Arabidopsis phosphatidylinositol 4‐phosphate 5‐kinase 2 contains a functional nuclear localization sequence and interacts with alpha‐importins

• Published in: The Plant journal : for cell and molecular biology 2017-12, Vol.92 (5), p.862-878
• Main Authors: Gerth, Katharina, Lin, Feng, Daamen, Franziska, Menzel, Wilhelm, Heinrich, Franziska, Heilmann, Mareike
• Format: Article
• Language: English
• Subjects: Active Transport, Cell Nucleus; alpha Karyopherins - metabolism; alpha‐importins; Amino acids; Arabidopsis; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Botany; Epidermis; Fluorescence; Immunofluorescence; Imports; Isoforms; Localization; Membrane trafficking; Meristem - metabolism; Nuclear Localization Signals - genetics; Nuclear Localization Signals - metabolism; Nuclear transport; Nuclei (cytology); nucleus; Phosphates; Phosphatidylinositol 4-phosphate; Phosphatidylinositol 4-phosphate 5-kinase; Phosphoinositides; Phosphotransferases (Alcohol Group Acceptor) - genetics; Phosphotransferases (Alcohol Group Acceptor) - metabolism; PI4P 5‐kinases; Plant growth; Plant Roots - metabolism; Plants (botany); Ribulose-bisphosphate carboxylase; subcellular localization; Ubiquitin; Yeast
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/tpj.13724

Summary The Arabidopsis phosphoinositide kinase PIP5K2 has been implicated in the control of membrane trafficking and is important for development and growth. In addition to cytosolic functions of phosphoinositides, a nuclear phosphoinositide system has been proposed, but evidence for nuclear phosphoinositides in plants is limited. Fluorescence‐tagged variants of PIP5K2 reside in the nucleus of Arabidopsis root meristem cells, in addition to reported plasma membrane localization. Here we report on the interaction of PIP5K2 with alpha‐importins and characterize its nuclear localization sequences (NLSs). The PIP5K2 sequence contains four putative NLSs (NLSa–NLSd) and only a PIP5K2 fragment containing NLSs is imported into nuclei of onion epidermis cells upon transient expression. PIP5K2 interacts physically with alpha‐importin isoforms in cytosolic split‐ubiquitin‐based yeast two‐hybrid tests, in dot‐blot experiments and in immuno‐pull‐downs. A 27‐amino‐acid fragment of PIP5K2 containing NLSc is necessary and sufficient to mediate the nuclear import of a large cargo fusion consisting of two mCherry markers fused to RubisCO large subunit. Substitution of basic residues in NLSc results in reduced import of PIP5K2 or other cargoes into plant nuclei. The data suggest that PIP5K2 is subject to active, alpha‐importin‐mediated nuclear import, consistent with a nuclear role for PIP5K2 in addition to its reported cytosolic functions. The detection of both substrate and product of PIP5K2 in plant nuclei according to reporter fluorescence and immunofluorescence further supports the notion of a nuclear phosphoinositide system in plants. Variants of PIP5K2 with reduced nuclear residence might serve as tools for the future functional study of plant nuclear phosphoinositides. Significance Statement The data presented in this study indicate biologically relevant nuclear import of PIP5K2 according to several independent criteria, and serve as a base for functional studies of nuclear phosphoinositides in plants. Our findings might be relevant for the interpretation of future and previously reported mutant phenotypes and localization data that have so far not been linked to potential nuclear roles of phosphoinositides.