Abstract
The edible mushroom Agrocybe aegerita produces a ribotoxin-like protein known as Ageritin. In this work, the gene encoding Ageritin was characterized by sequence analysis. It contains several typical features of fungal genes such as three short introns (60, 55 and 69 bp) located at the 5′ region of the coding sequence and typical splice junctions. This sequence codes for a precursor of 156 amino acids (~17-kDa) containing an additional N-terminal peptide of 21 amino acid residues, absent in the purified toxin (135 amino acid residues; ~15-kDa). The presence of 17-kDa and 15-kDa forms was investigated by Western blot in specific parts of fruiting body and in mycelia of A. aegerita. Data show that the 15-kDa Ageritin is the only form retrieved in the fruiting body and the principal form in mycelium. The immunolocalization by confocal laser scanning microscopy and transmission electron microscopy proves that Ageritin has vacuolar localization in hyphae. Coupling these data with a bioinformatics approach, we suggest that the N-terminal peptide of Ageritin (not found in the purified toxin) is a new signal peptide in fungi involved in intracellular routing from endoplasmic reticulum to vacuole, necessary for self-defense of A. aegerita ribosomes from Ageritin toxicity.
Highlights
Ageritin is a monomeric protein isolated from Agrocybe aegerita fruiting bodies that inhibits protein synthesis in vitro [1]
In the Agrocybe aegerita AAE-3 gene database collecting all the identified coding sequences derived from the analysis of the A. aegerita AAE-3 genomic sequence [17], the sequence encoding for Ageritin protein is identified with the gene ID: AAE3_01767 [13], Figure S1
The results shows that the apparent 17-kDa cross-reactive protein band in mycelium is not Ageritin with the additional peptide of 21 amino acid residues at N-terminal region (Figure S2)
Summary
Ageritin is a monomeric protein isolated from Agrocybe aegerita fruiting bodies that inhibits protein synthesis in vitro [1] This enzyme catalyzes the cleavage of a specific phosphodiester bond of larger rRNA. The specific hydrolysis due to its ribonuclease action occurs in the Sarcin Ricin Loop (SRL), which is necessary for the binding of EF-G or EF-2 elongation factors on the ribosome in prokaryotes and eukaryotes, respectively [2]. This rRNA endonuclease enzymatic activity releases a 460 nt-fragment (α-fragment) at 3 end of the 28S RNA [3]. Ageritin represents the first member of novel specific ribonuclease family retrieved in basidiomycetes for which our group proposed the name of ribotoxin-like proteins [1,6,7]
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