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

The P30 movement protein (MP) of tomato mosaic tobamovirus (ToMV) is synthe sized 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 ph osphorylation. MP mutants in which serine was replaced by alanine at positi ons 37 and 238 (LQ37A238A) or at position 37 only (LQ37A) were not phosphor ylated, and mutant viruses did not infect tobacco or tomato plants. By cont rast, mutation of serine 238 to alanine did not affect the infectivity of t he virus (LQ238A). To investigate the subcellular localization of mutant MP s, we constructed viruses that expressed each mutant MP fused with the gree n fluorescent protein (GFP) of Aequorea victoria. Wild-type and mutant LQ23 8A MP fusion proteins showed distinct temporally regulated patterns of MP-G FP localization in protoplasts and formation of fluorescent ring-shaped inf ection sites on Nicotiana benthamiana. However mutant virus LQ37A MP-GFP di d not show a distinct pattern of localization or formation of fluorescent r ings. Pulse-chase experiments revealed that MP produced by mutant virus LQ3 7A was less stable than wild-type and LQ238A MPs. MP which contained threon ine 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 localiza tion and stability of the MP, which is necessary for the protein to functio n.