Uncoupling the Threading and Unfoldase Actions of Plasmodium HSP101 Reveals Differences in Export between Soluble and Insoluble Proteins

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. 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.