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Multiple serine phosphorylation sites on the 30 kDa TMV cell-to-cell movement protein synthesized in tobacco
A Haley1, T Hunter, P Kiberstis
1MRC Laboratory of Molecular Biology, Cambridge, UK.
Abstract:
p30, the protein required for cell-to-cell movement of tobacco mosaic virus (TMV), has a slightly reduced mobility on SDS-polyacrylamide gels when isolated by immunoprecipitation from TMV-infected protoplasts compared with that of p30 translated from viral RNA in vitro. Further investigation established a probable cause for the difference in mobility between the two: protoplasts incorporate [32P]orthophosphate into p30 at multiple sites, predominantly as phosphoserine. Tryptic peptide mapping reveals at least five internal phosphopeptides in p30, besides the C-terminal tryptic phosphopeptide already reported, involving at least two distinct domains of the protein (at residues 61-114 and residues 212-231), which may be substrates for different protein kinases. These structural results are consistent with a three-domain model for the TMV movement protein with two regulatory domains similar to that recently proposed on genetic grounds for dianthovirus movement proteins.
Insights
Tobacco mosaic virus (TMV) movement protein p30 is phosphorylated in infected plant cells, affecting its mobility. This post-translational modification involves multiple sites and suggests regulatory domains within the TMV protein.
Area of Science:
- Plant virology
- Molecular biology
- Protein biochemistry
Background:
- The p30 protein is essential for cell-to-cell movement of Tobacco Mosaic Virus (TMV) within plants.
- Understanding p30's structure and modifications is key to deciphering viral spread mechanisms.
Purpose of the Study:
- To investigate the cause of differential mobility of TMV p30 protein on SDS-PAGE.
- To characterize the post-translational modifications of p30 in infected plant cells.
Main Methods:
- Immunoprecipitation of p30 from TMV-infected protoplasts.
- In vitro translation of p30 from viral RNA.
- Analysis of protein mobility on SDS-polyacrylamide gels.
- Tryptic peptide mapping and phosphopeptide analysis using [32P]orthophosphate.
Main Results:
- TMV p30 isolated from infected protoplasts showed reduced mobility compared to in vitro translated p30.
- Protoplasts incorporate [32P]orthophosphate into p30 at multiple sites, primarily as phosphoserine.
- Tryptic peptide mapping identified at least five internal phosphopeptides in p30, located in distinct domains (residues 61-114 and 212-231).
Conclusions:
- Phosphorylation is a significant post-translational modification of the TMV p30 movement protein in vivo.
- These modifications suggest the presence of regulatory domains within p30, potentially targeted by different protein kinases.
- The findings support a three-domain model for TMV movement protein, with similarities to dianthovirus movement proteins.