Retrograde Transport of Tobacco Phytaspase Is Mediated by Its Partner, Tubby-like F-Box Protein 8

Phytaspases, plant cell death-promoting and proprotein-processing proteolytic enzymes of the plant subtilase family, display aspartate (caspase-like) cleavage specificity and a very unusual retrograde trafficking from the apoplast to the cell interior upon induction of death-inducing stresses. To determine the underlying molecular mechanisms, we performed a search for tobacco phytaspase (<i>Nt</i>Phyt) interactors using an in vivo cross-linking approach in <i>Nicotiana tabacum</i> plants. Tobacco Tubby-like F-box protein 8 (named Tubic hereafter) was identified as an <i>Nt</i>Phyt interactor, with formation of the cross-linked complex being only efficient under the oxidative stress conditions. Direct interaction of the two proteins was further corroborated in the in vitro experiments. Analysis of Tubic-EGFP behavior in plant cells revealed that Tubic is a membrane-associated and fairly unstable protein. Furthermore, we showed that <i>Nt</i>Phyt and Tubic are capable of negatively affecting one another in plant cells. On the other hand, down-regulation of Tubic in Tubic-silenced plants impaired specifically the retrograde transport of <i>Nt</i>Phyt upon the induction of oxidative stress, testifying to a critical role of Tubic in this process. Our study, thus, contributes to understanding of the mechanisms of <i>Nt</i>Phyt retrograde trafficking in plant cells subjected to stress.