PIP5K: Role in Plant Nuclear Function and Characterization of MORN Domain Structure.

摘要

Phosphoinositides (inositol phospholipids and inositol phosphates, PI) are important molecules in cellular signaling, biotic and abiotic stress responses in animals and plants (Balla et al., 2009; Im et al., 2011). The cytosolic animal PI pathway is well characterized (Balla et al., 2009), and novel nuclear functions for phosphoinositides have been discovered (Barlow et al., 2010; Bunce et al., 2006). The PI pathway in plants is less well characterized and the functions of plant PIP5Ks are not well elucidated. However, several plant specific features have been described. Specifically, the ratio of phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) to PtdIns4P is lower in plants than animals (Boss et al., 2006) and the Arabidopsis thaliana phosphatidylinositol 4--phosphate 5 kinase (PIP5K) family has subfamily B with a unique N-terminal membrane occupation and recognition nexus (MORN) domain (Mueller-Roeber and Pical, 2002) This thesis focuses on the plant nuclear PI pathway and the PIP5K MORN domain.;The initial hypothesis of this dissertation was that one or more plant PIP5K proteins localize to the nucleus and affect plant nuclear functions. Chapter 1 summarizes what is currently known about the plant nuclear PI pathway. Isolation of the nuclear PIP5K proteins was not possible for a number of reasons: plant PIP5K activity in the nucleus was low, antibodies to the PIP5K proteins were not useful in plant cells, the size of the nuclear PIP5K is not determined and can vary between 45 to 90 kDa, and isolation of large quantities of nuclei from plant cells is difficult.;A different approach to understanding the role of the nuclear PI pathway in plants was needed. The human PIP5K1alpha (HsPIP5K1alpha) is known to localize to the plasma membrane and the nucleus in human cells. We hypothesized that expression of HsPIP5K1alpha in plants could increase nuclear PIP5K, nuclear PtdIns(4,5)P2, and affect nuclear functions. In Chapter 2 we describe the effects of expressing HsPIP5K1alpha in Nicotiana tobacum NT-1 cells on nuclear lipids and nuclear functions.;Two plant PIP5K proteins, with the unique N-terminal MORN domain, have been localized to the nucleus by GFP fluorescence (AtPIP5K9 (Lou et al., 2007) and OsPIP5K1(Ma et al., 2004)). The MORN domain has been associated with the nucleus and nuclear division in the parasite Toxoplasma gondii (Gubbels et al., 2006). It is likely that the MORN domain of the plant PIPKs affects their subcellular localization and function, we hypothesized that bioinformatic analysis of the plant MORN domain would reveal information about the potential function and origin of the PIPK MORN domain. In Chapter 3, we present analyses suggesting that the plant MORN domain proteins originated from one ancestral protein and that there are significant differences between the PIP5K protein MORN domain and other MORN domain proteins.