Identification of a novel gene similar to both vesicular monoamine and acetylcholine transporters
The D. melanogaster
genome contains orthologs of all known vesicular neurotransmitter transporters, including genes similar to VGLUT
(Daniels et al., 2004
; Fei et al., 2010
; Greer et al., 2005
; Kitamoto et al., 1998
). We searched the genomic database for genes similar to Drosophila VMAT
) to identify additional, potentially novel vesicular transporters. We identified a gene similar to both DVMAT
that localizes to cytogenetic region 95A on chromosomal arm 3R. DVMAT
localize to cytogenetic regions 50B (2R) and 91C (3R), respectively. We found that CG10251 shows 35.8% similarity to DVMAT and 30.2% similarity to DVAChT (Fig S1
). In comparison, DVMAT and DVAChT share 35.5% similarity. The long open reading frame of CG10251 contains 12 predicted transmembrane domains similar to both mammalian and Drosophila
VMAT and VAChT.
RNA and protein expression
To confirm that CG10251
RNA is expressed in vivo
, we probed Northern blots of adult fly heads and bodies (). We detected a major band migrating at just above the 2 kb marker and a minor species at 5 kb. We also detected the ~2 kb species in bodies, but at low levels relative to heads. We observe similar enrichment in heads for DVMAT
and other neurotransmitter transporters (Greer et al., 2005
; Romero-Calderon et al., 2007
). The size of the major CG10251
mRNA species was similar to the cDNA we obtained with RT-PCR (2.2 kB), suggesting that we identified the full extent of the major CG10251
transcript. Repeated trials of 5′ and 3′ RACE did not reveal additional exons (not shown); thus, the minor 5 kB species likely represents an mRNA precursor, although we cannot rule out the possibility of a low-abundance splice variant.
Fig 1 Expression of CG10251 mRNA, protein, and subcellular localization. A) Northern blots show expression of CG10251 mRNA in heads and, to a lesser extent, in bodies. B) PCR using a panel of cDNAs from various developmental stages (see labels in panel C) confirmed (more ...)
We performed PCR with a commercially available cDNA panel representing various developmental stages and a CG10251-specific primer set (). Our data suggest that CG10251 is primarily expressed during adulthood and late larval stages rather than during embryonic development. We have attempted in situ hybridization of embryos for CG10251 expression, but have been unable to detect a signal (not shown), possibly due to the low levels of CG10251 mRNA at this stage.
We next generated an antibody against the CG10251 carboxy terminus and probed homogenates of S2 cells transfected with CG10251 cDNA to test its activity (). Cells expressing CG10251 (+) showed a broad 70 kD signal whereas untransfected S2 cells (−) did not. We probed homogenates of adult heads and detected a band at 70 kD, as well as additional bands at the top of the gel that may represent non-specific cross reactivity (). A faint band immediately above the major band suggests that a portion of CG10251 may undergo post-translational modification. This species was more visible in biochemically fractionated samples (see below). Another faint band at 40 kD may represent a degradation product. We confirmed the specificity of the antiserum with the CG10251 mutant (see below). We thus demonstrated the in vivo expression of both CG10251 mRNA and protein.
The similarity of CG10251 to DVMAT and DVAChT suggests that it, too, might encode a vesicular transporter. CG10251 localized to intracellular membranes at steady-state when expressed in S2 cells, and in vitro endocytosis assays revealed that CG10251 internalized from the cell surface as we have observed for DVMAT and DVGLUT (not shown). We therefore tested whether the protein would also localize to synaptic vesicles (SVs) in vivo. Relatively low expression of endogenous CG10251 made it difficult to detect in initial biochemical fractionation experiments (not shown). To facilitate these analyses we created a fly transgene expressing an HA-tagged version of the protein and used the pan-neuronal elav-Gal4 driver. To determine whether CG10251 localizes to SVs, we applied homogenates from flies expressing CG10251 to a glycerol velocity gradient. A portion of CG10251 peaked in fractions containing the peak for SV marker cysteine string protein (CSP, fractions 11–13, ). These data suggest that at least a fraction of the protein localizes to SVs, consistent with the prediction from sequence analysis that CG10251 is a vesicular transporter.
We also performed sucrose density fractionation to determine whether CG10251 might localize to other types of secretory vesicles, in particular large dense core vesicles (LDCVs). We found that while some CG10251 co-localized with CSP in light fractions, most of the immunoreactivity was found in heavier fractions, some coincident with a fusion protein containing mammalian Atrial Natriuretic Factor (ANF, ), a marker for LDCVs (Rao et al., 2001
). These data suggest that CG10251 likely localizes to LDCVs as well as SVs, similar to mammalian VMAT2, which preferentially localizes to LDCVs in cultured cells and in vivo
(Nirenberg et al., 1995
Localization in the larval nervous system
To localize CG10251 in vivo
, we labeled whole mounts of 3rd
instar larval brain and ventral ganglia. A small subset of cells in the ventral ganglia expressed CG10251 (). We did not detect significant co-localization with 5HT, TH, or Ddc (not shown). Thus, CG10251 is unlikely to store either dopamine or serotonin, in contrast to DVMAT, which localizes to these cell types (Greer et al., 2005
). Other aminergic transmitters in the fly include octopamine and tyramine; however, DVMAT is likely responsible for their transport as well (Greer et al., 2005
) and both localize to large midline cells (Monastirioti et al., 1995
; Nagaya et al., 2002
). We did not detect cells expressing CG10251 at the midline.
Fig 2 Localization of PRT in the larval ventral ganglion. A–C: One to two cell bodies per hemisegment express PRT and localize to lateral aspects of the ventral nerve cord. Processes project medially into the neuropil in a complex pattern that runs (more ...)
In the larval brain, we observed robust expression of CG10251 in the MBs (). To confirm localization to cells in the MBs, we expressed mCD8-GFP
with the MB driver 0K107-Gal4
(Connolly et al., 1996
) (), and co-labeled larval brains for CG10251 (). We observed overlap in the medial and vertical lobes that make up the axonal projections of the KCs, the calyces or dendritic bundles, as well as the KC bodies (). These data indicate that CG10251 is expressed by at least a subset of the KCs intrinsic to the MBs and therefore may be responsible for storage of neurotransmitter in these cells. In light of this expression pattern and the proposed transport function of CG10251 we have renamed the gene portabella
) and refer to the CG10251 protein as PRT.
We note that subsets of KCs did not appear to be labeled (asterisks, ) by the PRT antibody. A similar pattern has been reported for several developmental markers expressed in KCs (Noveen et al., 2000
) suggesting that PRT may be expressed at a relatively late stage during differentiation and perhaps only in a subpopulation of KCs.
We observed PRT expression in at least one bilateral extrinsic neuron projecting ipsilaterally to the vertical and medial lobes of the larval MBs (arrowheads, ). The location and projections from this cell appear similar to that described for a neuron expressing the amnesiac peptide, which is critical for memory formation in Drosophila
(Waddell et al., 2000
). However, co-labeling experiments suggest the extrinsic neurons expressing PRT are distinct from those expressing amnesiac (Fig S2
). Expression of PRT in these cells and four other small clusters in the larval brain is shown schematically in .
Localization in the adult nervous system
nervous system undergoes extensive remodeling during metamorphosis, resulting in adult MBs that are morphologically distinct from the larval structures. In the adult, each vertical lobe of the MB can be recognized as distinct α and α′ lobes and the medial lobes include distinct β, β′ and γ lobes (Crittenden et al., 1998
). We observed strong PRT expression in the adult MBs, including labeling of all 5 lobes (). Relative to the lobes, labeling of the calyx and KC bodies was less intense in the adult than the larva (). This pattern likely reflects the localization of the protein to secretory vesicles that are concentrated in the axons and less abundant in mature dendrites and cell bodies.
Fig 3 PRT expression in adult mushroom bodies, central complex and thoracic ganglion. PRT is expressed in the medial lobes β, γ (A, B), and β′ (A), the peduncle (ped, D–F) and the vertical lobes α and α′ (more ...)
We also detected PRT expression in the peduncle, formed by KC axons before they branch into the lobes (). PRT was not distributed uniformly throughout the peduncle and a portion of the core was weakly labeled ( and data not shown). This pattern suggests that PRT may not be expressed in all KCs although further experiments will be needed to confirm this. Several additional cell bodies near the MBs express PRT () as well as 1 cluster of 2–3 cells in the subesophageal ganglion that project medially toward the esophogeal foramen (arrows, ).
During metamorphosis there is also extensive development of the central complex (CCX), a midline structure just posterior to the MB medial lobes involved in motor activity (Strauss, 2002
) and visual memory (Liu et al., 2006
). PRT labeling of the adult brain revealed that it is expressed in components of the CCX including the neuropil of the ellipsoid and fan shaped bodies ().
We also detected PRT expression in two bilaterally symmetric clusters of two and three cells each near the medial aspect of the optic lobe that project outward toward the medulla (asterisks, ). The cartoon in summarizes the PRT expressing cells in the adult. Other than the KCs there are approximately 56 labeled cell bodies. For comparison, the adult brain contains approximately 300 dopaminergic and 106 serotonergic cells (Monastirioti, 1999
To complete our survey of the adult central nervous system, we also labeled the thoracic ganglion and found 3 clusters with 2–4 cells each that lie along the ventral midline (). This expression pattern was not sexually dimorphic ().
Generation of a prt mutant
To investigate the function of PRT, we generated a mutant fly. A survey of the public database revealed a previously generated line with a SUPor-P element inserted into the 5′ UTR of prt (). Line KG07780 was obtained from the Bloomington Drosophila Stock Center (Indiana University) and we confirmed that the SUPor-P element was located 118 bp upstream of the predicted initiating methionine (not shown). We used imprecise excision to generate a prt mutation. Lines were screened by PCR with primers flanking the P element insertion. In CantonS (CS) we detected a major product that migrated at 1.2 kb, consistent with the size predicted by the primary sequence (). In one line, the major band migrated at 400 bp, consistent with an 850 bp deletion (). We designated this allele prt1. We immunolabeled adult brains to determine whether prt1 mutants produce any residual protein and failed to detect any labeling of the MBs or elsewhere (). These data confirm the specificity of the antiserum to PRT. In addition, the size of the deletion and the absence of residual protein suggest that prt1 is either a severe hypomorph or null mutation (see also deficiency analysis below).
Fig 4 The prt1 mutation. A) The relative position of the prt gene is shown in cytological region 95A8 on the 3rd chromosome. The shaded box at the top of the figure represents an approximately 850 bp deletion created with imprecise excision of a P-element (“P” (more ...)
For both mammals and invertebrates, developmental perturbations of neurotransmitter metabolism can have neuroanatomical sequelae (Budnik et al., 1989
; Lawal et al., 2010
; Levitt et al., 1997
). We therefore analyzed the morphology of the MBs and CCX in the prt1
mutant and found it grossly intact in paraffin sections of adult brains stained with H&E ( and data not shown). To rule out more subtle neuroanatomical changes, we performed volumetric analyses of the MB calyx and CCX (ellipsoid body + fan shaped body). We detected no difference in either calyx or CCX volume between CS and prt1
(), indicating that prt1
does not result in significant anatomical defects.
To further examine changes in the function of the MBs and other tissues expressing PRT, we investigated prt1 mutant behavior. We first outcrossed the prt1 flies for 6 generations into the wild-type strain CS. Outcrossing removed a closely linked mutation that reduced viability and fertility (not shown), and all behavioral experiments were performed using the outcrossed lines. The outcrossed prt1 flies were viable, fertile, and showed no obvious external morphological defects.
The relatively high level of PRT expression in the MBs as compared to other structures suggests it may play a role in olfactory classical conditioning, known to require the MBs (Davis, 2011
). We used a modified T-maze to test olfactory classical conditioning as previously described (de Belle and Heisenberg, 1994
). As controls for these experiments, we first established that prt1
flies had normal avoidance of both electric shock and the odors used to test learning (). We next tested olfactory learning. We found that prt1
mutants have a learning defect, evidenced by a decreased performance index immediately following training (). The performance indices for prt1
were also reduced at 30 minutes and 6 hours after training (). The difference between CS and prt1
was consistent over short-term (30 minutes) and middle-term (6 hours) phases of memory, suggesting normal memory decay in prt1
Fig 5 prt1 mutants show a learning deficit. A) prt1 mutants show normal avoidance of electric shock (90V) and the odor concentrations used for olfactory learning assays, B) octanol (10−4) and C) benzaldhyde (2 × 10−4). Bars represent (more ...)
We next tested prt1
behavior using several other well-established assays. Performance indices for negative geotaxis and fast phototaxis were equivalent to those of wild-type flies (Fig S3A, B
) indicating that gross locomotor activity and the response to both mechanical stimuli and visible light are intact in prt1
. We did find a modest impairment in courtship behavior. While prt1
males performed all of the necessary courting rituals, they spent less time courting ().
Fig 6 prt1 reproductive behavior. A) prt1 mutant males spent 26% less time courting (*, p = 0.0182). B) The top two panels show normal copulatory positions seen in wild-type flies while the bottom two panels show improper positioning exemplary of prt1. C) (more ...)
In the course of performing courtship assays, copulation was observed. Normally, males mount the female from behind, curling their abdomen upwards to allow coupling. Once coupled, the male maintains a forward-facing orientation, in the same direction as the female (, top pictures, Movie S1
). Copulation typically lasts ~20 minutes (Jagadeeshan and Singh, 2006
). For the first several minutes of this period, wild-type pairs may move forward or adjust positions, but for the remainder of this time the flies remain essentially motionless.
Copulation in prt1
mutants differs dramatically. Similar to wild-type flies, prt1
males mount the female and curl their abdomen to begin copulation. However, after coupling, the prt1
male continuously struggles to maintain his orientation and can be seen in a variety of different positions relative to the female (, bottom pictures, Movie S2
). We quantified the amount of time that prt1
males spent in a position distinct from that usually seen in wild-type flies as a percentage of the total copulation duration (). While CS flies primarily remain centered on the dorsal abdomen of the female, prt1
flies spent nearly half of copulation severely misaligned. Although the genitalia of the male and female remain in contact, the male can be positioned perpendicular to the normal axis or rotated nearly 180 degrees from horizontal. Moreover, during copulation, prt1
mating pairs move about the observation chamber, with the female dragging the male behind. In cross-genotype mating experiments, prt1
males mated to CS females showed defective copulation whereas CS males mated to prt1
females did not (). Thus, the prt1
males were primarily, if not exclusively, responsible for the defect in copulation.
To determine whether the change in the males’ position was due to a defect in genital morphology, we examined both the prt1
male and female genitalia using scanning electron microscopy. We found that the external genitalia of prt1
males and females were indistinguishable from wild-type (Fig S3D, E
). We also examined the prt1
males’ sex combs, specialized foreleg structures used to grasp the female during copulation (Ahuja and Singh, 2008
; Ng and Kopp, 2008
). The morphology of prt1
sex combs was intact in scanning electron micrographs (Fig S3F
) without obvious gaps between bristles, although the number of bristles in the prt1
sex combs was slightly lower than controls (Fig S3G
) (Ahuja and Singh, 2008
; Tokunaga, 1961
We employed deficiency analysis to help determine the severity of the prt1 sexual phenotype (). Two deficiency lines that uncover the prt locus were used and the extent of their chromosomal deletions is represented in . The copulatory phenotype seen in the prt1 homozygote was replicated in both the prt1/Df(3R)mbc-30 and prt1/Df(3R)Exel6195 transheterozygotes (). It is possible that the prt1 copulation phenotype cannot get measurably worse, and further deficiency analysis using other aspects of the prt1 phenotype will be necessary to more precisely assess the severity of the prt1 allele. However, in light of our current data showing that the severity of the phenotype seen in prt1/Df was equivalent to that of the prt1 homozygotes, and our molecular analysis showing undetectable levels of PRT protein, we conclude that the prt1 allele is either a severe hypomorph or a null mutation.
mutants were surprisingly fecund given their contorted mating positions. They were able to produce approximately half the number of offspring as CS flies ( and S3C
). Either insemination occurred during periods when the male was correctly oriented, or wild-type position is not required for insemination. The total duration of copulation was also decreased in prt1
(). It is perhaps surprising that the decrease in copulation time and fecundity were not more severe given the tremendous struggling observed in mating pairs.
Fig 7 Genetic rescue of copulation defect (see legends on right for genotypes). A) The combination of the Da-Gal4 driver with a UAS-prt transgene rescued the copulation phenotype (columns 6 & 7, p < 0.01 and 0.05, respectively); driver or UAS-prt (more ...)
Rescue of prt1 copulation phenotype
To confirm that the copulation defects we observed were due to prt1 rather than another spurious mutation, we performed genetic rescue experiments with UAS-prt transgenes. The circuitry involved in copulation is not known. Therefore, to maximize our chances of expressing prt1 in the relevant tissue we used the broadly distributed driver daughterless-Gal4 (Da-Gal4). As controls, we tested the Da-Gal4 driver alone and the UAS-prt transgenes alone, and none rescued the copulation deficit (). In contrast, when Da-Gal4 was used in combination with a UAS-prt transgene on the 3rd chromosome we saw rescue of the copulation phenotype (). We replicated these results using a UAS-prt transgene on the 2nd chromosome ().
Similarly, we found that the decrease in copulation duration was rescued using Da-Gal4 and either of these UAS-prt transgenes, but not by Da-Gal4 or UAS-prt alone (). Taken together, these data confirm that prt plays a critical role in an important but poorly described aspect of D. melanogaster sexual behavior.
While the MBs have been previously linked to courtship behavior (O’Dell et al., 1995
; Sakai and Kitamoto, 2006
), we wanted to explore whether the MBs could also be involved in copulatory behavior. To this end we performed genetic rescue experiments using OK107-Gal4
, a driver commonly used for expression in the MBs (e.g. Connolly et al., 1996
combined with a UAS-prt
transgene on either the 2nd
chromosome resulted in rescue of the copulation phenotype while the controls did not ().
We found that the decrease in copulation duration was also rescued using OK107-Gal4 and either of these UAS-prt transgenes (); the 2nd chromosome UAS-prt transgene alone also appeared to rescue copulation duration, presumably due to leaky expression of PRT. Although we cannot rule out the possibility that other cells expressing OK107-Gal4 are responsible for these effects, these data suggest that the MBs play a critical role in copulatory behavior.
At present we do not know the PRT substrate and suggest that it may be an unknown neurotransmitter. The absence of candidates prevents the validation of PRT’s proposed transport activity using most standard biochemical assays. We therefore employed a genetic approach to test the hypothesis that PRT might function as a vesicular transporter in vivo
. For mammalian VMATs and VAChT, a wealth of data have identified specific residues required for either transport activity or substrate recognition (See (Parsons, 2000
) for review and Supplementary Information
). A number of these important residues are conserved in DVMAT, DVAChT and PRT. These include aspartates (D) in the first and tenth transmembrane domains (TM1 and TM10) of DVMAT, DVAChT and PRT (Fig S1
). For both rat VMAT2 and VAChT, mutation of the aspartate in TM10 abolishes transport activity, while mutation of the aspartate in TM1 of VMAT2, but not VAChT, inhibits transport (Kim et al., 1999
; Merickel et al., 1997
; Merickel et al., 1995
We used site-directed mutagenesis to convert these homologous sites to alanine (D59A or D483A) and expressed each in vivo as a UAS transgene. Both UAS-prtD59A and UAS-prtD483A showed robust expression on Western blots (not shown); however, neither rescued the prt1 phenotype (). Thus, residues conserved in VMAT2, DVMAT and PRT, and required for VMAT2 activity, are also required for PRT function. Furthermore, the aspartate in TM1 required for both PRT and VMAT2 transport activity is not required for VAChT. These data support the idea that PRT functions as a vesicular transporter more similar to VMATs than VAChT.
Fig 8 Point mutants of conserved aspartates fail to rescue copulation defect. Transgenic expression with a ubiquitous driver (Da-Gal4) rescued the prt1 copulation phenotype with wild-type PRT (8th column, p < 0.001) but not with the D59A (9th column) (more ...)
To obtain additional insight into the structural requirements for PRT activity, we turned our attention to another, more ambiguous site. Mutation of a conserved aspartate in TM11 of either VMAT2 or VAChT blocks transport activity (Kim et al., 1999
; Merickel et al., 1997
); however, PRT contains an uncharged glutamine in TM11 (Q521, star, Fig S1B
). The presence of a nonconserved glutamine at this site in PRT suggested that it might not be essential for its activity. Indeed, in contrast to PRT mutants D59A and D483A, the Q521A mutant partially rescued the prt1
mutant phenotype ().
Together our data suggest that PRT likely functions as a vesicular transporter similar to the VMATs. However, PRT did not display appreciable affinity for known substrates such as dopamine or serotonin in in vitro transport assays with DVMAT as a positive control (not shown). Moreover, PRT localizes to cells that do not express any of the enzymes required for the synthesis of known monoamines (see below). These data along with the differential structural requirements for activity support the possibility that PRT recognizes a substrate distinct from either VMATs or VAChT.