Mutant/mutation
The protein expresses a C-terminal HA/streptavidine tagged form of HSP101. In addition, it expresses a reporter comprised of the N-terminal leader of the knob-associated histidine-rich protein (KAHRP leader [KL]), including its hydrophobic signal sequence and PEXEL motif, conjugated to GFP and placed under the transcriptional control of the constitutive HSP70 promoter

Protein (function)
Plasmodium parasites must export proteins into their erythrocytic host to survive. Exported proteins must cross the parasite plasma membrane (PPM) and the parasitophorous vacuolar membrane (PVM) encasing the parasite to access the host cell. Crossing the PVM requires protein unfolding and passage through a translocon, the Plasmodium translocon of exported proteins (PTEX). In this study, we provide the first direct evidence that heat shock protein 101 (HSP101), a core component of PTEX, unfolds proteins for translocation across the PVM by creating transgenic Plasmodium parasites in which the unfoldase and translocation functions of HSP101 have become uncoupled.

Phenotype
To study HSP101 function and dissect how it contributes to protein export, two transgenic parasite lines were generated in the rodent malaria parasite Plasmodium berghei, termed Pb101HA/KLGFP and Pb101HA+2AmCh/KLGFP. To create these parasites, the endogenous hsp101 locus was replaced with full-length hsp101 fused at its 3' end to a triple hemagglutinin (HA) and streptavidin epitope and incorporating a heterologous 3' untranslated region (3' UTR).
Previous studies of Plasmodium falciparum and P. berghei have demonstrated that HSP101 can accommodate these tags at its C terminus without impacting parasite growth or protein export. Utilizing the same construct, an exported reporter cassette was incorporated downstream of the hsp101 locus of both lines. This reporter comprised the N-terminal leader of the knob-associated histidine-rich protein (KAHRP leader [KL]), including its hydrophobic signal sequence and PEXEL motif, conjugated to GFP and placed under the transcriptional control of the constitutive HSP70 promoter . The Pb101HA+2AmCh/KLGFP line also harbors an mCherry coding sequence, which is separated from the HSP101 coding sequence by the foot and mouth disease virus 2A skip peptide, to produce a polycistronic HSP101-HA-2A-mCherry mRNA .
We anticipated that the 2A skip peptide would produce separate HSP101-HA and mCherry proteins. However, Western blot analysis of HSP101 revealed that separation by the 2A peptide was inefficient in the Pb101HA2AmCh/KLGFP line. Instead, two forms of HSP101 protein were generated, corresponding to HSP101-HA alone (Pb101HA) or a larger HSP101-HA2A-mCherry protein (Pb101HA2AmCh).
We have previously published a similar approach to generate parasite expressing PbEXP2-2A-FRT in which EXP2 and mCherry were also generated from a polycistronic mRNA. In that case, cleavage of the 2A peptide was very efficient, giving rise to separate EXP2 and mCherry polypeptides.
Since HSP101 oligomerizes into hexamers to associate with the PTEX complex, we examined whether conjugation of HSP101 with a bulky 28-kDa mCherry protein impaired the ability of HSP101 to be incorporated into PTEX. Immunoprecipitation of mCherry using red fluorescence protein (RFP)-TRAP resulted in the pulldown of Pb101HA2AmCh together with Pb101HA. Importantly, Pb101HA2AmCh also immunoprecipitated with EXP2. Together, this indicates that Pb101HA2AmCh is incorporated into HSP101 hexamers and into the PTEX complex.
We then examined the solubility of Pb101HA2AmCh using carbonate extraction assays. HSP101 is peripherally associated with the PVM and thus is normally released into the supernatant fraction only after treatment with carbonate. We found that Pb101HA2AmCh has a solubility profile similar to that of Pb101HA, indicating that the addition of mCherry to Pb101HA did not change the solubility profile of the protein. This result is consistent with Pb101HA2AmCh incorporating into the PTEX complex at the PVM, rather than being present as a soluble protein in the PV, in which case HSP101HA2AmCh would be present in the hypotonic supernatant fraction.
Ii was further shown that:
- Pb101HA2AmCh/KLGFP parasites fail to export the GFP reporter and show reduced parasite growth.
- Pb101HA2AmCh/KLGFP can export native proteins.
- Soluble reporter proteins are directly affected by the efficiency of HSP101 function.
- PEXEL proteins with TMDs are exported without unfolding by PTEX

Additional information
From the paper:
In this study, we provide the first direct evidence that heat shock protein 101 (HSP101), a core component of PTEX, unfolds proteins for translocation across the PVM by creating transgenic Plasmodium parasites in which the unfoldase and translocation functions of HSP101 have become uncoupled. Strikingly, while these parasites could export native proteins, they were unable to translocate soluble, tightly folded reporter proteins bearing the Plasmodium export element (PEXEL) across the PVM into host erythrocytes under the same conditions. In contrast, an identical PEXEL reporter protein but harboring a transmembrane domain could be exported, suggesting that a prior unfolding step occurs at the PPM. Together, these results demonstrate that the export of parasite proteins is dependent on how these proteins are presented to the secretory pathway before they reach PTEX as well as their folded status. Accordingly, only tightly folded soluble proteins secreted into the vacuolar space and not proteins containing transmembrane domains or the majority of erythrocyte-stage exported proteins have an absolute requirement for the full unfoldase activity of HSP101 to be exported.

Other mutants