Phosphorylation and/or presence of serine 37 in the movement protein of tomato mosaic tobamovirus is essential for intracellular localization and stability in vivo

Abstract

The P30 movement protein (MP) of tomato mosaic tobamovirus (ToMV) is synthesized in the early stages of infection and is phosphorylated in vivo. Here, we determined that serine 37 and serine 238 in the ToMV MP are sites of phosphorylation. MP mutants in which serine was replaced by alanine at positions 37 and 238 (LQ37A238A) or at position 37 only (LQ37A) were not phosphorylated, and mutant viruses did not infect tobacco or tomato plants. By contrast, mutation of serine 238 to alanine did not affect the infectivity of the virus (LQ238A). To investigate the subcellular localization of mutant MPs, we constructed viruses that expressed each mutant MP fused with the green fluorescent protein (GFP) of Aequorea victoria. Wild-type and mutant LQ238A MP fusion proteins showed distinct temporally regulated patterns of MP-GFP localization in protoplasts and formation of fluorescent ring-shaped infection sites on Nicotiana benthamiana. However mutant virus LQ37A MP-GFP did not show a distinct pattern of localization or formation of fluorescent rings. Pulse-chase experiments revealed that MP produced by mutant virus LQ37A was less stable than wild-type and LQ238A MPs. MP which contained threonine at position 37 was phosphorylated, but the stability of the MP in vivo was very low. These studies suggest that the presence of serine at position 37 or phosphorylation of serine 37 is essential for intracellular localization and stability of the MP, which is necessary for the protein to function.

FIG. 1

Genome organization of ToMV. The positions of primers used for site-directed mutagenesis are indicated in relation to the ToMV genome and the tryptic peptide map of the MP. (A) Genome organization of ToMV and positions of primers used to construct MP-GFP fusion proteins and to construct mutants with mutations of the MP gene. (B) Schematic representation of the MP gene and positions of primers for constructing mutants with mutations of the MP gene. (C) Predicted tryptic peptides of wild-type ToMV MP. Amino acid sequences are depicted by the one-letter code. Possible tryptic peptides are represented by gaps and underlines following lysine and arginine residues; peptides are numbered 1 to 39 (italicized) in order from the N to C terminus (below the amino acid sequences). Peptides including serine residues (bold and underlined) are presented in large type; those not including serine residues are presented in smaller type. Some of the amino acid differences observed between Ls1, Ltb1, and 2^a (indicated in parentheses) are indicated by arrowheads, with substituted amino acids shown below the arrowheads. A possible partially digested peptide which corresponds to peptides 38 plus 39 is also shown in the bottom row. Bold numbers shown above the amino acid residues indicate the residues into which we introduced substitutions to alanine. Regions I and II are postulated by Saito et al. (39).

FIG. 2

Phosphopeptide analysis of in vivo-phosphorylated MP. In vivo ³²P-labeled MP was immunoprecipitated with anti-MP antisera, gel purified, and subjected to hydrolysis with 6 N HCl. Hydrolyzed products were spotted with phosphoserine, phosphothreonine, and phosphotyrosine and electrophoresed. Following electrophoresis, ninhydrin reactions were used to locate the positions of standard phosphoamino acids (right), after which the plate was exposed to an imaging plate (left).

FIG. 3

2-D analysis of tryptic peptides of in vivo-phosphorylated MP. In vivo ³²P-labeled MP was immunoprecipitated with anti MP antisera, gel purified, and subjected to trypsin digestion. The trypsinized peptides were lyophilized and spotted onto TLC plates. Electrophoresis was carried out in the first dimension (horizontal) and was followed by chromatography in the second dimension (vertical) (4). (Left) Phosphopeptide map of MP of wild-type ToMV (L); (middle) map of ToMV 2^a; (right) a mixed sample of L and 2^a phosphopeptides. Arrowheads indicated phosphopeptide spots; arrowheads of wild-type ToMV are labeled 1, 2, and 3; arrowhead of ToMV 2^a is labeled 4. The right panel indicates that one phosphopeptide spot (arrowhead 2) of the wild-type MP has the same mobility as the sole spot (arrowhead 4) of ToMV 2^a protein.

FIG. 4

In vivo phosphorylation of alanine mutants. Transcripts of wild-type construct (W3), LQ238A, LQ18A238A, LQ37A238A, LQ75A238A, and LQ89A238A were inoculated into BY-2 protoplasts. Each MP was immunoprecipitated with anti-MP antisera and subjected to SDS-PAGE (10% polyacrylamide). Lane Mock contains immunoprecipitate from uninfected protoplasts. Radioactive bands were detected by a Fuji Image Analyzer. The bottom panel is a result of Western analysis of MP accumulation in the respective protoplasts with anti-MP antibody as described previously (44).

FIG. 5

In vivo phosphorylation of mutant MPs. (Top) In vivo phosphorylation analysis in which wild-type (W3), LQ238A, LQ37A, LQ37A238A, LQ37T, and LQ37T238A transcripts were inoculated into BY-2 protoplasts and checked for susceptibility to phosphorylation as in Fig. 4. Lane Mock contains immunoprecipitate from uninfected protoplasts. (Bottom) Protoplasts inoculated with W3 and mutants were labeled with [³⁵S]methionine-[³⁵S]cysteine at 8 h p.i. for 10 min, and ³⁵S-labeled MP bands were detected by a Fuji Image Analyzer.

FIG. 6

Visualization of MP-GFP fusion proteins. Wild-type, LQ238A, LQ37A, LQ37T, and LQ37E MPs fused with GFP were expressed from ToMV constructs. Fluorescence microscopy observations were done from 9 to 24 h p.i. in protoplasts. Bars, 10 μm. The protein localizes to bodies associated with cortical ER and to microtubules (MT) in protoplasts.

FIG. 7

Stability of wild-type and mutant MPs analyzed by pulse-chase experiments. Wild-type (Wt) and mutant transcripts were inoculated into BY-2 protoplasts that were cultured in the presence of actinomycin D for 8 h after inoculation. [³⁵S]methionine-[³⁵S]cysteine protein-labeling mixture was added to the culture medium for 10 min. The protoplasts were cultured and sequentially harvested immediately or 1, 4, 16, or 28 h after labeling. Proteins were extracted from protoplasts and subjected to electrophoresis in 10% polyacrylamide gels containing SDS. ³⁵S-labeled MP bands were detected and quantitated by a Fuji Image Analyzer. The intensities of bands were normalized relative to label in MP prior to the chase (set at 100%). This experiment was performed three times, and the data given are means and standard deviations.

FIG. 8

Visualization of fluorescent rings produced by MP-GFP fusion viruses. Wild-type, LQ37A, and LQ37T MP-GFP fusion proteins were expressed from ToMV constructs. Fluorescence microscopy observations were made at 3 days p.i. in plant line H₃Nb-3, a transgenic MP(+) line derived from N. benthamiana. The widths of rings are indicated by opposing arrows. Exposure times of micrographs of the ring produced by wild-type, LQ37A, and LQ37T MP-GFP fusion virus were 20, 272, and 32 s, respectively. Bars, 0.5 cm (top) and 0.2 cm (bottom).