XPORT is Required for TRP and Rhodopsin Expression
By screening the Zuker collection of EMS-mutagenized Drosophila
(Koundakjian et al., 2004
) we identified a novel mutant, xport1
, which displayed an abnormal electroretinogram (ERG) compared to wild-type (). The xport1
mutant had a transient response during prolonged light stimulation, which was indistinguishable in amplitude and time-course from the transient receptor potential (trp
) phenotype observed in the trp343
null mutant. Photoreceptor cells that lack TRP protein are unable to sustain a steady-state current, via TRPL, due to reduced Ca2+
influx. More specifically, low levels of Ca2+
result in a failure of Ca2+
and PKC dependent inhibition of PLC. Consequently, uncontrolled PLC activity depletes its substrate (microvillar PIP2
) leading to premature closure of TRPL channels (Gu et al., 2005
; Hardie et al., 2001
). Consistent with the transient light response, xport1
displayed a severe reduction in TRP protein levels compared to wild-type (). Interestingly, Rh1 protein levels were also severely reduced in the xport1
mutant (). The mutation in xport1
is recessive, as the heterozygotes were normal for both TRP and Rh1 protein (). Therefore, xport
is required for the proper expression of both TRP and Rh1.
Characterization of the xport1 Mutant in Drosophila
To identify the xport
locus, we first narrowed the cytogenetic location by deficiency mapping to 92B3-92C1 on the third chromosome, corresponding to 26 loci spanning 145 kb of DNA ( and S3A
). We identified a C to T substitution at nucleotide position 145 within the coding region of CG4468, causing a premature stop codon at glutamine49 (Figure S3A
To confirm that CG4468 was the xport
locus, we restored wild-type function by introducing a wild-type copy of CG4468 into the genome of the xport1
mutant. TRP and Rh1 protein expression were restored to wild-type levels in the rescue line (). These data confirm that CG4468 is, indeed, the xport
locus. In addition, we found that eye-specific expression of three independent CG4468 RNAi transgenes leads to a severe reduction in TRP by Western blot analysis (Figure S1
We analyzed electrical responses to light in the xport1 mutants by whole cell voltage-clamp recordings of dissociated ommatidia. In wild-type photoreceptors, brief flashes elicited rapid macroscopic inward currents mediated by TRP and TRPL channels (). In xport1 mutants, response amplitudes were ~20-fold reduced (), consistent with a severe reduction in TRP and Rh1. Sensitivity was restored to wild-type levels in xport1 flies expressing the wild-type xport cDNA rescue construct ().
If TRP channel expression was completely eliminated in the xport1 mutants, then we would predict that the residual response in xport1 would be eliminated in a trpl302;xport1 double mutant. Instead, we found a residual response in the trpl302;xport1 double mutant (), but sensitivity was now reduced ~500-fold with respect to wild-type and 25-fold with respect to xport1 ().
To determine whether the residual response in trpl302
was mediated by TRP channels, we measured the reversal potential (Erev
) of the light response. Erev
in wild-type and rescue flies represented the mixed contribution of both TRP and TRPL channels and was approximately 11 mV (Figure S2A
). As predicted, Erev
mutants was negatively shifted compared to wild-type and indistinguishable from that measured in trp343
mutants. However, Erev
for the trpl302
double mutant was similar to that measured in trpl302
mutants, indicating that the residual response was mediated by TRP channels.
TRP and TRPL channels can also be distinguished by their sensitivity to La3+, which completely blocks TRP channels, while leaving TRPL channels unaffected. In wild-type and rescue flies, La3+ (50 μM) blocked approximately 80% of the light-induced current, leaving a residual response mediated by TRPL channels (, wild-type data not shown). In xport1 mutants, La3+ had no detectable blocking action, indicating that most of the response was mediated by TRPL channels (). In the trpl302;xport1 double mutant, the response was completely blocked by perfusion with La3+ (), again confirming that TRP channels mediated this residual response.
Because sensitivity to light in the trp343
mutant was much greater than in the xport1
mutant (), the near complete loss of TRP channels in xport1
can only partially account for the 20-fold observed reduction in sensitivity (5% of wild-type sensitivity). It seemed likely that the additional loss of sensitivity would be accounted for by the reduction in Rh1 content. To test this, we measured effective quantum efficiency (Q.E.), which should be proportional to Rh1 concentration, by counting quantum bumps in response to dim flashes such that ~50% of the flashes contained no effective photons and induced no response (Figure S2B and S2C
, failures). In xport1
mutants, Q.E. was reduced on average by approximately 8-fold compared to wild-type (Figure S2D
). However, bump amplitude (3.6 pA), although smaller than in wild-type flies, was indistinguishable from that measured in trp343
mutants (Figures S2E and S2F
). This indicates that the loss of sensitivity in xport1
mutants can be fully accounted for by a drastic reduction in TRP channels combined with an ~8-fold reduction in visual pigment concentration. Both bump amplitude and Q.E. were fully rescued by expression of the wild type xport
cDNA rescue construct in the xport1
mutant (Figures S2D–F
Taken together, these data indicate an ~60-fold reduction in TRP channel activity (1.7% of wild-type levels) and imply an ~8-fold reduction in Rh1 content (12% of wild-type levels) in the xport1 mutant. These estimates are consistent with the levels of TRP and Rh1 detected by immunoblotting (). While XPORT is required for both TRP and Rh1, TRP and Rh1 expression are not dependent upon one another. TRP protein levels were wild-type in the ninaEI17 (Rh1) null mutant () and Rh1 protein levels were wild-type in the trp343 null mutant (). Therefore, XPORT provides a novel biosynthetic link between TRP and its GPCR, Rh1.
XPORT is a Novel Eye-Specific Secretory Pathway Protein
locus is comprised of 2 exons and 1 intron (Figure S3A
). The 953 base pair transcript encodes a 116 amino acid protein ( and S3A
) that was detected as a 14 kD band in wild-type flies (). Consistent with the presence of a premature stop codon, XPORT protein was reduced in xport1
heterozygotes and completely absent in xport1
homozygotes as well as in flies harboring the xport1
allele in trans
to Df(3R)BSC636 (). XPORT expression was completely restored in the rescue line (). Although TRP and Rh1 require XPORT protein for their expression, XPORT is expressed normally in both the trp
(Rh1) null mutants ().
Drosophila XPORT is an Eye-Specific, Resident Secretory Pathway Membrane Protein
The XPORT protein is predicted to be a Type II transmembrane protein with a single C-terminal transmembrane domain and a cytosolic N-terminal globular domain (, S3A and S3B
). Consistent with this prediction, following centrifugation of a total cell homogenate from wild-type heads, XPORT was absent from the soluble fraction and exclusively present in the membrane pellet (). XPORT was solubilized by suspension of the membrane pellet in SDS. Following subsequent centrifugation, XPORT was detected entirely in the supernatant and was absent from the pellet, confirming that XPORT is an integral membrane protein.
XPORT is highly conserved among 12 Drosophila
species as well as among other Diptera, including two mosquito genera, Anopheles
and Culex. Drosophila
XPORT is also conserved in the Jerdon’s jumping ant and honeybee (Hymenoptera) as well as in the red flour beetle (Coleoptera) (Figure S3C
). While XPORT is highly conserved among insect species, there are currently no vertebrate counterparts in the NCBI database. Although XPORT lacks an obvious vertebrate homolog, it has a small recognizable motif that displays 46% amino acid identity and 62% similarity with the KH domain of a DnaJ-like protein from Chlamydomonas reinhardtii
(). DnaJ proteins, also known as Hsp40s, are members of a large family of highly diverse co-chaperones that bind Hsp70 via a 70 amino acid J-domain, and assist in the folding and quality control of a vast array of client proteins (Kampinga and Craig, 2010
). While DnaJ and DnaJ-like chaperones are defined by the presence of the J-domain, XPORT lacks this domain.
The KH domain is a nucleic acid recognition motif that binds single stranded RNA or DNA with low affinity. KH domains contain a “GXXG” loop that is key to nucleotide binding and this motif is also present in XPORT (). KH-domain proteins perform a wide variety of cellular functions and have been implicated in regulation of transcription and translation (Valverde et al., 2008
). To distinguish between these two possibilities, we performed Northern blot analysis. The trp
(Rh1) transcript levels were indistinguishable from wild-type in the xport1
mutant (), indicating that XPORT functions post-transcriptionally for TRP and Rh1.
Certain Hsp70/DnaJ chaperone complexes, as well as calnexin, have been shown to specifically associate with ribosomes to ensure the proper folding of newly synthesized polypeptide chains as they exit the ribosome during translation (Craig et al., 2003
; Delom and Chevet, 2006
; Hundley et al., 2005
; Jaiswal et al., 2011
). Members of this ribosome-tethered chaperone network are conserved from yeast through humans and are thought to serve as the first line of defense against protein misfolding. Consistent with a role for XPORT in the early stages of TRP and Rh1 biosynthesis, XPORT protein was detected in the peri-nuclear ER in all 8 photoreceptor cells (, R8 cell not shown). XPORT’s labeling pattern was similar to that of the known chaperones, calnexin and NinaA (Figure S4
). Therefore, XPORT may exhibit co-translational chaperone function at the early stages of TRP and Rh1 biosynthesis at the ribosome. XPORT has ideal predicted topology for positioning its KH and “GXXG” motifs on the cytosolic face of the ER, where ribosomes reside.
Just like TRP and Rh1, XPORT is eye-specific. By Northern blot analysis, the xport, ninaE (Rh1) and trp transcripts were detected in wild-type heads but were absent in bodies and in heads from flies lacking eyes (eya1) (). Furthermore, by immunocytochemistry, XPORT was detected exclusively in the photoreceptor cell bodies, but was not detected in the lamina, medulla, lobula, lobula plate or brain, compared to the synaptic protein, synapsin ().
XPORT not only localized to the perinuclear ER, but was also detected more extensively in the secretory pathway () unlike the inositol 1,4,5-trisphosphate receptor (IP3
R), which was highly restricted to the perinuclear ER (Figure S4
). This makes XPORT ideally situated to function as a chaperone in the early as well as in the later stages of TRP and Rh1 biosynthesis.
XPORT is Critical for Trafficking of TRP and Rh1
In wild-type flies, the TRP channel specifically resides within the rhabdomere for its function in phototransduction (, top). In contrast, TRP protein was severely mislocalized in all eight photoreceptor cells in the xport1 mutant. It was detected throughout the secretory pathway with very little labeling in the rhabdomeres (, bottom). These data are consistent with the electrophysiological analyses showing that there is very little functional TRP (1.7%) present in the xport1 mutant (). Therefore, successful transport of TRP to the rhabdomeres of all eight photoreceptors requires XPORT.
XPORT is Critical for Trafficking of TRP and Rh1
We investigated the kinetics of Rh1 maturation in the xport1 mutant using transgenic flies harboring an epitope-tagged Rh1 under the control of a heat-inducible promoter (hsp70). In wild-type flies, Rh1 was initially synthesized as immature high-molecular weight (MW) glycosylated forms that were processed down to the mature form by 14 hours. By 24 hours, the vast majority of Rh1 was detected in the mature low-MW form (, top). In the xport1 mutant, Rh1 was also initially detected as immature high-MW forms that were partially processed to the mature form. In contrast to wild-type flies, in the xport1 mutant, Rh1 disappeared rapidly between 16–24 hours, indicating that Rh1 was degraded (, bottom). Therefore, XPORT is required for the proper maturation and stability of newly synthesized Rh1.
In wild-type flies, Rh1 was precisely localized to the rhabdomeres for its role in phototransduction (, top). In contrast, in the xport1
mutant, Rh1 was abnormally retained in the ER and secretory pathway with only some Rh1 present in the rhabdomeres (, bottom). This is consistent with the electrophysiological analyses demonstrating that there is a small amount of functional Rh1 (~12%) present in the xport1
mutant (Figure S2D
). Therefore, like TRP, successful transport of Rh1 through the secretory pathway and efficient delivery of Rh1 to the rhabdomere also requires XPORT.
XPORT Promotes TRP and Rh1 Transport to the Cell Surface in S2 Cells
Consistent with XPORT residing in the secretory pathway of photoreceptor cells (), XPORT was detected in the perinuclear ER and secretory pathway of Drosophila
S2 cells transfected with xport
(). Likewise, in cells singly transfected with either trp
(Rh1), the proteins were detected in the secretory pathway in a perinuclear and/or punctate fashion (, −X). However, when trp
were co-expressed with xport
, TRP and Rh1 proteins were now detected at the cell surface (, +X). These results were quantified by analyzing over a 100 cells for each condition (Table S1
) and cell surface labeling of TRP was confirmed by co-localization with a plasma membrane marker, wheat germ agglutinin (WGA) (, bottom row). These data demonstrate that XPORT promotes the transport of TRP and Rh1 to the cell surface in S2 cells, consistent with a role for XPORT as a chaperone for TRP and Rh1.
XPORT is Required by TRP in the Ocelli
In addition to the compound eye, Drosophila have two additional light-sensing organs: the adult ocelli and the larval Bolwig’s organ. Phototransduction in ocelli likely occurs via a signaling pathway very similar to the compound eye, utilizing the ocellar-specific opsin, Rh2, the G-protein (DGq), norpA-encoded PLC, TRP channels, arrestin1 (Arr1) and arrestin2 (Arr2). To investigate the potential role of XPORT in Drosophila ocelli, we examined the expression of XPORT and TRP in both wild-type and xport1 mutants. shows that XPORT and TRP were both expressed in wild-type ocelli. TRP protein was reduced in the xport1 mutant, while Arr1 was normal. These results suggest that XPORT is specifically required for TRP in the ocelli.
XPORT is Required by TRP in the Ocelli
XPORT is Uniquely Required by TRP and Rh1
Given that mutations in xport
severely disrupt the biosynthesis of TRP and Rh1, we examined the expression of additional photoreceptor cell proteins in the xport1
mutant. shows that other proteins critical for phototransduction and rhabdomere stability, including the G-protein alpha subunit (Gqα), PLCβ (norpA
), the TRPL channel, Arr1, Arr2, chaoptin and NinaA were all expressed at normal levels. We also investigated expression levels of proteins involved in calcium regulation and synaptic transmission. We determined that calnexin, the Na+
exchanger (CalX), the sarco/endoplasmic reticulum Ca2+
-ATPase (SERCA), as well as the synaptic proteins, synapsin and syntaxin, were all expressed normally in the xport1
mutant (Figure S5
). Finally, we determined that the minor opsins, Rh3, Rh4, and Rh5, were properly localized to their rhabdomeres in the R7 and R8 cells in the xport1
mutant (). These results demonstrate that XPORT is specifically required by TRP and Rh1 and is not required for expression of other photoreceptor cell proteins.
XPORT is Specifically Required by TRP and Rh1
XPORT in Bolwig’s Organ
In larvae, Bolwig’s organ is comprised of two bilateral groups of 12 photoreceptor cells that each express one of two R8-cell-type opsins, Rh5 and Rh6 (Malpel et al., 2002
). Phototransduction in Bolwig’s organ is thought to involve similar components as the adult visual cascades, including norpA
-encoded PLC (Busto et al., 1999
; Malpel et al., 2002
). To investigate the potential role of XPORT in Bolwig’s organ, we examined the expression of XPORT, TRP, TRPL, Rh1 and Rh5 in both wild-type and xport1
mutants. shows that XPORT was expressed in Bolwig’s organ. Interestingly, TRP was not detected in Bolwig’s organ, indicating that it is not used for larval phototransduction. In contrast, TRPL was detected in Bolwigs organ (), suggesting that it may function as the primary channel in the larvae. Consistent with previous reports, Rh1 was not detected in Bolwig’s organ () whereas Rh5 was detected () (Busto et al., 1999
; Kaneko et al., 1997; Malpel et al., 2002
; Sawin-McCormack et al., 1995). Interestingly, both Rh5 and TRPL are expressed normally in the xport
mutant indicating that, as in the adult, XPORT is not required for Rh5 or TRPL expression in Bolwig’s organ (). Therefore, although XPORT is expressed in Bolwig’s organ, it does not appear to be required for visual processes in the larvae.
Mutations in xport Lead to Retinal Degeneration
TRP and Rh1 are key components of phototransduction and mutations that alter the function of either of these proteins lead to severe retinal pathology and retinal degeneration. Consistent with the finding that XPORT is essential for TRP and Rh1 protein expression, xport1
mutants displayed an early-onset retinal degeneration. In 1-day-old xport1
mutants grown on a 12:12 light-dark cycle, the rhabdomeres were diminished in size () compared to wild-type flies (). In agreement with this reduction in rhabdomere size, the membrane surface area in the xport1
mutant, as measured by whole-cell capacitance, was almost halved indicating a significant reduction in microvillar surface area (Figure S6
). Furthermore, the photoreceptor cells displayed extensive ER membrane accumulations and dilated Golgi (), consistent with aggregation of TRP and Rh1 in the secretory pathway. At 2 weeks, the xport1
mutant photoreceptor cells were severely degenerated. The rhabdomeres of all eight photoreceptors were vastly reduced and many were completely missing (). To assess whether the retinal degeneration was enhanced by light stimulation of phototransduction, we reared the xport1
mutant for 2 weeks in constant darkness. Dark-reared flies still showed ER membrane accumulations and dilated Golgi (), but now exhibited nearly normal rhabdomere morphology (). Therefore activation of phototransduction by light enhances the retinal degeneration in xport1
mutants. The retinal pathology was fully rescued by the expression of wild-type XPORT in the xport1
The xport1 Mutant Undergoes a Severe Retinal Degeneration
Mechanisms of Retinal Degeneration in xport Mutants
Mechanisms of Retinal Degeneration in xport1
The molecular mechanisms underlying retinal degeneration are diverse and have been well studied in the Drosophila
visual system. Two well-characterized mechanisms involve either 1) accumulation of Rh1 in the secretory pathway due to defective folding/trafficking or 2) unregulated Ca2+
levels due to defective phototransduction (Colley, 2010
; Rosenbaum et al., 2006
; Wang and Montell, 2007
). The finding that light significantly enhanced the retinal degeneration in the xport1
mutant is contrasted to other known mutants defective in Rh1 maturation, for which the retinal degeneration is light-independent (Colley et al., 1991
; Colley et al., 1995
; Kurada and O’Tousa, 1995
; Webel et al., 2000
). However, the xport1
mutant is unique in that it displays defects in both protein trafficking and TRP channel function. Loss of TRP channel expression can lead to a retinal degeneration unrelated to protein trafficking (Wang and Montell, 2007
). In this instance, the retinal degeneration is light-dependent and is triggered by defects in calcium influx through the light-sensitive TRP channels. Given that the retinal degeneration in xport1
is light-enhanced, we investigated the relative contribution of protein trafficking defects versus the lack of TRP channel function to the overall retinal degeneration. To accomplish this, we took advantage of two retinal degeneration mutants, ninaE318
mutant exhibited a severe reduction in Rh1 and displayed defects in Rh1 transport through the secretory pathway (Figures S7A and S7C
). However, TRP protein levels were wild-type in ninaE318
). Therefore, ninaE318
exhibits a retinal degeneration that is due solely to defects in protein trafficking. In contrast, the trp343
mutant was null for TRP protein (Figure S7B
) but Rh1 levels were wild-type and Rh1 specifically localized to the rhabdomeres (Figures S7A and S7C
). Therefore, trp343
mutants undergo a retinal degeneration that is independent of protein trafficking defects. The xport1
mutant displays a combination of protein accumulation in the secretory pathway (like ninaE318
) and a severe reduction in TRP protein (like trp343
Like the xport1 mutant, the ninaE318 mutant displayed considerable ER membrane accumulations and dilated Golgi at 1-day-old (). In contrast, the trp343 null mutant showed no sign of secretory pathway membrane accumulations (). The secretory pathway defects were light-independent, as the membrane accumulations were still present in 1-day-old xport1 and ninaE318 mutants that had been reared in constant darkness ().
At two weeks, trp343 displayed a severe retinal degeneration () that was comparable to that observed in the xport1 mutant (). In contrast, the ninaE318 mutant exhibited milder pathology at two weeks (). As was shown for xport1 (), the retinal degeneration was significantly attenuated in trp343 mutants reared in constant darkness for 2 weeks (). Taken together, these results indicate that the retinal degeneration in the xport1 mutant is due to the combined detrimental effects of protein aggregation in the secretory pathway and misregulation of Ca2+ levels in the absence of TRP. More specifically, the light-independent membrane accumulations in xport1 are likely the result of defects in TRP and Rh1 trafficking, while the light-enhanced retinal degeneration is likely due to the near complete loss of TRP channels in the rhabdomere.
XPORT Participates in a Coordinated Pathway for Rh1 Biosynthesis
Given that two other chaperones, namely NinaA and calnexin, are also essential for Rh1 maturation and trafficking (Colley et al., 1991
; Rosenbaum et al., 2006
), XPORT may play a critical role in a conserved protein-processing pathway with these chaperones. To investigate the temporal sequence of calnexin, NinaA and XPORT chaperone activity for Rh1, we conducted genetic epistatic analyses by generating double mutant flies. In all three single mutants, Rh1 was severely reduced (). However, in the ninaAP269
mutant, a substantial amount of Rh1 was detected in the immature high MW form. The ninaAP269
double mutant displayed severely reduced levels of Rh1 in the mature low molecular weight form, a phenotype characteristic of the calnexin1
mutation alone (). These results demonstrate that calnexin functions upstream of NinaA in Rh1 biosynthesis. Consistent with this finding, calnexin was entirely digested by both endoglycosidase H (Endo H) and peptide N-glycosidase F (PNGase F) (Figure S4
). Endo H selectively cleaves high mannosyl residues on glycoproteins that have not yet been processed in the Golgi and thus Endo H sensitivity implicates glycoproteins as ER residents. Therefore, calnexin is restricted to the ER. NinaA, however, was only partially digested by Endo H and fully digested by PNGase F (Figure S4
). These results support previous findings that while some NinaA protein resides in the ER, NinaA is also retained by Rh1 as it travels through the distal compartments of the secretory pathway. Accordingly, NinaA is not entirely restricted to the ER (Colley et al., 1991
). XPORT was insensitive to both enzymes, indicating that it is not glycosylated (Figure S4
). Hence, for XPORT, Endo H sensitivity was not informative. To evaluate the epistatic relationship between xport
, we generated a ninaAP269; xport1
double mutant and again, examined Rh1 expression. The ninaAP269;xport1
double mutant displayed severely reduced levels of Rh1 with most of the Rh1 present in the immature high molecular weight form (). This phenotype is characteristic of the ninaAP269
mutation alone and suggests that NinaA functions upstream of XPORT in Rh1 biosynthesis. Taken together, these data suggest that calnexin, NinaA, and XPORT function in a coordinated pathway ensuring the proper folding, quality control and maturation of Rh1 during biosynthesis. We propose that calnexin functions upstream of NinaA which, in turn, functions upstream of XPORT during Rh1 biosynthesis (). Interestingly, neither calnexin nor NinaA are required for the biosynthesis of the TRP channel, as TRP protein is expressed normally in the cnx
mutants (Figure S8
XPORT Functions as Part of a Coordinated Pathway for Rh1 Biosynthesis
XPORT Associates with TRP and Rh1
Consistent with XPORT’s function as a chaperone for TRP and Rh1, XPORT physically associates with both TRP and Rh1. Rh1 was isolated in a stable complex with XPORT and this association was specific, as Rh1 did not bind to or elute from the XPORT antibody column in the absence of XPORT protein (). TRP was also isolated in a stable complex with XPORT (). Further support for the specificity of these interactions was obtained by investigating several other photoreceptor cell proteins. Like all neurons, photoreceptors are polarized and, therefore, protein trafficking occurs in two directions: to the rhabdomeres and to the synapse. We investigated whether XPORT was required for the transport of the synaptic vesicle proteins synapsin and syntaxin. Neither protein interacted with XPORT, as both were found entirely in the unbound fraction in both wild-type and xport1
mutant tissue (). We also assessed the interaction between XPORT and two other chaperones involved in Rh1 biosynthesis, calnexin and NinaA. Neither calnexin nor NinaA interacted with XPORT, as both proteins were detected entirely in the unbound fraction in both wild-type and xport1
mutant tissues (). That XPORT does not associate with synapsin, syntaxin, NinaA or calnexin is consistent with the finding that these proteins do not require XPORT for their biosynthesis, as they were all expressed at wild-type levels in the xport1
mutant ( and S5
). Furthermore, these results support the notion that calnexin, NinaA and XPORT sequentially interact with Rh1 during its biosynthesis in a step-wise fashion, as opposed to functioning as components of a macromolecular chaperone complex.
Although XPORT was not detected in a complex with calnexin and NinaA, it is possible that XPORT functions as part of an Hsp complex. Given that XPORT displays amino acid identity with a DnaJ-like protein, we first investigated whether the Hsp70 protein was present in a complex with XPORT, TRP and Rh1. Indeed, Hsp70 was detected in the bound fraction of wild-type tissue, but it was also detected in the bound fraction of the xport1 mutant tissue (). Due to the binding of Hsp70 in the absence of XPORT, we were unable to determine whether Hsp70 was truly part of the XPORT complex. To further investigate the potential interaction between XPORT and the Hsp family, we examined whether Hsp90 or Hsp27 were present in the complex. Hsp90 and Hsp27 represent two other highly conserved chaperones that function, together with Hsp70, to promote protein folding and prevent protein aggregation. Indeed, both Hsp90 and Hsp27 were specifically isolated in a stable complex with XPORT, with no binding detected in the xport mutant (). These results suggest that XPORT may serve as a chaperone in conjunction with the Hsp family.