The highly charged region of plant beta-type phosphatidylinositol 4-kinase is involved in membrane targeting and phospholipid binding.
In Arabidopsis thaliana and Oryza sativa, two types of PI 4-kinase (PI4Ks) have been isolated and functionally characterized. The alpha-type PI4Ks (approximately 220 kDa) contain a PH domain, which is lacking in beta-type PI4Ks (approximately 120 kDa). Beta-type PI4Ks, exemplified by Arabidopsis AtPI4Kbeta and rice OsPI4K2, contain a highly charged repetitive segment designated PPC (Plant PI4K Charged) region, which is an unique domain only found in plant beta-type PI4Ks at present. The PPC region has a length of approximately 300 amino acids and harboring 11 (AtPI4Kbeta) and 14 (OsPI4K2) repeats, respectively, of a 20-aa motif. Studies employing a modified yeast-based "Sequence of Membrane-Targeting Detection" system demonstrate that the PPC(OsPI4K2) region, as well as the former 8 and latter 6 repetitive motifs within the PPC region, are able to target fusion proteins to the plasma membrane. Further detection on the transiently expressed GFP fusion proteins in onion epidermal cells showed that the PPC(OsPI4K2) region alone, as well as the region containing repetitive motifs 1-8, was able to direct GFP to the plasma membrane, while the regions containing less repetitive motifs, i.e. 6, 4, 2 or single motif(s) led to predominantly intracellular localization. Agrobacterium-mediated transient expression of PPC-GFP fusion protein further confirms the membrane-targeting capacities of PPC region. In addition, the predominant plasma membrane localization of AtPI4Kbeta was mediated by the PPC region. Recombinant PPC peptide, expressed in E. coli, strongly binds phosphatidic acid, PI and PI4P, but not phosphatidylcholine, PI5P, or PI(4,5)P2 in vitro, providing insights into potential mechanisms for regulating sub-cellular localization and lipid binding for the plant beta-type PI4Ks.
Lou
Ying
Y
National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science (SiBS), Chinese Academy of Sciences, Shanghai 200032, P.R. China.
Ma
Hui
H
Lin
Wen-Hui
WH
Chu
Zhao-Qing
ZQ
Mueller-Roeber
Bernd
B
Xu
Zhi-Hong
ZH
Xue
Hong-Wei
HW
D019870:1-Phosphatidylinositol 4-Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D019470:Biolistics / Q000379:methods; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019697:Onions / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D012275:Oryza / Q000201:enzymology; D010743:Phospholipids / Q000378:metabolism*; D019441:Plant Epidermis / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011485:Protein Binding; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
D010743:Phospholipids; D010940:Plant Proteins; D049452:Green Fluorescent Proteins; D019870:1-Phosphatidylinositol 4-Kinase