Analysis of an autoproteolytic activity of rice yellow mottle virus silencing suppressor P1
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Isabel Weinheimer
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 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.
©2010 by Walter de Gruyter Berlin New York
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- Highlight: Signal Transduction and Disease
- HIGHLIGHT: SIGNAL TRANSDUCTION AND DISEASE
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- Ubiquitin ligase complexes: from substrate selectivity to conjugational specificity
- MAP3K1 functionally interacts with Axin1 in the canonical Wnt signalling pathway
- Signal transduction by the atopy-associated human thymic stromal lymphopoietin (TSLP) receptor depends on Janus kinase function
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- Peptides from the Mycobacterium tuberculosis Rv1980c protein involved in human cell infection: insights into new synthetic subunit vaccine candidates
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