Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes

This article has been updated

Abstract

To mediate its survival and virulence, the malaria parasite Plasmodium falciparum exports hundreds of proteins into the host erythrocyte1. To enter the host cell, exported proteins must cross the parasitophorous vacuolar membrane (PVM) within which the parasite resides, but the mechanism remains unclear. A putative Plasmodium translocon of exported proteins (PTEX) has been suggested to be involved for at least one class of exported proteins; however, direct functional evidence for this has been elusive2,3,4. Here we show that export across the PVM requires heat shock protein 101 (HSP101), a ClpB-like AAA+ ATPase component of PTEX. Using a chaperone auto-inhibition strategy, we achieved rapid, reversible ablation of HSP101 function, resulting in a nearly complete block in export with substrates accumulating in the vacuole in both asexual and sexual parasites. Surprisingly, this block extended to all classes of exported proteins, revealing HSP101-dependent translocation across the PVM as a convergent step in the multi-pathway export process. Under export-blocked conditions, association between HSP101 and other components of the PTEX complex was lost, indicating that the integrity of the complex is required for efficient protein export. Our results demonstrate an essential and universal role for HSP101 in protein export and provide strong evidence for PTEX function in protein translocation into the host cell.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: HSP101 is essential for development of asexual and sexual blood stages.
Figure 2: HSP101 is required for export of PEXEL and PNEP proteins.
Figure 3: HSP101 is required for activation of PSAC but not trafficking of CLAG3 to the RBC periphery.
Figure 4: Inactivated HSP101DDD dissociates from the PTEX complex.

Change history

  • 31 July 2014

    A panel label in Fig. 2e has been corrected

References

  1. Boddey, J. A. & Cowman, A. F. Plasmodium nesting: remaking the erythrocyte from the inside out. Annu. Rev. Microbiol. 67, 243–269 (2013)

    CAS  Article  PubMed  Google Scholar 

  2. de Koning-Ward, T. F. et al. A newly discovered protein export machine in malaria parasites. Nature 459, 945–949 (2009)

    CAS  ADS  Article  PubMed  PubMed Central  Google Scholar 

  3. Bullen, H. E. et al. Biosynthesis, localization, and macromolecular arrangement of the Plasmodium falciparum translocon of exported proteins (PTEX). J. Biol. Chem. 287, 7871–7884 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Riglar, D. T. et al. Spatial association with PTEX complexes defines regions for effector export into Plasmodium falciparum-infected erythrocytes. Nature Commun. 4, 1415 (2013)

    ADS  Article  Google Scholar 

  5. Iwamoto, M., Bjorklund, T., Lundberg, C., Kirik, D. & Wandless, T. J. A general chemical method to regulate protein stability in the mammalian central nervous system. Chem. Biol. 17, 981–988 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Muralidharan, V., Oksman, A., Iwamoto, M., Wandless, T. J. & Goldberg, D. E. Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag. Proc. Natl Acad. Sci. USA 108, 4411–4416 (2011)

    CAS  ADS  Article  PubMed  PubMed Central  Google Scholar 

  7. Muralidharan, V., Oksman, A., Pal, P., Lindquist, S. & Goldberg, D. E. Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers. Nature Commun. 3, 1310 (2012)

    ADS  Article  Google Scholar 

  8. Silvestrini, F. et al. Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum. Mol. Cell. Proteomics 9, 1437–1448 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Matthews, K. et al. The Plasmodium translocon of exported proteins (PTEX) component thioredoxin-2 is important for maintaining normal blood-stage growth. Mol. Microbiol. 89, 1167–1186 (2013)

    CAS  Article  PubMed  Google Scholar 

  10. Marti, M., Good, R. T., Rug, M., Knuepfer, E. & Cowman, A. F. Targeting malaria virulence and remodeling proteins to the host erythrocyte. Science 306, 1930–1933 (2004)

    CAS  ADS  Article  PubMed  Google Scholar 

  11. Hiller, N. L. et al. A host-targeting signal in virulence proteins reveals a secretome in malarial infection. Science 306, 1934–1937 (2004)

    CAS  ADS  Article  PubMed  Google Scholar 

  12. Russo, I. et al. Plasmepsin V licenses Plasmodium proteins for export into the host erythrocyte. Nature 463, 632–636 (2010)

    CAS  ADS  Article  PubMed  PubMed Central  Google Scholar 

  13. Boddey, J. A. et al. An aspartyl protease directs malaria effector proteins to the host cell. Nature 463, 627–631 (2010)

    CAS  ADS  Article  PubMed  PubMed Central  Google Scholar 

  14. Rock, E. P. et al. Comparative analysis of the Plasmodium falciparum histidine-rich proteins HRP-I, HRP-II and HRP-III in malaria parasites of diverse origin. Parasitology 95, 209–227 (1987)

    CAS  Article  PubMed  Google Scholar 

  15. Spielmann, T. et al. A cluster of ring stage-specific genes linked to a locus implicated in cytoadherence in Plasmodium falciparum codes for PEXEL-negative and PEXEL-positive proteins exported into the host cell. Mol. Biol. Cell 17, 3613–3624 (2006)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Crabb, B. S. et al. Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress. Cell 89, 287–296 (1997)

    CAS  Article  PubMed  Google Scholar 

  17. Culvenor, J. G., Day, K. P. & Anders, R. F. Plasmodium falciparum ring-infected erythrocyte surface antigen is released from merozoite dense granules after erythrocyte invasion. Infect. Immun. 59, 1183–1187 (1991)

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Boddey, J. A. et al. Role of plasmepsin V in export of diverse protein families from the Plasmodium falciparum exportome. Traffic 14, 532–550 (2013)

    CAS  Article  PubMed  Google Scholar 

  19. Grüring, C. et al. Uncovering common principles in protein export of malaria parasites. Cell Host Microbe 12, 717–729 (2012)

    Article  PubMed  Google Scholar 

  20. Mundwiler-Pachlatko, E. & Beck, H. P. Maurer's clefts, the enigma of Plasmodium falciparum. Proc. Natl Acad. Sci. USA 110, 19987–19994 (2013)

    CAS  ADS  Article  PubMed  PubMed Central  Google Scholar 

  21. Blisnick, T. et al. Pfsbp1, a Maurer’s cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton. Mol. Biochem. Parasitol. 111, 107–121 (2000)

    CAS  Article  PubMed  Google Scholar 

  22. Heiber, A. et al. Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export. PLoS Pathog. 9, e1003546 (2013)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Miller, L. H., Baruch, D. I., Marsh, K. & Doumbo, O. K. The pathogenic basis of malaria. Nature 415, 673–679 (2002)

    CAS  Article  PubMed  Google Scholar 

  24. Külzer, S. et al. Parasite-encoded Hsp40 proteins define novel mobile structures in the cytosol of the P. falciparum-infected erythrocyte. Cell. Microbiol. 12, 1398–1420 (2010)

    Article  PubMed  Google Scholar 

  25. Klemba, M., Beatty, W., Gluzman, I. & Goldberg, D. E. Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum. J. Cell Biol. 164, 47–56 (2004)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Yeoh, S. et al. Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes. Cell 131, 1072–1083 (2007)

    CAS  Article  PubMed  Google Scholar 

  27. Morahan, B. J. et al. Functional analysis of the exported type IV HSP40 protein PfGECO in Plasmodium falciparum gametocytes. Eukaryot. Cell 10, 1492–1503 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Nguitragool, W. et al. Malaria parasite clag3 genes determine channel-mediated nutrient uptake by infected red blood cells. Cell 145, 665–677 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Besteiro, S., Michelin, A., Poncet, J., Dubremetz, J. F. & Lebrun, M. Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion. PLoS Pathog. 5, e1000309 (2009)

    Article  PubMed  PubMed Central  Google Scholar 

  30. Jackson, K. E. et al. Selective permeabilization of the host cell membrane of Plasmodium falciparum-infected red blood cells with streptolysin O and equinatoxin II. Biochem. J. 403, 167–175 (2007)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Liu, J., Gluzman, I. Y., Drew, M. E. & Goldberg, D. E. The role of Plasmodium falciparum food vacuole plasmepsins. J. Biol. Chem. 280, 1432–1437 (2005)

    CAS  Article  PubMed  Google Scholar 

  32. Balu, B., Shoue, D. A., Fraser, M. J., Jr & Adams, J. H. High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac. Proc. Natl Acad. Sci. USA 102, 16391–16396 (2005)

    CAS  ADS  Article  PubMed  PubMed Central  Google Scholar 

  33. Hall, R. et al. Antigens of the erythrocytes stages of the human malaria parasite Plasmodium falciparum detected by monoclonal antibodies. Mol. Biochem. Parasitol. 7, 247–265 (1983)

    CAS  Article  PubMed  Google Scholar 

  34. Anders, R. F. et al. in Molecular Strategies of Parasitic Invasion (eds Goodman, H., Agabian, N. & Noguiera, N. ) 333–342 (Alan R. Liss, 1987)

  35. Kumar, N., Koski, G., Harada, M., Aikawa, M. & Zheng, H. Induction and localization of Plasmodium falciparum stress proteins related to the heat shock protein 70 family. Mol. Biochem. Parasitol. 48, 47–58 (1991)

    CAS  Article  PubMed  Google Scholar 

  36. Hawthorne, P. L. et al. A novel Plasmodium falciparum ring stage protein, REX, is located in Maurer’s clefts. Mol. Biochem. Parasitol. 136, 181–189 (2004)

    CAS  Article  PubMed  Google Scholar 

  37. Eksi, S. et al. Identification of a subtelomeric gene family expressed during the asexual-sexual stage transition in Plasmodium falciparum. Mol. Biochem. Parasitol. 143, 90–99 (2005)

    CAS  Article  PubMed  Google Scholar 

  38. Frankland, S. et al. Serum lipoproteins promote efficient presentation of the malaria virulence protein PfEMP1 at the erythrocyte surface. Eukaryot. Cell 6, 1584–1594 (2007)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Francis, S. E., Banerjee, R. & Goldberg, D. E. Biosynthesis and maturation of the malaria aspartic hemoglobinases plasmepsins I and II. J. Biol. Chem. 272, 14961–14968 (1997)

    CAS  Article  PubMed  Google Scholar 

  40. Ragge, K. et al. In vitro biosynthesis and membrane translocation of the serine rich protein of Plasmodium falciparum. Mol. Biochem. Parasitol. 42, 93–100 (1990)

    CAS  Article  PubMed  Google Scholar 

  41. Grüring, C. & Spielmann, T. Imaging of live malaria blood stage parasites. Methods Enzymol. 506, 81–92 (2012)

    Article  PubMed  Google Scholar 

  42. Ginsburg, H., Kutner, S., Krugliak, M. & Cabantchik, Z. I. Characterization of permeation pathways appearing in the host membrane of Plasmodium falciparum infected red blood cells. Mol. Biochem. Parasitol. 14, 313–322 (1985)

    CAS  Article  PubMed  Google Scholar 

  43. Kirk, K., Horner, H. A., Elford, B. C., Ellory, J. C. & Newbold, C. I. Transport of diverse substrates into malaria-infected erythrocytes via a pathway showing functional characteristics of a chloride channel. J. Biol. Chem. 269, 3339–3347 (1994)

    CAS  PubMed  Google Scholar 

  44. Babbitt, S. E. et al. Plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state. Proc. Natl Acad. Sci. USA 109, E3278–E3287 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by National Institutes of Health grants AI047798 to D.E.G., T32-AI007172 to J.R.B. and AI099156 to V.M. We thank J. McBride, D. Cavanagh and EMRR for anti-EXP2 antibody, J. Adams and ATCC (MR4) for anti-BiP antibody, D. Taylor for anti-HRP2 antibody, R. Anders for anti-RESA antibody, C. Braun-Breton for anti-SBP1 antibody, K. Williamson for anti-PfGECO and anti-Pfs16 antibodies, T. Spielmann for anti-REX2, anti-REX3 and anti-MSRP6 antibodies, L. Tilley for anti-REX1 and anti-PfEMP1 antibodies, S. Desai for anti-CLAG3 antibody, A. Cowman for anti-KAHRP antibody, J. Przyborski and K. Lingelbach for anti-SERP antibody, W. Beatty for assistance with electron microscopy, B. Vaupel and T. Butler for technical assistance and P. Sigala and N. Spillman for suggestions.

Author information

Authors and Affiliations

Authors

Contributions

J.R.B., V.M. and D.E.G. conceived and designed experiments. J.R.B. performed the majority of the experiments and V.M. performed some experiments. V.M. and A.O. generated the HSP101DDD strains. J.R.B and A.O. performed the gametocyte analysis. J.R.B. and D.E.G. analysed the data and wrote the manuscript. All authors discussed and edited the manuscript.

Corresponding author

Correspondence to Daniel E. Goldberg.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Generation of HSP101DDD strains.

a, Schematic of strategy to generate HSP101DDD parasites. 3′F, 3′ flank for homologous recombination; BSD, blasticidin S deaminase; TMP, trimethoprim; HA, haemagglutinin tag; DDD, DHFR destabilization domain. b, Diagnostic PCR showing integration of the DDD fusion in the two independent clones 13F10 and 14G11. Primers shown as black arrows in a. Image is representative of two independent experiments. c, Southern blot showing integration of plasmid pHSP101-HDB occurred at the intended genomic locus. Expected NcoI digestion products and sizes are indicated in blue, red and orange in a. The 7.3-kb band in 13F10 and 14G11 indicates the presence of concatemers commonly observed in P. falciparum. Image is representative of one experiment. d, Western blot with anti-HA antibodies detects a 124 kDa band in clones 13F10 and 14G11. Image is representative of two independent experiments. e, IFA of acetone-fixed 13F10 parasites showing colocalization of HSP101DDD and EXP2 at the PVM. Scale bar, 5 µm. Images are representative of two independent experiments.

Extended Data Figure 2 Characterization of HSP101DDD strains.

a, TMP dose-response for 13F10 and 14G11 parasites grown for 96 h was measured. Error bars represent s.d. of three technical replicates. b, Western blot on lysates from asynchronous 13F10 parasites grown with or without TMP for 24 h. BiP serves as a loading control. Blotting with anti-HA antibody shows no decrease in HSP101DDD protein levels relative to the BiP loading control. Images are representative of two independent experiments. c, Quantification of new ring parasitaemia with or without TMP. In a parallel experiment to that shown in Fig. 1d (but beginning with lower parasitaemia cultures), new rings were counted in Giemsa-stained smears of synchronous 13F10 parasites where TMP was removed or not during the proceeding trophozoite stage. No significant difference in the resulting new ring parasitaemia was observed with or without TMP, indicating no difference in re-invasion efficiency–TMP. Error bars represent s.d. of three technical replicates. Data are representative of two independent experiments. d, Giemsa-stained smears of synchronous 13F10 parasites grown with or without TMP from Fig. 1d. Reintroduction of TMP after 24 or 48 h restored progression through the intraerythrocytic cycle. After 72 h without TMP, most parasites appeared as dead, pyknotic forms. Images are representative of three independent experiments. Original magnification × 1,000.

Extended Data Figure 3 HSP101 function is critical for early stage gametocyte development.

Giemsa-stained smears from day nine post gametocyte induction as quantified in Fig. 1e. TMP was removed from parallel samples at 24-h intervals beginning just before reinvasion (0 h) following gametocytogenesis induction. Data are representative of 4 independent experiments. While control (+TMP) gametocytaemia varied between experiments, a similar effect on gametocyte formation was observed in each experiment following TMP removal. Original magnification × 1,000.

Extended Data Figure 4 Localization and quantification of exported proteins following HSP101DDD inactivation.

af, IFA and immunoelectron microscopy of ring-stage 13F10 parasites with or without TMP. TMP was removed in late schizont stage and parasites were allowed to reinvade and grow 18–24 h before fixation with paraformaldehyde (a) or acetone (bd, f). Immunoelectron microscopy fixation (e) is detailed in methods. A similar export block with accumulation of the exported protein at the parasite periphery was observed in each case when TMP was removed. The soluble PEXEL-containing protein REX3 (a) is normally exported into the host RBC cytosol. The PEXEL-containing KAHRP protein (b) is normally exported through Maurer’s clefts to knob structures at the cytoplasmic face of the infected RBC membrane. The PEXEL-containing protein RESA (c) is normally exported to the RBC periphery. SERP is a marker for the PV. SBP1 (d) is an integral membrane PNEP normally exported to the Maurer’s clefts. In the absence of TMP, blocked SBP1 colocalizes with EXP2. e, Immunoelectron microscopy showing localization of SBP1 in ring-stage parasites with or without TMP. MC, Maurer’s cleft. Scale bars, 500 nm. Images are representative of one experiment. REX2 (f) is an integral membrane PNEP normally exported to the Maurer’s clefts. g, Quantification of export block by IFA for exported proteins shown here and in Fig. 2c–f. Export was scored as complete (all signal in the host cell), partial (signal within the host cell but also within the PV) or no export (signal only seen within the PV and not in the host cell). Example images of each scoring scenario are given for REX1 in Extended Data Fig. 5a. In the case of PfEMP1, cells were scored as having PfEMP1 signal at the RBC periphery (complete export) or not (no export) due to the fact that some PfEMP1 signal is always seen within the PV under normal export conditions (see Fig. 2e, +TMP). Error bars represent s.d. of three technical replicates. Data are representative of at least two independent experiments. h, IFA of PfGECO in paraformaldehyde-fixed, stage I gametocytes 36 h post invasion with or without TMP. Pfs16 is a gametocyte-specific PVM marker. All IFA scale bars, 5 µm. All IFA images (ad, f, h) are representative of two independent experiments.

Extended Data Figure 5 Reactivation of export does not require new protein synthesis.

a, b, TMP was removed in late schizonts and parasites were allowed to reinvade and develop for 18 h before TMP add back with or without 10 µg ml−1 cycloheximide (CHX). Parasites were acetone-fixed 24 h later and processed for IFA. Export was scored as complete (no REX1 retained within the EXP2-labelled PVM), partial (REX1 in the host cell and retained within the PVM) or no export (no REX1 signal beyond the PVM). Error bars represent s.d. of three technical replicates. Data are representative of two independent experiments. Scale bar, 5 µm. c, Metabolic labelling with [35S]methionine/cysteine, performed as previously described44, confirms that CHX treatment conditions inhibit new protein synthesis. Parasite proteins were TCA-precipitated and incorporated radioactivity was determined through scintillation counting. Error bars represent s.d. of three technical replicates.

Extended Data Figure 6 Maturation of plasmepsin 2 is not affected by inactivation of HSP101.

Western blot showing normal maturation of plasmepsin 2 (PM2) in asynchronous parasites after 24 h –TMP. BiP serves as a loading control. Maturation requires proPM2 trafficking through the PV before internalization to the digestive vacuole where maturation occurs25. Images are representative of two independent experiments.

Extended Data Figure 7 DDD targeted to the PV independent of HSP101 does not interfere with parasite growth or export.

a, Schematic of the PV-targeted GFP–DDD fusion protein consisting of a signal peptide appended to a GFP–DDD fusion with a C-terminal HA epitope tag and expressed under the control of the HSP86 promoter. The predicted size of the fusion protein after signal peptide cleavage is 46 kDa. Live imaging of GFP demonstrates PV targeting of the fusion protein. Images are representative of two independent experiments. b, Western blot showing GFPDDD is not degraded in the absence of TMP. Synchronized GFPDDD parasites were grown 72 h with or without TMP before purification over Percoll and treatment with tetanolysin to release the RBC cytosol but not the PV contents. The GFPDDD fusion was detected with anti-HA antibodies. BiP serves as a loading control. Images represent one experiment. c, Growth analysis of asynchronous GFPDDD parasites shows no growth defect −TMP. Error bars represent s.d. of three technical replicates. Data are representative of two independent experiments. d, IFA showing no defect in export in GFPDDD parasites in the absence of TMP. No difference was observed in export of ring-specific (REX1) and the trophozoite-specific (MSRP6) exported proteins in the presence or absence of TMP. All scale bars, 5 µm. Images represent one experiment.

Extended Data Figure 8 Localization of PTEX components is unchanged under export blocking conditions.

a, Immunoelectron microscopy showing localization of HSP101DDD in ring-stage parasites with or without TMP. TMP was removed or not from synchronous late schizonts and parasites were allowed to re-invade and develop for 18 h before fixation. Scale bars, 500 nm. Images represent one experiment. b, c, IFA of 13F10 parasites showing co-localization between HSP101DDD or EXP2 and the PV marker SERP with or without TMP, indicating that localization of these PTEX components is not altered under export blocking conditions. TMP treatment was performed as in a before fixation with acetone and processing for IFA. d, e, IFA of ring-stage 13F10 parasites expressing a PTEX150–Flag fusion. The upper panel (d) shows co-localization with HSP101DDD. The lower two panels (e) show that PTEX150 remains at the PVM during export block and partially colocalizes with blocked SBP1. TMP treatment and fixation as in b, c. All IFA scale bars, 5 µm. All IFA images (be) are representative of two independent experiments.

Extended Data Figure 9 Immunoprecipitation bead controls indicate target-specific interactions.

Replicate IP experiments to those shown in Fig. 4c, e with bead controls included. a, b, Purification of RESA by HSP101DDD or EXP2 (a) and purification of EXP2 by PTEX150Flag (b) is specific to the target antibodies. IP experiments were performed in parallel by incubating equivalent portions of lysate supernatant input with beads alone or beads and the indicated IP antibodies. S, supernatant input. E, elution. Data are representative of two independent experiments.

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Beck, J., Muralidharan, V., Oksman, A. et al. PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes. Nature 511, 592–595 (2014). https://doi.org/10.1038/nature13574

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature13574

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing