The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane.
Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle.Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER.These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.
Affiliation: Infectious Diseases Section, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. email@example.com
Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM-organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH(2)-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30-amino acid (aa) NH(2)-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.
- Intracellular Membranes/metabolism*
- Membrane Proteins/metabolism*/physiology*
- Protozoan Proteins/metabolism*/physiology*
- Amino Acid Sequence
- CHO Cells
- Cells, Cultured
- Endoplasmic Reticulum/metabolism
- Green Fluorescent Proteins
- Luminescent Proteins/metabolism
- Microsomes, Liver/metabolism
- Mitochondria, Liver/metabolism
- Models, Genetic
- Molecular Sequence Data
- Nuclear Matrix/metabolism
- Protein Binding
- Protein Structure, Tertiary
- Protein Transport
- Subcellular Fractions
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fig4: ROP2hc translocation is not affected by treatments blocking import to the matrix. (A) Effect of treatments known to inhibit import to the matrix and the mAb T34A7, on ROP2hc, and OTC import. The standard import assay, and all treatments except for that of temperature (0°C) were conducted by incubating mitochondria with the appropriate 35S-Met–labeled substrate in an reticulocyte lysate on ice for 15 min, followed by import for 20 min at 30°C. The effect of temperature was determined by the 15-min pretreatment on ice followed by an additional 20 min at 0°C. ATP depletion was achieved by pretreatment with apyrase (5 U/ml) on ice for 15 min followed by import for 20 min at 30°C with apyrase present. Dissipation of the membrane potential (ΔΨ) was achieved by pretreatment on ice with CCCP (2 mM), followed by import for 20 min at 30°C in the presence of CCCP. The effect of the mAb T34A7 on import was determined by preincubating the reticulocyte lysate with the ascites at 1:20 dilution for 15 min on ice before addition to mitochondria and import at 30°C. Treatment with anti-TOM 20 antibody was performed by its addition to the import mixture (reticulocyte lysate with mitochondria) at 1:10 dilution for 15 min on ice followed by incubation at 30°C for 20 min. None of the treatments blocked the binding of ROP2hc to the mitochondrial pellet (ROP2hc, lanes 1, 5, 9, 13, 17, 21, and 25). Insertion and/or translocation across the MOM was determined by the generation of the 17-kD protease-protected fragment in the mitochondrial pellet in the presence of exogenous trypsin, the position of which is shown by the arrowhead. Neither incubation at 0°C (ROP2hc, P, lane 7, arrowhead), ATP depletion (ROP2hc, P, lane 11, arrowhead), or the dissipation of the ΔΨ (ROP2hc, P, lane 15, arrowhead), affected the generation of the protease protected fragment indicating ROP2hc does not use the matrix targeted pathway. In contrast, preincubation of the reticulocyte lysate with the mAb T34A7 (epitope included in aa 98–127 of ROP2hc) inhibited the generation of the ROP2hc protease-protected fragment (ROP2hc, P, lane 19, arrowhead) but failed to inhibit the import and processing of OTC (OTC, lane 19). Incubation in the presence of anti-TOM20 ablated the import and processing of OTC (OTC, lane 23) but failed to affect ROP2hc translocation (ROP2hc, lane 23). The “no treatment” control for the anti-TOM20 assay for ROP2hc is in lanes 21–24. As a control for all the treatments tested, OTC import and processing from the precursor to the mature (arrowhead) form was blocked by incubation at 0°C (OTC, lane 7), apyrase (OTC, lane 11), CCCP (OTC, lane 15), and anti-TOM20 (OTC, lane 23). For OTC, all the treatments (except for T34A7) revealed only the precursor form (p) when trypsin treatment was excluded (lanes 5 and 6, 9 and 10, 13 and 14, 21 and 22). In this experiment, the inhibition by apyrase was incomplete (OTC, lanes 13–16). (B) Pretreatment of mitochondria with trypsin fails to inhibit the translocation of ROP2hc across the MOM. The use of PK to assess ROP2hc translocation into mitochondria results in the generation of a slightly smaller (15-kD) protease protected fragment in the standard import assay (lane 4, arrowhead). The generation of this fragment is not affected by trypsin pretreatment of mitochondria indicating a trypsin-sensitive surface receptor is not involved in ROP2hc translocation (lane 8, arrowhead). Trypsin pretreatment completely abolished the import and processing of OTC as only the precursor form is observed in the absence of PK (lanes 15 and 16). Upon the addition of PK to nontrypsinized mitochondria, only the mature (m) form is detected in the pellet (lane 13). The band in the supernatant fraction represents contamination from the pellet as it is comprised entirely of the processed, mature (m) form (lane 14). The effect of trypsin pretreatment was examined by treating mitochondria with trypsin (1 mg/ml final concentration) for 15 min on ice, inactivation with SBTI as above, and reisolation by centrifugation. Pelleted mitochondria were resuspended in import buffer + SBTI to 20 mg/ml organelle protein and a standard import reaction performed. Import was assessed using protection from proteinase K as described in the experimental procedures.
In light of the potential mitochondrial matrix targeting signal (aa 98–127) in ROP2hc, we examined the effects of treatments known to block matrix import. We examined the consequences of temperature (0°C), ATP depletion (apyrase), dissipation of the membrane potential across the inner membrane (carboxyl cyanide m-chlorophenylhydrazone [CCCP]), and the requirement for trypsin- sensitive receptors on the mitochondrial surface such as TOM20 (reviewed in Neupert, 1997), on ROP2hc translocation. In addition, the ability of anti-TOM20 antibodies to block import was tested. In control experiments, the import and processing of the matrix-targeted protein OTC was blocked at 0°C (Fig. 4 A, OTC, lanes 5–8), and was significantly inhibited by treatment with apyrase (Fig. 4 A, OTC, lanes 9–12) or CCCP (Fig. 4 A, OTC, lanes 13–16). As predicted, the mAb T34A7 against aa 98–127 of ROP2hc, did not affect either the import or processing of OTC (Fig. 4 A, OTC, lanes 17–20). In contrast, pretreatment of mitochondria with a chicken anti-TOM20 antibody completely blocked both the import and processing of OTC (Fig. 4 A, OTC, lanes 21–24), as described previously (Goping et al., 1995).