ORIGINAL ARTICLE Sun Yong Jeong Æ Nancy Peffer Æ Iris Meier Phosphorylation by protein kinase CKII modulates the DNA-binding activity of a chloroplast nucleoid-associated protein Received: 9 December 2003 / Accepted: 10 January 2004 / Published online: 17 February 2004 Ó Springer-Verlag 2004 Abstract Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein–DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this localization is unknown. MFP1 is a DNA-binding, coiled-coil protein associated with the thylakoid membranes of mature chloroplasts. It is also a component of nucleoids, suggesting a function at the interface of the chloroplast genome and the photosyn- thetic membranes. Several thylakoid proteins are phos- phorylated 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 chloro- plast transcription complex. Here, we show evidence for the phosphorylation of MFP1 in purified chloroplasts from tobacco (Nicotiana tabacum L.). We demonstrate that the DNA-binding domain of MFP1 is a substrate for CKII and that phosphorylation by CKII inhibits DNA binding. Using site-directed mutagenesis, we identify a conserved twin CKII site in the DNA-binding domain that is required for the inhibition of DNA binding. Phosphorylation of MFP1 by chloroplast CKII as a possible means to modulate its DNA-binding activity is discussed. Keywords Chloroplast Æ DNA-binding protein Æ Nicotiana Æ Protein kinase CKII Abbreviations CKII: Casein kinase II Æ MFP1: MAR-binding filament-like protein 1 Æ PTK: Plastid transcription kinase Introduction Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein–DNA complexes called nucle- oids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thy- lakoid membranes in mature chloroplasts, but the mechanism for this relocalization is unknown (Miyam- ura et al. 1986; Liu and Rose 1992; Sato et al. 1993). The nuclear-encoded protein MFP1 (MAR-binding filament- like protein 1) from tomato (LeMFP1) was identified by its ability to bind to matrix attachment region DNA (Meier et al. 1996). MFP1 is an 80-kDa, predominantly coiled-coil protein with a C-terminal DNA-binding do- main. It was subsequently shown to be localized in plastids and associated with thylakoid membranes, with the DNA-binding domain oriented towards the stroma. In mature Arabidopsis chloroplasts, MFP1 is a major DNA-binding activity which binds to all tested chloro- plast DNA fragments without detectable sequence specificity (Jeong et al. 2003). The expression of MFP1 is tightly correlated with the accumulation of thylakoid membranes. Importantly, MFP1 is associated in vivo with nucleoids, suggesting a function at the interface between chloroplast nucleoids and the thylakoid mem- brane system (Jeong et al. 2003). Phosphorylation has been shown to play a major role in modulating the function and DNA-binding activity of many nuclear proteins, including transcription factors and proteins involved in chromatin organization (e.g., Dang et al. 1994; Armstrong et al. 1997; Hoffmann et al. 1998). Recently, a chloroplast protein (cpCK2a) with high similarity to the alpha subunit of casein kinase II (CKII) has been reported as a component of the chlo- roplast transcription apparatus (Ogrzewalla et al. 2002). cpCK2a has the functional characteristics of a S. Y. Jeong Æ N. Peffer Æ I. Meier (&) Plant Biotechnology Center and Department of Plant Biology, The Ohio State University, 244 Rightmire Hall, 1060 Carmack Road, Columbus, OH 43210-1002, USA E-mail: meier.56@osu.edu Tel.: +1-614-2928323 Fax: +1-614-2925379 Present address: N. Peffer Bristol-Myers Squibb, Wilmington, DE 19880, USA Planta (2004) 219: 298–302 DOI 10.1007/s00425-004-1215-8