Subcellular localization of host and viral proteins associated with tobamovirus RNA replication - PubMed (original) (raw)

Subcellular localization of host and viral proteins associated with tobamovirus RNA replication

Yuka Hagiwara et al. EMBO J. 2003.

Abstract

Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identified to be necessary for efficient intracellular multiplication of tobamoviruses. AtTOM1 interacts with the helicase domain polypeptide of tobamovirus-encoded replication proteins and with AtTOM2A, suggesting that both AtTOM1 and AtTOM2A are integral components of the tobamovirus replication complex. We show here that AtTOM1 and AtTOM2A proteins tagged with green fluorescent protein (GFP) are targeted to the vacuolar membrane (tonoplast)-like structures in plant cells. In subcellular fractionation analyses, GFP-AtTOM2A, AtTOM2A and its tobacco homolog NtTOM2A were predominantly fractionated to low-density tonoplast-rich fractions, whereas AtTOM1-GFP, AtTOM1 and its tobacco homolog NtTOM1 were distributed mainly into the tonoplast-rich fractions and partially into higher-buoyant-density fractions containing membranes from several other organelles. The tobamovirus-encoded replication proteins were co-fractionated with both NtTOM1 and viral RNA-dependent RNA polymerase activity. The replication proteins were also found in the fractions containing non-membrane-bound proteins, but neither NtTOM1 nor the polymerase activity was detected there. These observations suggest that the formation of tobamoviral RNA replication complex occurs on TOM1-containing membranes and is facilitated by TOM2A.

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Figures

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Fig. 1. Confocal laser scanning micrographs of A.thaliana epidermal cells transiently expressing (A) GFP, (B) AtTOM1–GFP and (C) GFP–AtTOM2A. A single optical section of the cell is shown on the left, and a projection of 10 × 1 µm confocal optical sections through the cell is shown on the right. Note that the leaf epidermal cells have a ‘jigsaw-puzzle-like’ appearance and possess huge vacuoles that occupy most of the intracellular space. Scale bars, 25 µm.

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Fig. 2. Confocal laser scanning micrographs of transgenic BY-2 cells expressing (A) AtTOM1–GFP, (B) GFP–AtTOM2A, (C) GFP–VM23, (D) GmMan1–GFP, (E) GFP–PAQ1 and (F) mGFP5ER. A single optical section through several cells is displayed on the lower left, and its Nomarski image is shown on the upper left. A high-magnification image is shown on the right. Scale bars, 10 µm.

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Fig. 3. Confocal laser scanning micrographs of FM4-64-treated transgenic BY-2 cells expressing (A–D) AtTOM1–GFP, (E–H) GFP–AtTOM2A and (I–L) GFP–VM23. Cells are shown at ∼20 min (A, E, I) and 10 h (B–D, F–H, J–L) after labeling. The GFP and FM4-64 signals are shown as green and red, respectively. In the merged images (A, D, E, H, I, L), the overlapping signals of GFP and FM4-64 appear as yellow. Scale bars, 10 µm.

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Fig. 4. Equilibrium iodixanol density-gradient analysis of the TOM proteins in BY-2 cells. Lysates from non-transgenic BY-2 cells (B, D, E and G) and transgenic BY-2 cells expressing (A) AtTOM1–GFP, (C) GFP–AtTOM2A, (F) GFP–VM23, (H) mGFP5ER, (I) GmMan1– GFP and (J) GFP–PAQ1 were subjected to density gradient centrifugation. Fractions (1–18, from top to bottom of a gradient) were analyzed by SDS–PAGE and immunoblotting. An anti-GFP antibody was used to detect the GFP-fused proteins. NtTOM1 (B), NtTOM2A (D), V-PPase (E) and Sec61p (G) were detected with the corresponding antibodies. The top (fraction 1), middle (fractions 9–11) and sample-loaded (fractions 16–18) parts are referred to as Fraction T, Fraction M and Fraction L, respectively, as shown below the panels. The positions of molecular mass markers (kDa) are indicated on the right.

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Fig. 5. Equilibrium iodixanol density-gradient analysis of the 130K/180K proteins and the viral RdRp activity of TMV-L-inoculated BY-2 protoplasts. Lysates from mock- or TMV-L-inoculated protoplasts harvested at 6 h.p.i. were subjected to density-gradient centrifugation. Fractions were collected and analyzed by SDS–PAGE and immunoblotting. (A) Immunoblot analysis of Sec61p, V-PPase and the TOM proteins in mock-inoculated protoplasts. (B and C) Immunoblot analysis of Sec61p, V-PPase, TOM proteins, and the 180K and 130K replication proteins in TMV-L-inoculated protoplasts (see text for definition of ‘Infect 1’ and ‘Infect 2’). The RF RNA synthesized in the RdRp assay of each fraction is shown in the bottom panel of (B) and (C). The band intensity for NtTOM1, the 130K protein and the RF RNA was quantitated and represented graphically.

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