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
© Copyright Policy
fig8: Interaction of ROP2hc and its derivatives with murine liver mitochondria and ER. (A) Mitochondria and ER-enriched microsomes were prepared as described in Materials and methods. After the loading of 100 μg total protein, the mitochondrial proteins COXI and COXIII were detected exclusively in the mitochondrial but not on the ER preparation. The ER was detected using anti-KDEL and anti-calnexin antibodies. Both antibodies illuminated their targets in the ER prep. Although no KDEL signal was visible in the mitochondrial prep, a trace calnexin signal was apparent. This is likely due to the mitochondrion-associated ER or MAM fraction, which is highly enriched in liver (Vance, 1990). (B) The binding of ROP2hc, Δ98–127ROP2hcGFP, aa 98–127GFP, and GFP to mitochondria and ER was determined without further treatment and after extraction with 0.1 M Na2CO3, pH11.5 (Carbonate +), as indicated. Equivalent volumes of the organelle pellets (P) and supernatants (S) were resolved by SDS-PAGE and exposed by fluorography. Both ROP2hc and its derivatives bind efficiently to both mitochondria and ER. ROP2hc binding to mitochondria is resistant to extraction with carbonate (top panel). In contrast, binding to ER is not as strong and exhibits both extractable- and carbonate-resistant binding (top panel). Deletion of the proposed 30-aa NH2-terminal mitochondrial targeting signal (Δ98–127ROP2hcGFP) does not significantly affect binding to either mitochondria or ER (second panel). Furthermore, Δ98–127ROP2hcGFP is partially extracted from mitochondria by carbonate treatment, but is completely resistant to extraction from ER (second panel). The 30-aa NH2-terminal signal promotes the binding of the passenger protein GFP to mitochondria but not ER (third panel). Carbonate treatment promotes the fractionation of aa 98–127GFP into the pellet fraction with both organelles (third panel). This suggests the protein may be precipitated due to its basic nature by the carbonate treatment. Finally, the soluble passenger protein GFP exhibits no binding to either mitochondria or ER under any conditions (bottom panel). (C) Based on sucrose floatation gradients, the apparent membrane association of ROP2hc to mitochondria and ER is not due to neither the precipitation or aggregation of the protein. ROP2hc bound to organelle membranes in the absence (NT, no treatment) and after carbonate extraction (Carbo) was incorporated into 55% sucrose and a continuous sucrose gradient (55–20%) established as described in the Materials and methods. In the absence of carbonate extraction (Mito-NT, ER-NT), the majority of the signal in all cases floated into the gradient, coinciding with the distribution of the organelles based on Coomassie blue staining of the gels (unpublished data) indicating true membrane association. After carbonate extraction (Mito-Carbo, ER-Carbo) ROP2hc continues to float into the gradient, indicating its association with organelle membranes in maintained and resistant to extraction with carbonate. Had the protein been extracted or merely precipitated, a significant signal would be observed in fractions 1 and 2 (load fractions). This indicates ROP2hc interacts with both mitochondria and ER with high affinity behaving like an integral membrane protein. (D) Neither ROP2hc nor Δ98–127ROP2hc are cotranslationally imported into canine microsomes. Cotranslational import of ROP2hc, Δ98–127ROP2hc, and E. coli BLA was performed as described in the Materials and methods. Although both ROP2hc (lane 1) and Δ98–127ROP2hc (lane 4) were synthesized, they were not translocated either partially or completely into the ER lumen and remain protease sensitive (PK, lanes 2 and 5). In contrast, BLA was efficiently imported into the microsomes and processed from the precursor (p, lane 7) to the mature form (m, lanes 7 and 8). Only the mature form is protease protected (PK, lane 8) and the protection is sensitive to detergent treatment (PK, Tx, lane 9), indicating the microsomes are import competent.
Next, we compared the binding of the ROP2 derivatives tested in vivo, to purified organelles in vitro. Mitochondria and ER-enriched microsomes were prepared from murine livers (see Materials and methods). By immunoblot, the mitochondrial proteins COXI and COXIII were detected exclusively in the mitochondrial preparations (Fig. 8 A). Both anti-KDEL and anticalnexin antibodies strongly recognized the ER preparation (Fig. 8 A). Although no KDEL signal was visible in the mitochondrial preparation, a trace calnexin signal was apparent (Fig. 8 A), likely due to the mitochondrion-associated ER or mitochondrion-associated membrane (MAM) fraction, which is highly enriched in liver (Vance, 1990).