PTEX is a multiprotein complex that comprises at least five protein components. Elsworth, Matthews et al. analysed the requirement for the core PTEX components HSP101 (which is a putative chaperone) and PTEX150 by generating a Plasmodium berghei line (Pbi101KD) in which HSP101 was under the transcriptional control of an anhydrotetracycline (ATc)-regulated transactivator. Knockdown of HSP101 by the addition of ATc caused a major reduction in parasite growth. Surface labelling of parasite antigens on Pbi101KD parasites that had been harvested from infected mice that were pretreated with ATc showed a considerable reduction in the export of surface proteins compared with Pbi101KD parasites from mice that had not been pretreated. Two categories of parasite proteins have been proposed to be exported through PTEX: those that contain a conserved pentameric Plasmodium export element motif (PEXEL) and those that do not (PEXEL-negative exported proteins (PNEPs)). Immunofluorescence analysis showed that the export of both PEXEL and PNEP proteins was blocked when HSP101 expression was decreased by ATc treatment. Finally, the authors used an inducible ribozyme system to generate a conditional PTEX150 knockdown Plasmodium falciparum line, which demonstrated that PTEX150 is required for blood-stage growth, and its loss led to a block in the export of PEXEL and PNEP proteins.
Beck, Muralidharan et al. used an autoinhibition strategy to reversibly inhibit the function of P. falciparum HSP101, which involved fusing the carboxyl terminus of HSP101 to a dihydrofolate reductase-based destabilization domain (DDD) and a haemagglutinin tag (HSP101DDD). The DDD is stabilized by the presence of trimethoprim (TMP), and TMP removal from synchronized P. falciparum cultures caused a block in parasite growth and an accumulation of late ring-stage parasites during the early phase of the asexual development cycle. Removal of TMP during gametocytogenesis induction experiments inhibited gametocyte development, which suggests that HSP101 is essential for protein export during both sexual and asexual blood-stage development in P. falciparum. Immunofluorescence assays were used to assess the range of protein substrates that are exported by PTEX, and vacuolar accumulation of both PEXEL and PNEP proteins was observed in the absence of HSP101 expression. This indicates that PTEX is involved in the translocation of PNEP and PEXEL proteins, which is consistent with the findings of Elsworth, Matthews et al. Beck, Muralidharan et al. went on to investigate the mechanism responsible for the block in protein export and found that, when HSP101DDD was destabilized by the removal of TMP, the interaction between HSP101DDD and PTEX150 was severely reduced. The authors suggest that destabilized HSP101DDD dissociates from the PTEX complex, and they present a model in which HSP101 recognizes and drives the translocation of exported proteins.
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