3. Results 51
3.1.1. Export requirements for PNEPs with a SP and a TMD
3.1.1.1. PF08_0004
PF08_0004 deletion constructs PF08_0004 contains a SP and a TMD and as a GFP fusion protein localizes to the Maurer’s clefts with additional staining of the parasite periphery, potentially caused by the GFP tag that could partially interfere with efficient trafficking (Heiber et al., 2013). To narrow down the part of the protein necessary for protein export it was divided into 4 parts. The contribution of these parts in export was assessed by individually deleting or replacing them in an episomally expressed (under the crt-promoter) modified PF08_0004 construct fused to GFP. Parts 1-3 covered the entire region N-terminal of the TMD and were individually deleted. Part 4 covered the entire C-terminus and was replaced with a myc epitope tag (see figure 3.1, A). Each part consisted of 16-25 amino acids (aa), the SP comprising 25 aa and the TMD 23 aa. The 7 aa just N-terminal of the TMD were not modified as they contain positively charged lysines, shown to be important for correct membrane topology of TMDs (von Heijne, 1992). Figure 3.1 B shows representative fluorescence images of the cell lines expressing the different deletion constructs. Deletion of part 1 (PF08_0004∆1-GFP), part 3 (PF08_0004∆3-GFP) and replacement of part 4 with a myc-tag (PF08_0004∆4myc-(PF08_0004∆3-GFP) had the most deleterious effect on protein export, with the fusion protein almost exclusively localizing to the parasite periphery. PF08_0004∆2-GFP and PF08_0004∆3-GFP both showed a
weak punctate fluorescence signal in the host cell with a prominent signal in the parasite periphery, suggesting low levels of export. The level of fluorescence in the host cell was higher for PF08_0004∆2-GFP compared to PF08_0004∆3-GFP and this was the best exported version of the modified PF08_0004 proteins, although export was still low.
To exclude effects on protein export induced by the complete deletion of protein parts, the sequence of part 1 and 3, whose deletions resulted in the strongest export decrease, was replaced with a randomly scrambled version of this region. The resulting constructs PF08_0004d1scr-GFP and PF08_0004d3scr-GFP have a similar phenotype as their coun-terparts with the corresponding deletions. In summary these results show that all the domains are necessary for the efficient export of PF08_0004.
In other known PNEPs the N-terminus is necessary or sufficient for protein export (Külzer et al., 2012; Grüring et al., 2012; Heiber et al., 2013). To test whether this was also the case for PF08_0004, a minimal construct was generated, containing the SP, part 1 and a small fraction of part 2 to assess a possible capacity to drive export independent of the TMD. This construct localized to the PV and in structures resembling the TVN, demonstrating that the N-terminus is not sufficient to mediate export.
GFP
SP 1 3 TM 4
PF08_0004∆2-GFP
GFP
SP 1 2 3 TM 4
GFP
SP 2 3 TM 4
PF08_0004∆1-GFP
PF08_0004∆3-GFP
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SP 1 2 TM 4
PF08_0004∆4myc-GFP
GFP
SP 1 2 3 TM myc
GFP
SP 2 3 TM 4
PF08_0004d1scr-GFP
GFP
SP 1 2 TM 4
PF08_0004d3scr-GFP
PF08_0004d1+2trunc-GFP
SP 1 2 GFP
PF08_0004-GFP
GFP DIC/DAPI DIC/GFP/DAPI
A B
3 1
Figure 3.1.: Deletion constructs of PF08_0004. A Schematic showing the subdivision of PF08_0004 into 4 parts (not to scale). (SP) signal peptide, (TM) transmembrane region.
B Live cell images of 3d7 parasites expressing GFP-fusion constructs under the crt-promoter.
Nuclei were stained with DAPI. Construct schematics and names are shown above the respective images. Scrambled parts are represented by a striped pattern. (scr) scrambled (DIC) differential interference contrast. Scale bar: 5µm.
The PF08_0004 SP and TMD PF08_0004 seemed to be strongly affected in export by all deletions tested and therefore behaved differently to the previously analyzed PNEPs.
In other PNEPs the type of TMD affects export with only PNEP TMDs permitting ex-port (Haase et al., 2009; Grüring et al., 2012; Saridaki et al., 2009). To test if PF08_0004 resembled the other PNEPs in this respect, it was tested whether a PNEP TMD could replace its TMD. For this the TMD of the PNEP REX2 was chosen and inserted into PF08_0004 to replace the original TMD (PF08_0004-Rex2TM-GFP) (figure 3.2). The resulting construct was not exported and localized to the parasite periphery. One possi-bility for the entirely different behavior of PF08_0004 to other PNEPs may be an altered membrane topology compared to the conventional PNEPs. Using the TMHMM Server v.
2.0 the localization and orientation of the TMDs in the different constructs was predicted (figure 3.3). For the topology prediction the sequences were used without the SP (if present) and without GFP, as depicted in the schematics. In PF08_0004 the N-terminus is predicted to be inside (the parasite cytoplasm) while for REX2 it is predicted to be outside. This is in accordance with the "positive inside rule", saying that the domain with the net positive charge flanking a TMD faces the cytoplasm (von Heijne, 1992). Surpris-ingly, when the flanking regions were replaced with amino acids flanking the REX2 TMD (PF08_0004Rex2TM+Rex2flanks) the predicted topology did not change, although the probability value of the prediction decreased (figure 3.3). The corresponding GFP-fusion construct (PF08_0004 Rex2 TM+Rex2 flanks-GFP) showed a punctate localization in the host cell and staining in the parasite periphery, indicating that the REX2 flanking regions could at least partially rescue the export of PF08_0004-Rex2TM-GFP (figure 3.2). Completely removing the TMD flanking amino acids (PF08_0004 Rex2TM+no flanks-GFP) resulted in a mostly perinuclear localization of the construct, typical for the ER (figure 3.2). This was probably not due to an altered topology, as the construct was predicted to have the same topology as PF08_0004 (figure 3.3).
Next, the PF08_0004 TMD was replaced with the TMD of the non-exported protein mTRAP (PF08_0004-mTRAP TM-GFP), which resulted in a staining in the parasite periphery, similar to PF08_0004-Rex2TM-GFP. Replacement of the C-terminal half of the TMD with the original TMD sequence (PF08_0004-mTRAP TM-N-GFP) could partially rescue the export (figure 3.2), indicating that the C-terminal part of the TMD could contribute to export.
PFL0065w is another PNEP identified by Heiber et al., which contains a SP and a TMD. Because of the similar structure it was reasoned that the PFL0065w TMD of this protein might better correspond to the TMD of PF08_0004 and was used to replace the PF08_0004 TMD. The resulting construct, expressed as a GFP fusion (PF08_0004-PFL0065wTM-GFP), showed no export but staining at the parasite periphery (figure
GFP
SP 1 2 3 4
PF08_0004-Rex2TM-GFP
PF08_0004-Rex2TM+Rex2Eflanks-GFP
GFP
SP 1 2 3 Rex2TM 4
PF08_0004-Rex2TM+noEflanks-GFP
GFP
SP 1 2 3 4
GFP
SP 1 2 3 nonETM 4
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SP 1 2 3 mTRAPTM 4
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SP 1 2 3 PFLTM 4
GFP SP 1 2 3 mTRTMTM 4
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Rex2 1-20
PF08_0004-mTRAP-TM-GFP
PF08_0004-mTRAP-TM-N-GFP
PF08_0004-PFL0065wTM-GFP
PF08_0004-nonTM-GFP
PF08_0004∆SP-GFP
PF08_0004-SERA7SP+12aa-GFP
PF08_0004∆SP-Rex2E1-20-GFP GFP DIC/DAPI DIC/GFP/DAPI
Rex2E TM
TM
TM
Rex2E TM Rex2E
TM
SERA7
Figure 3.2.: The role of the SP and TMD in the export of PF08_0004. Live cell images of 3d7 parasites expressing GFP-fusion constructs under the crt-promoter. Nuclei were stained with DAPI. Construct schematics and names are shown above the respective images.
(SP) signal peptide, (TM) transmembrane region, (DIC) differential interference contrast. Scale bar: 5µm.
TM
PF08_0004-mTRAPqTM-N
PF08_0004-mTRAPqTM PF08_0004-Rex2qTM+noqflanks PF08_0004-Rex2qTM+Rex2qflanks PF08_0004-Rex2qTM
PF08_0004 Rex2
PF08_0004-PFL0065wqTM
PF08_0004-randomqseqqTM
PFL0065w
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1 TM 21 2 3 Rex2TM 4
1 2 3 TM 4
1 2 3 Rex2TM 4
1 2 3 4
1 2 3 mTRAPTM 4
1 2 3 mTRTMTM 4
1 2 3 PFLTM 4
1 2 3 nonqTM 4
Rex2TM
Rex2TM
Figure 3.3.: Predicted TMDs in PF08_0004, REX2 and PFL0065w constructs. Mem-brane topology predictions generated by the TMHMM Server v. 2.0 are shown for PF08_0004, REX2, PFL0065w and derived constructs(A-J). Sequences used for the predictions do not in-clude the SPs and GFP. Red bars show the TMD probability, the horizontal red bar represents the TMD. The blue line shows the probability for a part of the protein to face the outside (initially the ER lumen), the purple line for facing the cytoplasm. (SP) signal peptide, (TM) transmembrane region.
A
B
SERA7SP 1 2 3 TM 4 GFPGFP
SP 1 2 3 PFLTM 4
Figure 3.4.: PF08_0004 signal peptide cleavage site prediction. Signal peptide cleav-age predictions generated by the SignalP 3.0 Server (Bendtsen et al., 2004) are shown for A PF08_0004 and B PF08_0004SERA7SP+12aa. Only the SPs and part 1 were used as a se-quence input for better representation, in the schematics the rest of the protein is depicted in darker shades. The blue lines and red bars represent the cleavage probability/position, the green line shows the SP probability. Amino acids are shown in single letter code. (SP) signal peptide, (TM) transmembrane region
3.2). Similar to the other constructs with TMD replacements the predicted membrane topology resembled that of PF08_0004 (figure 3.3). Taken together these data indicate that also the TMD of PF08_0004 plays a crucial role in export of this PNEP and that in contrast to other PNEPs this function cannot be replaced by a TMD of a PNEP and also not by that of a non-exported protein. Only the additional replacement of the flanking regions of the REX2 PNEP TMD restored some export.
In the "classical" PNEP REX2 the TMD is necessary for entry into the secretory path-way (Haase et al., 2009). To investigate the influence of the PF08_0004 TMD for this process the TMD was replaced with a random amino acid sequence obtained by scrambling amino acids 301-321 of mTRAP. The resulting construct had no predicted TMD (figure 3.3, PF08_0004-nonTM) and localized to the parasite periphery and structures probably resembling the TVN, showing that the SP is sufficient for recruitment into the secretory pathway (see figure 3.2, PF08_0004-randomseqTM-GFP), but that such a construct is not exported. Surprisingly, a construct without a SP but with a TMD (PF08_0004∆SP-GFP) was efficiently exported and showed a punctate pattern in the host cell, suggesting that the TMD alone is also sufficient for secretory pathway recruitment and that the SP is not necessary for export. Hence, this resembled the situation in conventional PNEPs without a classical N-terminal SP.
To further investigate the role of the SP it was replaced with the PV-residents’ SERA7 SP, accidentally including the 12 aa downstream of the SP (PF08_0004 SERA7 SP+12aa-GFP). This construct showed almost no export and localized predominantly to the par-asite periphery (see figure 3.2). A prediction of the SP cleavage sites in PF08_0004 and PF08_0004 SERA7 SP+12aa using the SignalP 3.0 server (Bendtsen et al., 2004) shows that the first 6 aa of mature N-terminus of PF08_0004 SERA7 SP+12aa are QEKPPP, in contrast to DERKNF of PF08_0004 (see figure 3.4). It was shown, that a stretch of pro-lines can block the export of an otherwise exported protein (Ullrich, 2016), which could be an explanation for the observed phenotype, rather than a dependence on the PF08_0004 SP. The first 20 aa of REX2 can mediate export of a reporter construct (Grüring et al., 2012), and replacement of the PF08_0004 SP with those resulted in efficient export of the construct (see figure 3.2, PF08_0004∆SP-Rex2 1-20-GFP). These results indicate some influence of the mature N-terminus on export, similar to other PNEPs.
In summary, these results show that almost every change to the PF08_0004 sequence leads to loss of export, including even the exchange of the TMD for another PNEP TMD.
The exception is the SP that was found to be dispensable for export. So, in contrast to the other PNEPs investigated so far, the export region could not be narrowed down to a single part of the protein.