification: ×10. D:
immunohistochemical staining of a frontal section of a wild-type female
head. Anti-outer loop ( -OL) antibody was used to detect OPT1 within
head cavity. fb, Fat bodies; bl, -lobes of mushroom bodies.
Magnification: ×20.
|
|
Hybridization of antisense riboprobes to female abdomens revealed that
copious levels of opt1 are synthesized
within the nurse cells and deposited in the developing oocyte (Fig.
8, A and
B). The earliest visible message is
perinuclear to the oocyte, and, by early stage
10, the nurse cells are filled with transcript that is
excluded from their nuclei (Fig. 8, A
and B). In stage 11 follicles, the nurse cell-produced message is seen
transported through the ring canal into the oocyte in a pattern
characteristic for maternally loaded messages (Fig.
8B). Because the expression of the
adjacent roX1 gene is controlled by
the dosage compensation system, we examined whether ectopically
expressed msl-2 could eliminate
opt1 expression in the ovaries.
Females carrying the msl-2 transgene
have smaller ovaries than wild-type females, but the few developing
follicles that they produce contain
opt1 in the same pattern as seen in
wild-type follicles (data not shown). This suggests that
msl-2 does not downregulate
opt1 in the female germ line.

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Fig. 8.
Maternal OPT1 is localized to embryonic yolk spheres. In situ
hybridizations with opt1 antisense
riboprobe and immunohistochemical staining with -Cterm antibody are
shown to developing oocytes and embryos.
A: in situ hybridization to a sagittal
section of a wild-type female abdomen. oo, Oocyte; ncn, nurse cell
nucleus. B: in situ hybridization to
whole mount ovaries. C:
immunohistochemical staining of a sectioned late stage
9 oocyte. Punctate staining is seen surrounding
germinal vesicle (gv). D:
immunohistochemical staining of a sectioned stage
14 oocyte. Punctate OPT1 staining is now spread
throughout central ooplasm. E: lateral
view of a 0- to 1-h embryo after in situ hybridization.
F: lateral view of a 1- to 2-h embryo
after in situ hybridization. Signal is gone from cortical regions.
G: lateral view of a 3-h-old embryo
(blastula) after in situ hybridization.
Opt1 message is no longer detectable.
H: dorsal view a
stage 15 embryo after
immunohistochemical staining. -Cterm antibody was used to detect
OPT1. Staining is present in central yolk mass.
|
|
In early stage 9 follicles,
immunohistochemical staining for OPT1 produces an even staining
throughout the developing oocyte. By late stage
9, an additional punctate staining at the nurse cell-oocyte boundary near the germinal vesicle is detected (Fig. 8C). In slightly older oocytes, this
vesicular pattern spreads through the subcortical regions, and the even
background staining begins to fade from the ooplasm. By
stage 14, OPT1-containing vesicles are
detected throughout the ooplasm but not in the cortex; the general
ooplasm staining has disappeared by this stage (Fig. 8D). We measured the diameter of
these vesicles under ×100 magnification. Vesicles from late
stage 9 oocytes are 3.8 ± 0.3 µm in diameter, and stage
14 oocyte vesicles were 4.3 ± 0.4 µm. The
appearance and size of these vesicles correspond well with mature
-yolk spheres (20, 29, 30). At ×100 magnification, many
smaller vesicles are distinguishable within the cortex of
stage 10 oocytes. These smaller
vesicles range in size from ~100 nm close to the oocyte membrane to 1 µm in diameter near the central ooplasm boundary (data not shown).
Stage 10 follicle cells occasionally
display OPT1 immunoreactivity (data not shown). This infrequent
staining may denote a very transient expression of OPT1 within these
cells.
Opt1 transcripts remain abundant in
wild-type embryos <2 h old, but, during the formation of the
blastoderm, transcripts are eliminated from the cortical regions
of the embryo, and by the completion of cellularization,
opt1 can no longer be detected (Fig.
8, E-G). In the late stages of
embryogenesis, the developing midgut encircles and engulfs the central
yolk mass. We detected OPT1 in the central yolk mass during these late
stages (Fig. 8H). Although the
opt1 message is gone by 3 h after egg
laying, the protein perdures for at least 16 h and remains associated
with the yolk spheres.
Opt1 message is also detected in
premeiotic germ cell cysts of the testes (Fig.
9). The
opt1 message and protein are limited to a small number of cysts in wild-type males, suggesting that the gene
product is tightly regulated and limited to a specific stage of germ
cell development in this tissue. No significant differences are seen in
opt1 expression patterns in Canton-S, MB710, SxlM1,f3,
or Sxlf7,M1 males
(Fig. 9; data not shown). In XX pseudomales produced by mutations of
tra or
Sxl, testicular development and
spermatogenesis is initiated but never completed; the result is
incomplete gonads referred to as pseudotestis. Pseudotestis from
XX;tra1
pseudomales are smaller than those of wild-type males, and development of germ cells is abnormal (Fig. 9D).
Opt1 probes stain cysts darkly; however, occasional abnormally large cysts located in a more basal region of the testis are also seen hybridizing with
opt1 (Fig. 9D).

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Fig. 9.
Opt1 is expressed in male germinal
cysts. In situ hybridizations to whole mount testes and
immunohistochemical staining to sectioned testes with -OL antibody
are shown. A: wild-type Canton-S
testis, in situ hybridization. B:
lateral view of wild-type testis after immunohistochemical staining.
Testis is on right; midgut is on
left.
C: MB710 testis, in situ
hybridization. D:
XX;tra1
pseudotestis, in situ hybridization.
|
|
 |
DISCUSSION |
In this paper, we present the characterization of the
D. melanogaster
opt1 gene and a functional analysis of
the OPT1 protein. We also define the foci of OPT1 expression through in
situ hybridization and immunohistochemistry. The
opt1 gene encodes a proton-dependent oligopeptide transporter found at cytological position 3F immediately adjacent to the roX1 nuclear RNA gene
(3, 52). OPT1 is expressed in several epithelia including the apical
membranes of the midgut, rectum, and female reproductive tract. OPT1 is
also expressed at low levels throughout the central nervous system and
in the
-yolk spheres of the oocyte and developing embryo.
Opt1 gene structure.
We have shown that the opt1 gene
contains two alternative 5'-exons. The most upstream exon was
identified by Northern analysis and RT-PCR and found within four
full-length cDNAs. The second exon was identified by the GeneFinder
algorithm and subsequently verified by Northern analysis (76). During
the preparation of this paper, Amrein and Axel (3) reported the
sequence of an opt1 cDNA that
contained this second intron. The opt1
long transcript, containing the upstream alternative exon, is the most
abundant in both head and body. The promoter for this transcriptional
start site is therefore the most active.
OPT1 has proton-dependent dipeptide transport activity.
The opt1 gene product shares
significant sequence similarities to the PTR family of carrier proteins
(59, 78). The greatest similarities were to the mammalian pepT1 and
pepT2 proteins. These proteins transport di- and tripeptides across
membranes energized by an electrochemical proton gradient (9, 25, 46,
47, 69). We utilized a transient expression assay to examine the biochemical activity of the OPT1 protein. A similar assay system was
previously used for the characterization of human pepT1 and pepT2 (46,
47). We have shown that OPT1 has a high-affinity dipeptide transport
activity. OPT1-dependent alanylalanine uptake is also severely affected
by the pH of the cis-compartment;
active transport is seen at pH 6, severely reduced uptake at pH 7, and almost absent at pH 8. When the proton gradient was collapsed with
FCCP, very little transport occurred. Taken together, these data
support the proton dependence of dipeptide transport by OPT1. The
length of the peptides transported also appears to be selective; single
amino acids and tetraalanine are incapable of competing for
alanylalanine uptake in our assay. These data strongly suggest that
OPT1 transports primarily di- and tripeptides in vivo.
To examine possible substrate specificities, we utilized
-lactam
antibiotics. The benzylpenicillin family of antibiotics includes
ampicillin, carbenicillin, and penicillin G. These peptidomimetics have
almost identical structures, differing only at the
-substituent. The
ability of ampicillin and cefadroxil to inhibit alanylalanine transport
suggests that these molecules are substrates for OPT1 transport. The
side chains of these molecules are very dissimilar chemically from
alanylalanine and from each other. OPT1 may therefore have little
specificity for amino acid side chains. In contrast, the failure of
carbenicillin and benzylpenicillin to inhibit transport and the weak
inhibition found with captopril are consistent with a requirement for
an
-amino group in the peptide substrate of OPT1. This necessity for
the
-amino group is similar to the rabbit pepT2, which shares this
requirement; the rabbit pepT1 protein does not have a strict
requirement for an
-amino group (9, 10, 25, 27). Glutathione is an
extremely abundant dietary and cellular peptide (33, 58). Because
reduced glutathione is a
-glutamyl-linked tripeptide, in theory, it
was possible for this peptide to be a substrate for OPT1 transport.
Glutathione, however, is a poor inhibitor of alanylalanine transport
and is therefore an unlikely substrate in vivo. Consistent with this finding, dietary and interorgan glutathione uptake in humans is not
mediated by either pepT1 or pepT2 but through a distinct
Na+-dependent carrier protein
(33).
OPT1 and protein metabolism.
The presence of OPT1 within several epithelial membranes suggests a
general role in protein metabolism. The expression of OPT1 in the
midgut is consistent with a role in the absorption of dietary peptides.
The midgut is the site of almost all dietary protein digestion and
absorption (75). A carboxypeptidase, a trypsinlike activity, and at
least two dipeptidases have been identified in the
Drosophila midgut (40, 44, 86). The
position of OPT1 on the apical membrane would suggest that this protein is organized in the membrane for uptake of peptides, generated by the
digestive proteases, from the lumen of the midgut into the epithelia
cells. The absence of OPT1 in the basolateral membrane intimates that
the transported peptides are processed intracellularly, presumably by
the dipeptidase A and B activities previously identified in this tissue
(45, 44).
In most insects, including Drosophila,
the formation of primary urine by filtration and the active secretion
of selected substances occurs within the Malpighian tubules (48, 65).
The excreta are then deposited into the hindgut, where many filtered
metabolites are reabsorbed by the rectum (64). Small peptides in the
Drosophila hemolymph represent a
significant proportion of the total amino acids in adults (15, 18, 54).
OPT1 present in the rectal epithelia is probably involved in the
reabsorbtion of some of these peptides that are filtered through the
Malpighian tubules.
There are few indications of protein digestion within the female
reproductive tract of Drosophila.
During copulation, the male transfers several proteins and small
peptides, produced in his accessory glands, into the female
reproductive tract. These proteins elicit several changes in female
physiology and behavior, including decreases in life span and mating
receptivity and increases in egg laying and the efficient storage and
utilization of sperm in the seminal receptacles and spermatheca (12,
16, 37). At least 85 distinct accessory gland proteins and peptides
have been identified by two-dimensional electrophoresis (19, 81). One
such protein, Acp26Aa, has been shown to be proteolytically processed
within the female reproductive tract (55, 60). The cleavage of Acp26Aa
requires at least one additional product of the male accessory glands,
although cleavage does not occur until deposition in the female
reproductive tract (60). The Acp76Aa protein is also transfered into
the uterus during copulation; by 6 h after transfer, Acp76Aa is barely
detectable in the female reproductive tract (17). This protein is a
member of the serine protease inhibitor superfamily (17). Taken
together, these results demonstrate that, after copulation, digestion
of accessory gland proteins occurs within the female reproductive
tract. OPT1 may remove the resulting peptides from the sites of
proteolysis into the epithelium. It is worth noting that the female
reproductive tract of Drosophila has
four times more soluble dipeptidase activity than the alimentary tract
(44).
Besides epithelia, OPT1 is also expressed in cells of the fat body and
the neurons of the central nervous system. The fat bodies have the
function in flies analogous to that of the vertebrate liver. The human
pepT1 and both the rabbit pepT1 and pepT2 genes are expressed in the
liver, consistent with a conserved function (9, 46, 47). The role of
peptide transport in the central nervous system is poorly understood.
Interestingly, rabbit pepT1 and pepT2 messages are expressed in the
brain, suggesting a general role for PTR proteins within the central
nervous system (9, 25). Saturable uptake systems have been described
for several neuropeptides in the blood-brain barrier, although the
proteins have not yet been isolated (5, 7, 80). Most of these
neuropeptides appear to be too large for transport by PTR family
members. However, many neuropeptides are metabolized in the brain on
the cell surface (6, 13, 41). OPT1 may function in the absorption of
these metabolites. A modest increase in OPT1 expression is seen in the
- and
-lobes of the mushroom bodies. These lobes are a subset of
the axonal projections of the mushroom bodies (21). There are currently
few characterized neuropeptides in
Drosophila. Nevertheless, the amnesiac
gene encodes a PACAP-like peptide that may effect the cAMP-dependent
physiology of mushroom bodies (22, 24). Thus metabolites of the
amnesiac gene product represent possible substrates for OPT1 transport
within the mushroom body axons.
OPT1 in early development.
The OPT1 protein is located on the
-yolk spheres from their
formation in stage 10 oocytes
throughout embryogenesis. The
-yolk spheres are membrane-limited
vesicles containing crystalline arrays of the three distinct yolk
proteins (11). In the developing embryo, these yolk spheres are the
primary source of amino acids for protein synthesis. An aspartic
proteinase is active in the yolk spheres of mature oocytes (51). The
aspartic proteinase is thought to activate a cathepsin B-like
proteinase found within the spheres at the start of embryogenesis (50).
In contrast to the aspartic proteinase, this cathepsin B-like
proteinase readily cleaves the yolk proteins at pH 6, and its activity
increases throughout embryogenesis (50). The cathepsin B proteins are endoproteases with peptidyl dipeptidase activity (4). OPT1 is probably
required to transport the dipeptides generated by this protease into
the developing embryo.
An interesting question is how OPT1 is placed on the yolk sphere
membrane. The insect oocyte remains an excellent model cell for the
observation of intercellular trafficking. Yolk proteins enter the
oocyte from the hemolymph through receptor-mediated endocytosis (68,
83, 84). The clatherin-coated vesicles containing yolk protein are then
trafficked through tubular, transient vesicles, where the yolk proteins
disassociate from receptor (31, 67). Small vesicles containing free
yolk proteins will pinch off these tubular compartments and fuse with
immature or transitional
1-yolk
spheres (31, 67). The maturation of these spheres involves fusion with
Golgi vesicles and the formation of yolk protein crystals (20,
29-31). The mature
2-sphere moves from the cortex
into the central regions of the oocyte. The
2-yolk spheres of
Drosophila are ~3 µm in size and
number close to 104 in a
stage 14 oocyte (20). The small 0.1- to 1-µm OPT1-containing vesicles within the stage
10 oocyte cortex are correctly sized and positioned to
be the early transitional yolk spheres. It is probable that OPT1 is
deposited in these spheres by the fusion of Golgi vesicles, and the
larger 4-µm OPT1 vesicles are the maturing
1- and
2-yolk spheres that have left
the oocyte cortex and are awaiting proteinase activation. The
orientation of OPT1 on these yolks spheres would be appropriate for the
transport of peptides out of the vesicle and into the developing
embryo.
We have presented data that are consistent with OPT1 being an authentic
orthologue of both pepT1 and pepT2. The primary sequence similarities
between OPT1 and the pepT1 and pepT2 proteins are significant
throughout their entire lengths, and the predicted topologies are also
very well conserved. The phylogenetic tree suggests the pepT1-pepT2
split occurred after divergence from OPT1. The kinetic properties of
OPT1 are more like the pepT2 than pepT1 proteins. Specifically, the
high-affinity for dipeptides and the apparent requirement for an
-amino group for substrate recognition are properties shared with
the renal pepT2 but not pepT1 proteins. The expression of OPT1 in the
apical membranes of the midgut and rectum is directly analogous to the
expression of pepT1 on the brush-border membranes of the small
intestine and pepT2 in the renal proximal tubules, respectively. Thus
it is likely that OPT1 has the cognate pepT1 and pepT2 biological functions in Drosophila.
 |
ACKNOWLEDGEMENTS |
We thank S. Ahmed, B. Schroeder, and F. Villalba for technical
assistance.
 |
FOOTNOTES |
This work was supported by Grant DR-1344 from the Cancer Research Fund
of the Damon Runyon-Walter Winchell Foundation (G. Roman) and by
National Institute of Mental Health Grant 1-RO1-H/NS-55230 and the R. P. Doherty-Welch Chair in Science (R. L. Davis).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: R. L. Davis, Dept. of Cell Biology,
Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
Received 27 April 1998; accepted in final form 3 June 1998.
 |
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