Premium
Arabidopsis phosphatidylinositol 4‐phosphate 5‐kinase 2 contains a functional nuclear localization sequence and interacts with alpha‐importins
Author(s) -
Gerth Katharina,
Lin Feng,
Daamen Franziska,
Menzel Wilhelm,
Heinrich Franziska,
Heilmann Mareike
Publication year - 2017
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13724
Subject(s) - biology , importin , nuclear localization sequence , nuclear transport , arabidopsis , microbiology and biotechnology , nuclear pore , bimolecular fluorescence complementation , phosphatidylinositol 4,5 bisphosphate , nucleoporin , inner membrane , biochemistry , cell nucleus , phosphatidylinositol , kinase , cytoplasm , mutant , gene , mitochondrion
Summary The Arabidopsis phosphoinositide kinase PIP 5K2 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 PIP 5K2 reside in the nucleus of Arabidopsis root meristem cells, in addition to reported plasma membrane localization. Here we report on the interaction of PIP 5K2 with alpha‐importins and characterize its nuclear localization sequences ( NLS s). The PIP 5K2 sequence contains four putative NLS s ( NLS a– NLS d) and only a PIP 5K2 fragment containing NLS s is imported into nuclei of onion epidermis cells upon transient expression. PIP 5K2 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 PIP 5K2 containing NLS c is necessary and sufficient to mediate the nuclear import of a large cargo fusion consisting of two mC herry markers fused to Rubis CO large subunit. Substitution of basic residues in NLS c results in reduced import of PIP 5K2 or other cargoes into plant nuclei. The data suggest that PIP 5K2 is subject to active, alpha‐importin‐mediated nuclear import, consistent with a nuclear role for PIP 5K2 in addition to its reported cytosolic functions. The detection of both substrate and product of PIP 5K2 in plant nuclei according to reporter fluorescence and immunofluorescence further supports the notion of a nuclear phosphoinositide system in plants. Variants of PIP 5K2 with reduced nuclear residence might serve as tools for the future functional study of plant nuclear phosphoinositides.