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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fig3: Interaction of ROP2hc and its derivatives with mitochondria. (A) The NH2 terminus of ROP2hc contains a putative mitochondrial matrix targeting signal, as described in the text. The constructs ROP2hc80 and Δ98–127ROP2hc extend from aa 80-465 and aa 128–465, respectively, but are identical to ROP2hc in all other respects. All constructs for the expression of ROP2hc and its derivatives possess an engineered initiator methionine (M). The predicted amphipathic helix is underlined and hydroxylated residues marked with an asterisk. (B) Translocation of ROP2hc into mitochondria in vitro. Reticulocyte lysate containing 35S-Met–labeled ROP2hc was incubated with freshly isolated murine liver mitochondria 20 min at 30°C. The reaction was divided into three equal parts that received either no trypsin (−), trypsin (+), or trypsin (+) in addition to 0.1% Triton X-100 (Tx) on ice for 15 min. Trypsin treatment was stopped by the addition of SBTI, and the reactions diluted to 50 μl with IB containing SBTI. The mitochondrial pellet (P) and supernatant (S) fractions were obtained after centrifugation from all but the Triton X-100–solubilized fraction (Tx). ROP2hc binds to the mitochondrial pellet (lane 2). After the addition of exogenous trypsin, a specific trypsin-protected fragment (lane 4, arrowhead) is observed in the pellet fraction. The presence of bands in the 25–30-kD range in the trypsin-treated pellet (lane 4), supernatant (lane 5) and the reaction containing both trypsin and TX-100 (lane 6) indicates their protease resistance is not due to translocation across the MOM. The sequence of ROP2hc protein predicts 50 potential tryptic sites with the longest predicted fragment from complete digestion being 4-kD long (unpublished data). The mAb T34A7 recognizes an epitope included in aa 98–127, at the extreme NH2 terminus of ROP2hc (see text), and immunoprecipitates both full-length ROP2hc (lane 7) and the 17-kD trypsin-protected fragment from solubilized pellet fractions after import into mitochondria (lane 8, arrowhead). (C) Contribution of the NH2 terminus of ROP2hc in translocation into mitochondria in vitro. 35S-Met–labeled import substrates generated in vitro were incubated with import competent murine mitochondria as in B. The import of ROP2hc (aa 98–465), ROP2hc80 (aa 80–465), and Δ98–127ROP2hc was examined, in addition to the human OTC as a positive control. All the ROP2hc derivatives, including Δ98–127ROP2hc, bound to the mitochondrial pellet (lanes 1, 5, and 9). Trypsin treatment of the import reaction resulted (B), in the generation of the 17-kD protease-protected fragment in the ROP2hc pellet (lane 3, arrowhead). Protease treatment of the ROP2hc80 import reaction revealed a 19-kD protease-protected fragment associated with the mitochondrial pellet after trypsin treatment (lane 7, arrowhead). Deletion of aa 98–127 does not affect binding to mitochondria (lane 9), but does abolish the generation of a protease-protected fragment (lane 11). This indicates that insertion into and/or translocation across the MOM is dependent on the NH2-terminal 30 aa, although other determinants in the protein can mediate binding. The import competence of mitochondria was confirmed in all experiments using OTC as a positive control. OTC was efficiently converted from the presequence containing precursor (p) to the mature processed protein (m) as a consequence of translocation to the matrix. Only the processed, mature form of the protein (m), is protected from exogenous protease (lane 15).
Examination of the ROP2 protein sequence (Beckers et al., 1994) immediately downstream of aa 98 revealed features reminiscent of a mitochondrial matrix targeting signal (Fig. 3 A); these include a predicted positively charged amphipathic helix (Fig. 3 A, underlined), a high concentration of hydroxylated residues (Fig. 3 A, asterisk), and a relative lack of negatively charged aa (von Heijne, 1990; Neupert, 1997).