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Functional investigation of arabidopsis long coiled-coil proteins and subcellular localization of plant rangap1

Jeong, Sun Yong
http://rave.ohiolink.edu/etdc/view?acc_num=osu1086119855

Abstract Details

2004, Doctor of Philosophy, Ohio State University, Plant Biology.
Long alpha-helical coiled-coil proteins are involved in a variety of organizational and regulatory processes in eukaryotic cells. In contrast to yeast and animals, only few such proteins have been investigated in plants. Here, two plant long coiled-coil proteins were investigated in detail, MAR-binding filament-like protein1 (MFP1) and the putative Arabidopsis homolog of Tpr (translocated promoter region). MFP1 is a nuclear-encoded, long coiled-coil protein that is targeted to plastids. It accumulates to comparable levels in all tissues of Arabidopsis which contain green chloroplasts, regardless of age and organ identity, but is much less abundant in roots of both light-grown and dark-grown seedlings. MFP1 protein accumulation parallels chloroplast development during the greening of seedlings shifted from dark to light, suggesting that MFP1 expression is regulated in a tissue-specific and light-dependent manner. MFP1 is localized in chloroplasts both in suspension culture cells and in leaves, and it is associated with the stromal side of thylakoid membranes of mature chloroplasts. It is co-purified with nucleoids, suggesting a function at the interface of the chloroplast genome and the photosynthetic membranes. MFP1 comprises a major DNA-binding activity in Arabidopsis chloroplasts and binds to several regions of the chloroplast DNA with equal affinity. Several thylakoid proteins are phosphorylated by a protein kinase CKII-like activity, and the alpha subunit of a chloroplast-located CKII has recently been identified as a component of the chloroplast transcription complex. Chloroplast-localized MFP1 is phosphorylated in vivo, and in vitro by CKII and phosphorylation inhibits its DNA-binding activity. A tandem CKII site in the DNA-binding domain of MFP1 was identified which is involved in the phosphorylation-dependent loss of DNA-binding activity. These features of MFP1 make CKII-dependent phosphorylation a possible mode of regulating the DNA-binding activity of the protein in vivo. Together, these data suggest that MFP1 is an interesting candidate for an architectural protein, possibly involved in anchoring nucleoids to thylakoid membranes. Tpr is a long coiled-coil protein associated with the nuclear surface of the nuclear pore in animals and yeast, where it is involved in mRNA export. A putative Arabidopsis Tpr homolog was identified. A T-DNA insertion mutant in its gene has a pleiotropic phenotype including early flowering, reduced apical dominance and morphological alterations of leaves and inflorescences. The connection between the predicted function of Tpr and its mutant phenotype is currently being investigated. In animals and yeast, the small GTPase Ran is involved in nucleocytoplasmic transport, spindle formation, and nuclear envelope formation. These functions are controlled by a RanGTPase-activating protein (RanGAP) and a guanine nucleotide exchange factor (RCC1). Vertebrate RanGAP1 is conjugated with the ubiquitin-like protein SUMO. SUMOylation of RanGAP1 is required for nuclear envelope-association in interphase and for spindle and centromere association in mitosis. Plant RanGAP lacks the SUMOylated C-terminal domain of vertebrate RanGAP, but contains instead a plant-specific N-terminal WPP domain, which is necessary and sufficient for targeting the protein to the nuclear rim. By examining the localization of Arabidopsis RanGAP1 during the cell cycle in stably transformed tobacco BY-2 cells expressing AtRanGAP1-GFP, we found that AtRanGAP1 localizes to the nuclear rim during interphase and to the cell plate during cytokinesis. A WPP domain-GFP fusion behaves like full-length AtRanGAP1-GFP, while WPP-domain deletion abolishes all targeting, demonstrating that the WPP domain is necessary and sufficient for both targeting events. Point mutations in conserved residues of the WPP domain abolish targeting to the nuclear rim and the cell plate, suggesting that the same mechanism is involved in anchoring RanGAP1 in both locations. These results imply a novel function of AtRanGAP1 during cell cycle and suggest a role of the Ran cycle in controlling cell plate formation in plant cytokinesis.
Iris Meier (Advisor)
230 p.
Biology, Botany
CKII: Casein kinase II; MAR: Matrix attachment region; MFP1: MAR-binding filament-like protein1; MLP: Myosin-like protein; PTK: Plastid transcription kinase; Ran: Ras-related nuclear protein; RanGAP: Ran GTPase activating protein

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Citations

  • Jeong, S. Y. (2004). Functional investigation of arabidopsis long coiled-coil proteins and subcellular localization of plant rangap1 [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1086119855

    APA Style (7th edition)

  • Jeong, Sun Yong. Functional investigation of arabidopsis long coiled-coil proteins and subcellular localization of plant rangap1. 2004. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1086119855.

    MLA Style (8th edition)

  • Jeong, Sun Yong. "Functional investigation of arabidopsis long coiled-coil proteins and subcellular localization of plant rangap1." Doctoral dissertation, Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1086119855

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.