title: Analysis of an autoproteolytic activity of rice yellow mottle virus silencing suppressor P1.
abstract: Ectopically expressed rice yellow mottle virus P1 fusion proteins were found to be cleaved in planta and in Escherichia coli. Cleavage takes place in the absence of bacterial protease activity, indicating that the P1 fusion is autocatalytically processed independently of host factors. N-terminal sequencing of the C-terminal cleavage product of transiently expressed P1/GFP (green fluorescence protein) in Nicotiana benthamiana showed that the cleavage site is located between the first two amino acids (aa) downstream of the P1 sequence. Mutagenesis experiments revealed that a phenylalanine to valine substitution at position 157 of the P1 aa sequence impairs proper cleavage, which is nearly unaffected by replacement of phenylalanine with tyrosine. Deletion of methionine(159) (first GFP aa residue) appeared to not affect P1/GFP cleavage. N-terminal P1-tagging with GFP turned out to impair autocleavage, whereas a small His-tag could not fully prevent cleavage. Additionally, a modified P1/GFP carrying an N-terminal deletion of 81 aa was not cleaved. These findings indicate that this region is involved in the proteolysis mechanism and that large N-terminal fusion partners might affect correct folding of the P1 necessary for self-catalysis.
authors.affiliation: RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany.
authors.forename: Isabel
authors.initials: I
authors.lastname: Weinheimer
authors.affiliation: RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany.
authors.forename: Kajohn
authors.initials: K
authors.lastname: Boonrod
authors.affiliation: RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany.
authors.forename: Mirko
authors.initials: M
authors.lastname: Moser
authors.affiliation: RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany.
authors.forename: Michèle
authors.initials: M
authors.lastname: Zwiebel
authors.affiliation: RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Füllgrabe
authors.affiliation: RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany.
authors.forename: Gabi
authors.initials: G
authors.lastname: Krczal
authors.affiliation: RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Wassenegger
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D010942:Plant Viruses / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins