Mechanical stress-induced subcellular re-localization of N-terminally truncated tobacco Nt-4/1 protein

The Nicotiana tabacum 4/1 protein (Nt-4/1) of unknown function expressed in plant vasculature has been shown to localize to cytoplasmic bodies associated with endoplasmic reticulum. Here, we analyzed molecular interactions of an Nt-4/1 mutant with a deletion of 90 N-terminal amino acid residues (Nt-4/1d90) having a diffuse GFP-like localization. Upon transient co-expression with VAP27, a membrane protein known to localize to the ER, ER-plasma membrane contact sites and plasmodesmata, Nt-4/1d90 was concentrated around the cortical ER tubules, forming a network matching the shape of the cortical ER. Additionally, in response to mechanical stress, Nt-4/1d90 was re-localized to small spherical bodies, whereas the subcellular localization of VAP27 remained essentially unaffected. The Nt-4/1d90-containing bodies associated with microtubules, which underwent noticeable bundling under the conditions of mechanical stress. The Nt-4/1d90 re-localization to spherical bodies could also be induced by incubation at an elevated temperature, although under heat shock conditions the re-localization was less efficient and incomplete. An Nt-4/1d90 mutant, which had phosphorylation-mimicking mutations in a predicted cluster of four potentially phosphorylated residues, was found to both inefficiently re-localize to spherical bodies and tend to revert back to the initial diffuse localization. The presented data show that Nt-4/1 has a potential for response to stresses that is manifested by its deletion mutant Nt-4/1d90, and this response can be mediated by protein dephosphorylation.