Analysis of an autoproteolytic activity of rice yellow mottle virus silencing suppressor P1

Author:

Weinheimer Isabel1,Boonrod Kajohn1,Moser Mirko1,Zwiebel Michèle1,Füllgrabe Marc1,Krczal Gabi1,Wassenegger Michael1

Affiliation:

1. RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, Breitenweg 71, D-67435 Neustadt, Germany

Abstract

AbstractEctopically expressedrice yellow mottle virusP1 fusion proteins were found to be cleavedin plantaand inEscherichia 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) inNicotiana benthamianashowed 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 methionine159(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.

Publisher

Walter de Gruyter GmbH

Subject

Clinical Biochemistry,Molecular Biology,Biochemistry

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