Malaria is caused by the parasite Plasmodium falciparum. For part of its life cycle, this organism resides inside human red blood cells in a membrane-bound compartment called a vacuole. To survive, multiply and evade an immune response in this environment, P. falciparum must transport nutrients and proteins across the vacuolar membrane1. Writing in Nature, Ho et al.2 report the structure of the parasite PTEX complex, which resides on the vacuolar membrane and facilitates the export of proteins from the vacuole to the cytoplasm of red blood cells3. And in a paper in Nature Microbiology, Garten et al.4 reveal that the protein EXP2, which forms part of the PTEX protein-conducting channel located in the vacuolar membrane, can also form a channel that facilitates nutrient transfer across the membrane. These insights into the structure and function of key proteins that aid the survival of P. falciparum might help efforts to develop new antimalarial drugs.
PTEX consists of five proteins3: HSP101, PTEX150, EXP2, PTEX88 and TRX2. Multiple HSP101, PTEX150 and EXP2 molecules assemble to form the core part of PTEX3,5. It has been predicted that HSP101 unfolds proteins destined for export, and provides the energy needed for cargo to pass through the vacuolar-membrane-spanning part of the channel, which is proposed3,6 to consist of EXP2. PTEX150 is thought5 to have a structural role, connecting HSP101 and EXP2.
Reduced expression7 of HSP101 or PTEX150, or inhibition8 of the assembly of HSP101 into the PTEX complex, results in parasite death. PTEX is specific to species of the genus Plasmodium and is not made by humans. It is an attractive drug target because it provides the only known route by which parasite proteins enter the cytoplasm of a red blood cell. However, PTEX’s relative novelty offers few clues to how it functions. EXP2 synthesized in the laboratory can form protein channels in lipid bilayers9. However, there have been no reports of full-length HSP101 or PTEX150 having been successfully synthesized for use in in vitro experiments. This has prevented structural analysis of the proteins, or reconstitution of the core PTEX complex in lipid membranes, to determine how the complex assembles and functions.
Because of these experimental limitations, Ho et al.2 opted instead to extract PTEX directly from red blood cells containing the parasite. Then, using a technique called cryo-electron microscopy (cryo-EM), the authors captured two distinct structural conformations of the core PTEX complex in the process of exporting unfolded protein cargo — they called these conformations the ‘engaged’ and ‘resetting’ states. The cryo-EM analysis revealed that HSP101, PTEX150 and EXP2 assemble into an asymmetrical structure containing six molecules of HSP101, seven of PTEX150 and seven of EXP2. These structures closely align with models of the organization and size of PTEX that had been predicted from biochemical and protein-analysis experiments3,5.
Ho and colleagues found that the seven EXP2 molecules, which make up the protein channel in the lipid membrane, create a funnel shape, with the amino terminus of each molecule forming a transmembrane helix in the vacuolar membrane to provide an anchoring ‘stem’ (Fig. 1). The ‘mouth’ of EXP2 constitutes the bulk of the protein, and faces into the vacuole. This end of EXP2 contains a domain that tethers it to the carboxy-terminal domain of HSP101, situated directly on top. Only approximately 20% of the structure of PTEX150 could be determined. Nevertheless, this was sufficient to reveal that each PTEX150 molecule slots in between adjacent EXP2 molecules at the mouth of the EXP2 funnel, curling down towards the stem. Thus, PTEX150 provides a protective path for unfolded protein cargo transiting from HSP101 to EXP2.
Of the three proteins, HSP101 displayed the greatest structural difference between the engaged and resetting states of PTEX, and on this basis the authors propose a mechanism for how cargo is threaded through PTEX’s central cavity. In this model, domains of the six assembled HSP101 molecules form two ‘hands’ that work together to thread unfolded cargo through the PTEX150 and EXP2 funnel. In the engaged state of PTEX, both the ‘active’ and ‘passive’ hands of HSP101 grasp the unfolded cargo. The cargo is then fed downwards through the central cavity of PTEX in a spiral fashion as it passes from the active to the passive hand. In the resetting state, HSP101’s active hand moves upwards to grasp the next section of the cargo protein for transport, and the passive hand grips the cargo to prevent it from slipping backwards and away from the PTEX channel.
The cryo-EM structures provide insight into several crucial interactions between the PTEX components. These interactions are potentially required for assembly and optimal function of the complex, and could be tested using genetic approaches to validate the model. Ho and colleagues were unable to determine the structure of the N-terminal domain of HSP101 that binds the protein cargo. Thus, it is unclear how cargo is recognized by HSP101, and whether cargo proteins are unfolded by proteins known as chaperones before they reach PTEX. Given that unfolded proteins pass through PTEX, these cargo proteins would then need to be refolded to function, presumably by other chaperone proteins. However, because EXP2 does not extend into the cytoplasm of red blood cells, it is unclear how chaperone proteins in the host cell might be recruited to cargo exiting PTEX.
Garten et al.4 investigated EXP2 using in vitro experiments, and report that it has another role in addition to its function in PTEX. Previous experiments using electrophysiological techniques have shown that a channel exists in the vacuolar membrane of parasite-infected red blood cells through which nutrients such as amino acids and sugars can pass10, but the identity of this channel has been a mystery. In electrophysiological studies, Garten and colleagues demonstrated a direct relationship between the level of expression of EXP2 and the frequency of detection of the mysterious channel. When the authors generated a version of EXP2 that had a truncated C-terminal domain, which is located in the vacuole and is not required for protein export, this altered the voltage-response properties of the nutrient channel, leading the authors to conclude that EXP2 is indeed the elusive nutrient channel.
That EXP2 might have a role separate from its function in PTEX is consistent with evidence that EXP2’s gene-expression profile differs from that of the other PTEX components5. Moreover, the authors found that most EXP2 is not present in a complex with PTEX. Although EXP2 is essential for parasite survival11,12, the contribution of the EXP2 nutrient channel to parasite growth remains unknown. The channel could be characterized in detail if EXP2 was incorporated into lipid bilayers for in vitro experiments.
The studies by Ho, Garten and their respective colleagues offer a close look at how major P. falciparum proteins function. Interestingly, EXP2 is evolutionarily conserved among vacuolar-dwelling parasites called apicomplexans1. Perhaps the nutrient-transiting capacity of EXP2 was adapted by P. falciparum to generate a protein-conducting channel that evolved through the recruitment of other proteins such as HSP101 and PTEX150. EXP2 and PTEX are expressed throughout the life cycle of P. falciparum, so drugs that target them might be highly effective at tackling malaria. These new insights into the interactions between the components of PTEX offer exciting possibilities for the development of peptides or small molecules that might block the function of this complex.
Nature 561, 41-43 (2018)