MORN motifs in plant PIPKs are involved in the regulation of subcellular localization and phospholipid binding

Abstract

Multiple repeats of membrane occupation and recognition nexus (MORN) motifs were detected in plant phosphatidylinositl monophosphate kinase (PIPK), a key enzyme in PI-signaling pathway. Structural analysis indicates that all the MORN motifs (with varied numbers at ranges of 7-9), which shared high homologies to those of animal ones, were located at N-terminus and sequentially arranged, except those of OsPIPK1 and AtPIPK7, in which the last MORN motif was separated others by an ~100 amino-acid "island" region, revealing the presence of two kinds of MORN arrangements in plant PIPKs. Through employing a yeast-based SMET (sequence of membrane-targeting) system, the MORN motifs were shown being able to target the fusion proteins to cell plasma membrane, which were further confirmed by expression of fused MORN-GFP proteins. Further detailed analysis via deletion studies indicated the MORN motifs in OsPIPK1, together with the 104 amino-acid "island" region are involved in the regulation of differential subcellular localization, i.e. plasma membrane or nucleus, of the fused proteins. Fat Western blot analysis of the recombinant MORN polypeptide, expressed in Escherichia coli, showed that MORN motifs could strongly bind to PA and relatively slightly to PI4P and PI(4,5)P 2 . These results provide informative hints on mechanisms of subcellular localization, as well as regulation of substrate binding, of plant PIPKs.

A Dibasic Amino Acid Pair Conserved in the Activation Loop Directs Plasma Membrane Localization and Is Necessary for Activity of Plant Type I/II Phosphatidylinositol Phosphate Kinase

“…In contrast to our data (Figs. 2-4), for example, it has been reported that the MD, but not the activation loop containing the dibasic amino acid pair, regulates the plasma membrane localization of plant PIPKs (Ma et al, 2006;Im et al, 2007;Kusano et al, 2008). However, our findings closely resemble those of Kunz et al (2000) on the membrane-targeting machinery of mammalian type I PIPKs, in which the dibasic amino acid pair in the activation loop also acts as a determinant of plasma membrane localization.…”

“…1), of which the MD is thought to be a plasma membranelocalizing module (Ma et al, 2006;Im et al, 2007;Kusano et al, 2008). Our demonstration that the two moss PIPKs, GFP-PpPIPK1 and GFP-PpPIPK2, localized to plasma membranes (Saavedra et al, 2009) raised the obvious question of whether the MD truly regulates plasma membrane localization of plant PIPKs.…”

“…Despite the hypothesized involvement of the MD in the plasma membrane localization of OsPIPK1, AtPIP5K1, and AtPIP5K3 (Ma et al, 2006;Im et al, 2007;Kusano et al, 2008), plasma membrane localization of MD and CD could not be demonstrated using transgenic tobacco cells (Im et al, 2007). In order to determine whether the MD of AtPIP5K1 localizes to plasma membranes in protoplasts of P. patens, plasmids for the expression of Citrine fused with the full-length AtPIP5K1, MD (amino acids 1-301), and CD (amino acids 320-752) were created ( Fig.…”

“…Indeed, a regulatory role of the MORN domain was reported in the enzymatic activation of AtPIP5K1 (Im et al, 2007) and in root hair formation, but not in enzymatic activation, of AtPIP5K3 . Moreover, the MORN domain may play a role in the plasma membrane localization of OsPIPK1 from rice and AtPIP5K1 and AtPIP5K3 from Arabidopsis (Ma et al, 2006;Im et al, 2007;Kusano et al, 2008). However, stable transformation of tobacco (Nicotiana tabacum) cells to express an AtPIP5K1 MORN domain-GFP fusion did not allow visualization of the plasma membrane localization of this protein (Im et al, 2007).…”

Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth

“…S5 A), we identified several potential partners of this myosin. We focused on retinophilin/MORN4, a protein with four membrane occupation and recognition nexus (MORN) repeats, given its role in the membrane association and stabilization of protein complexes (Ma et al, 2006) and the reported in vitro interaction between MORN4 and myosin IIIa (Mecklenburg et al, 2015). We confirmed this interaction by coimmunoprecipitation assays and colocalization experiments in transfected HEK293 cells (Fig.…”