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ИСТИНА ФИЦ ПХФ и МХ РАН |
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Phytaspases belong to the plant subtilase family and are distinguished by their strict Asp cleavage specificity and by their unusual localization dynamics. In particular, upon cell death induction these initially apoplastic proteases are re-imported into the cell by means of clathrin-mediated endocytosis. To elucidate the underlying molecular mechanisms, we strived to identify the protein interactors of Nicotiana benthamiana phytaspase. Two independent approaches were utilized: in vivo cross-linking using a bifunctional reagent, and a proximity-dependent biotin-based identification (BioID). With the first one, a Tubby-like protein was identified as a phytaspase interactor, while mainly the soluble residents of the endoplasmic reticulum were identified using the BioID, calreticulin-3 in particular. Direct interaction of these proteins with phytaspase was confirmed in in vitro binding assays using purified proteins. Furthermore, we showed that calreticulin-3 is a phytaspase substrate, while the Tubby-like protein is not. Interestingly, down regulation of either the calreticulin-3 or Tubby-like protein mRNAs using the VIGS approach precluded stress-induced retrograde transport of phytaspase. Implications of our findings for the functioning of phytaspase and its interactors will be discussed. This work was supported by the Russian Science Foundation grants # 19-14-00010 and 22-14-00071.