BioID Reveals Novel Proteins of the
            <i>Plasmodium</i>
            Parasitophorous Vacuole Membrane

Schnider, Cilly Bernardette; Bausch-Fluck, Damaris; Brühlmann, Francis; Heussler, Volker; Burda, Paul‐Christian

doi:10.1128/msphere.00522-17

Cited by 44 publications

(44 citation statements)

References 38 publications

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“…These results show that although EXP2 function in protein export is preserved following depletion of EXP1, its organisation in the PVM is drastically altered. probe the PV in P. berghei (Schnider et al, 2018). In the present study, we present the first use of BioID2 in Plasmodium spp., which we fused to the endogenous copy of EXP2 or HSP101 to identify proteins at the luminal face of the PVM.…”

Section: Depletion Of Exp1 Results In Late Cycle Arrest and Pv/pvmmentioning

confidence: 92%

EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole

Nessel

Beck

Rayatpisheh

et al. 2020

Cellular Microbiology

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Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) that closely overlays the parasite plasma membrane. Although the PVM is the site of several transport activities essential to parasite survival, the basis for organisation of this membrane system is unknown. Here, we performed proximity labeling at the PVM with BioID2, which highlighted a group of single‐pass integral membrane proteins that constitute a major component of the PVM proteome but whose function remains unclear. We investigated EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 genome editing system to install the TetR–DOZI‐aptamers system for conditional translational control. Importantly, although EXP1 was required for intraerythrocytic development, a previously reported in vitro glutathione S‐transferase activity could not account for this essential EXP1 function in vivo. EXP1 knockdown was accompanied by profound changes in vacuole ultrastructure, including apparent increased separation of the PVM from the parasite plasma membrane and formation of abnormal membrane structures. Furthermore, although activity of the Plasmodium translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the translocon pore‐forming protein EXP2 but not the HSP101 unfoldase was substantially altered. Collectively, our results reveal a novel PVM defect that indicates a critical role for EXP1 in maintaining proper organisation of EXP2 within the PVM.

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Section: Depletion Of Exp1 Results In Late Cycle Arrest and Pv/pvmmentioning

confidence: 92%

EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole

Nessel

Beck

Rayatpisheh

et al. 2020

Cellular Microbiology

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“…GRA44, in contrast, contains a putative phosphatase domain that shares homology to a region of the Plasmodium serine/threonine phosphatase UIS2 (28% identity over 21% of the protein; BLASTP, v2.10.0ϩ), which has recently been shown to localize to the Plasmodium PVM in liver-stage parasites (39). Whether UIS2 plays a role in protein translocation in Plasmodium remains to be determined, but it would be surprising if it did, given that none of the other components of the complex known to promote translocation in Plasmodium (known as PTEX) so far studied play a role in translocation in Toxoplasma (2).…”

Section: Discussionmentioning

confidence: 99%

Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells

Cygan

Theisen

Mendoza

et al. 2020

mSphere

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Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins. IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.

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“…To date, the original E. coli BirA* (BioID) has been employed by four studies in Plasmodium spp ., including two that targeted BioID to the PV (49–52). Khosh-Naucke and colleagues targeted a signal peptide-GFP-BirA* fusion to the P. falciparum PV lumen (50) while Schnider and colleagues utilized a second copy of EXP1 with a C-terminal BirA* fusion to probe the PV in P. berghei (51). In the present study, we present the first use of BioID2 in Plasmodium spp ., which we fused to the endogenous copy of EXP2 or HSP101 to identify proteins at the luminal face of the PVM.…”

Section: Discussionmentioning

confidence: 99%

EXP1 is required for organization of the intraerythrocytic malaria parasite vacuole

Nessel

Beck

Rayatpisheh³

et al. 2019

Preprint

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word count: 250 22 23 24 Importance word count: 149 25 26 Abstract 27Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) 28 that closely overlays the parasite plasma membrane (PPM) and constitutes the barrier between 29 parasite and host compartments. The PVM is the site of several essential transport activities but 30 the basis for organization of this membrane system is unknown. We utilized the second-31 generation promiscuous biotin ligase BioID2 fused to EXP2 or HSP101 to probe the content of 32 the PVM, identifying known and novel candidate PVM proteins. Among the best represented 33 hits were members of a group of single-pass integral membrane proteins that constitute a major 34 component of the PVM proteome but whose function remains unclear. We investigated the 35 function of EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 36 genome editing system to install the TetR-DOZI-aptamers system for conditional translational 37 control. EXP1 knockdown was essential for intraerythrocytic development and accompanied by 38 profound changes in vacuole ultrastructure, including increased separation of the PVM and 39 PPM and formation of abnormal membrane structures in the enlarged vacuole lumen. While 40 previous in vitro studies indicated EXP1 possesses glutathione S-transferase activity, a mutant 41 version of EXP1 lacking a residue important for this activity in vitro still provides substantial 42 rescue of endogenous exp1 knockdown in vivo. Intriguingly, while activity of the Plasmodium 43 translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the 44 translocon pore-forming protein EXP2 was substantially altered. Collectively, our results reveal 45 a novel PVM defect that indicates a critical role for EXP1 in maintaining proper PVM 46 organization. 47 48 Importance 49 Like other obligate intracellular apicomplexans, blood-stage malaria parasites reside within a 50 membrane-bound compartment inside the erythrocyte known as the parasitophorous vacuole. 51Although the vacuole is the site of several transport activities essential to parasite survival, little 52 is known about its organization. To explore vacuole biology, we adopted recently developed 53 proteomic (BioID2) and genetic (CRISPR/Cpf1) tools for use in Plasmodium falciparum, which 54 allowed us to query the function of the prototypical vacuole membrane protein EXP1. 55Knockdown of EXP1 showed that a previously reported glutathione S-transferase activity cannot 56 fully account for the essential function(s) of EXP1 and revealed a novel role for this protein in 57 maintaining normal vacuole morphology and PVM protein arrangement. Our results provide new 58 insight into vacuole organization and illustrate the power of BioID2 and Cpf1 (which utilizes a T-59 rich PAM uniquely suited to the P. falciparum genome) for proximity protein identification and 60 genome editing in P. falciparum. 61 62 65principal barrier between the parasite and its host cell (1). Durin...

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BioID Reveals Novel Proteins of the Plasmodium Parasitophorous Vacuole Membrane

Cited by 44 publications

References 38 publications

EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole

EXP1 is required for organisation of EXP2 in the intraerythrocytic malaria parasite vacuole

Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells

EXP1 is required for organization of the intraerythrocytic malaria parasite vacuole

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