Parasite-encoded Hsp40 proteins define novel mobile structures in the cytosol of the P. falciparum-infected erythrocyte

Abstract

SummaryPlasmodium falciparum is predicted to transport over 300 proteins to the cytosol of its chosen host cell, the mature human erythrocyte, including 19 members of the Hsp40 family. Here, we have generated transfectant lines expressing GFP-or HA-Strep-tagged versions of these proteins, and used these to investigate both localization and other properties of these Hsp40 co-chaperones. These fusion proteins labelled punctate structures within the infected erythrocyte, initially suggestive of a Maurer's clefts localization. Further experiments demonstrated that these structures were distinct from the Maurer's clefts in protein composition. Transmission electron microscopy verifies a non-cleft localization for HA-Strep-tagged versions of these proteins. We were not able to label these structures with BODIPY-ceramide, suggesting a lower size and/or different lipid composition compared with the Maurer's clefts. Solubility studies revealed that the Hsp40-GFP fusion proteins appear to be tightly associated with membranes, but could be released from the bilayer under conditions affecting membrane cholesterol content or organization, suggesting interaction with a binding partner localized to cholesterol-rich domains. These novel structures are highly mobile in the infected erythrocyte, but based on velocity calculations, can be distinguished from the 'highly mobile vesicles' previously described. Our study identifies a further extra-parasitic structure in the P. falciparum-infected erythrocyte, which we name 'J-dots' (as their defining characteristic so far is the content of J-proteins). We suggest that these J-dots are involved in trafficking of parasiteencoded proteins through the cytosol of the infected erythrocyte.

Export of virulence proteins by malaria-infected erythrocytes involves remodeling of host actin cytoskeleton

“…57 It was suggested J-dots could be protein-cholesterol aggregates or vesicular structures of an unusual lipid composition. 30 This may explain absence of a visible membrane in our EM studies around vesicular structures we observed. PfPTP1 and SBP1 occur on these structures and it has been shown that PfEMP1 is found on structures with very similar characteristics.…”

“…[24][25][26] There are a number of different vesicles described in P falciparum including electron dense vesicles (EDVs), J-dots, and vesicle-like structures (VLSs), which could play a role in PfEMP1 trafficking. [27][28][29][30][31] We have characterized a novel PEXEL-containing protein that we have named P falciparum PfEMP1 trafficking protein.…”

Intracellular Protozoan Parasites of Humans: The Role of Molecular Chaperones in Development and Pathogenesis

“…Two of these exported PfHsp40 proteins were found to be associated with 701 cholesterol containing membranes [121]. They seem to be localized in punctuate, 702 highly mobile structures, termed J-dots.…”

Maurer's clefts, the enigma of Plasmodium falciparum

“…Additionaly, there is no known vesicular transport to the Maurer's clefts because none of the vesicles observed could be labeled with antibodies against any Maurer's clefts protein (21,36). Whether these proteins travel via a nontypical vesicular transport or as soluble chaperoned protein complexes (76,81,114) remains to be shown, and recently described J-dots carrying parasitederived HSP40 and HSP70 proteins might play a role in this process (49,50).…”

“…Only a few proteins are known to be trafficked to structures in the host cell without passing through the Maurer's cleft hub. These proteins include MAHRP2 at tether-like structures (48), heat shock protein 40 (HSP40) and HSP70-x in or at J-dots (49,50), and some soluble proteins in the cytosol, such as ring exported protein 3 (REX3) (40). Although most resident proteins are encoded by single-copy genes, such as membrane associated histidine rich protein 1 (MAHRP1) (41) or knob associated histidine rich protein (KAHRP) (38, 51), Maurer's clefts gain their importance as sorting stations of a number of exported protein families.…”