Plant Physiology Preview. Published on August 24, 2015, as DOI:10.1104/pp.15.01115
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Running title: Phosphorylation of Mitochondrial Precursor Protein
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Author to whom correspondence should be sent:
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Boon Leong LIM
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School of Biological Sciences, the University of Hong Kong, Pokfulam, Hong
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Kong, China.
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Tel: +852 22990826
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Email: [email protected]
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Research area: Membranes, Transport and Biogenetics
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Copyright 2015 by the American Society of Plant Biologists
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Phosphorylation and Dephosphorylation of the Presequence of pMORF3
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During Import into Mitochondria from Arabidopsis thaliana
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Yee-Song Law1, Renshan Zhang1, Xiaoqian Guan1, Shifeng Cheng1, Feng
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Sun1, Owen Duncan2, Monika W. Murcha2, James Whelan3, Boon Leong
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Lim1,4,*
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School of Biological Sciences, the University of Hong Kong, Pokfulam, Hong
Kong, China.
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Australian Research Council Centre of Excellence in Plant Energy Biology,
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University of Western Australia, 35 Stirling Highway, Crawley, Western Australia,
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6009, Australia.
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Department of Animal, Plant and Soil Science, School of Life Science,
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Australian Research Council Centre of Excellence in Plant Energy Biology, La
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Trobe University, Bundoora, Victoria, 3086, Australia.
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State Key Laboratory of Agrobiotechnology, The Chinese University of Hong
Kong, Shatin, Hong Kong, China.
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One-Sentence Summary:
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Phosphorylation of presequence by cytosolic kinases and dephosphorylation of
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presequence by outer mitochondrial membrane phosphatase are involved in the
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mitochondrial import process of pMORF3.
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Footnotes:
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This project was supported by the Seed Funding Program for Basic Research
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(201311159043) and the Strategic Research Theme on Clean Energy of the
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University of Hong Kong, the General Research Fund (HKU772710M and HKU
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772012M) and the Innovation and Technology Fund (Funding Support to Partner
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State Key Laboratories in Hong Kong) of the HKSAR, China.
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The author responsible for distribution of materials integral to the findings
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presented in this article in accordance with the policy described in the
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Instructions
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([email protected]).
for
Authors (www.plantphysiol.org) is: Boon Leong Lim
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Abstract
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The nuclear-encoded mitochondrial-targeted proteins, multiple organellar
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RNA editing factors (MORF3, MORF5, MORF6) interact with AtPAP2 (Purple
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Acid Phosphatase 2) located on the chloroplast and mitochondrial outer
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membranes in a presequence dependent manner. Phosphorylation of the
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presequence of the precursor MORF3 (pMORF3) by endogenous kinases in
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wheat germ translation lysate, leaf extracts, or STY kinases, but not in rabbit
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reticulocyte translation lysate, resulted in the inhibition of protein import into
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mitochondria. This inhibition of import could be overcome by altering
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threonine/serine residues to alanine on the presequence, thus preventing
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phosphorylation. Phosphorylated pMORF3, but not the phosphorylation deficient
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pMORF3, can form a complex with 14-3-3 proteins and HSP70. The
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phosphorylation deficient mutant of pMORF3 also displayed faster rates of import
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when translated in wheat germ lysates. Mitochondria isolated from plants with
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altered amounts of AtPAP2 displayed altered protein import kinetics. The import
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rate of pMORF3 synthesized in wheat germ translation lysate into pap2
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mitochondria was slower than that into wild-type mitochondria, and this rate
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disparity was not seen for pMORF3 synthesized in rabbit reticulocyte translation
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lysate, the latter translation lysate largely deficient in kinase activity. Taken
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together,
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dephosphorylation of pMORF3 during the import into plant mitochondria. These
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results suggest that kinases, possibly STY kinases, and AtPAP2 are involved in
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the import of protein into both mitochondria and chloroplasts, and provides a
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mechanism by which the import of proteins into both organelles may be
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coordinated.
these
results
support
a
role
for
the
phosphorylation
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and
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INTRODUCTION
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Chloroplasts and mitochondria are endosymbiotic organelles that are
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intimately involved in energy metabolism in plants (Araujo et al., 2014). The
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majority of proteins located in chloroplasts and mitochondria are encoded in the
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nucleus, translated in the cytosol and imported into the organelles (Murcha et al.,
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2014). For both chloroplasts and mitochondria, over 1000 different proteins are
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required to be specifically imported into each organelle. Furthermore, while most
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proteins are imported specifically into one organelle, a significant number of
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proteins are dual targeted to both via ambiguous targeting signals (Carrie et al.,
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2009; Ye et al., 2012; Ye et al., 2015). The sorting of proteins to chloroplast and
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mitochondria is achieved through the inclusion of targeting signals in newly
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synthesized proteins which act in combination with receptor domains present in
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outer membrane multi-subunit protein complexes to specifically direct proteins to
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their destination compartments (Jarvis, 2008; Shi and Theg, 2013; Murcha et al.,
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2014). Chloroplast targeting signals (transit peptides) and mitochondrial targeting
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signals (presequences) are recognized by receptors on the translocase of the
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outer chloroplast envelope (TOC) and translocase of the outer mitochondria
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membrane (TOM) respectively (Ye et al., 2015). In addition to the targeting
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signals and protein receptors it has been proposed that cytosolic chaperone
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proteins may also play a role in maintaining precursor proteins in an import
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competent state and may play a role in determining targeting specificity. However
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in plants, the role of cytosolic chaperones has only been characterized to some
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extent for protein import into chloroplasts (Jarvis, 2008; Fellerer et al., 2011;
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Flores-Perez and Jarvis, 2013; Lee et al., 2013; Schweiger et al., 2013), but not
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into mitochondria.
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In addition to cytosolic chaperone factors, three STY kinases are also
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involved to phosphorylate the transit peptides of several chloroplast precursor
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proteins, such as the precursor for the small subunit of ribulose 1, 5
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bisphosphate carboxylase/ oxygenase (pSSU) and precursor for the high
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chlorophyll fluorescence 136 (pHCF136), but not the presequence of the tobacco
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precursor for the β-subunit of the mitochondria ATP synthase (pF1β) (Martin et
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al., 2006; Lamberti et al., 2011; Lamberti et al., 2011). In addition, a pea 14-3-3
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protein has also been reported to bind to the phosphorylated-Ser on the transit
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peptide of pSSU but not to an SA mutant. The formation of the pSSU/14-3-
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3/HSP70 complex enhances the kinetics of the import of pSSU into chloroplasts,
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as free pSSU is imported relatively slowly (May and Soll, 2000). Phosphatase
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inhibitors, NaF and NaMoO4, inhibit pSSU import into plastids in a reversible
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manner, suggesting that the dephosphorylation of the phosphorylated transit
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peptide of pSSU is required for import (Flugge and Hinz, 1986; Waegemann and
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Soll, 1996). A model for pSSU recognition and TOC translocation has been
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proposed in which the transit peptide of pSSU is phosphorylated at Ser34 and the
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transit peptide binds to Toc33 and Toc159 to form a trimeric complex (Becker et
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al., 2004; Oreb et al., 2011). Hydrolysis of GTP at Toc33 dissociates it from the
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complex and an as yet unknown phosphatase dephosphorylates pSer34 on pSSU,
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allowing import to proceed through the combined action of Toc159 and Toc75
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(Becker et al., 2004; Oreb et al., 2011). Phosphorylation seems to affect import
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as phospho-mimicking transit peptides of pSSU and pHCF136 reduced their
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import rates into chloroplasts (Lamberti et al., 2011; Nickel et al., 2015).
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Unlike chloroplast import, there is no evidence that the phosphorylation and
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dephosphorylation of plant mitochondrial presequences is required for efficient
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protein import. In yeast (Saccharomyces cerevisiae) Tom22 functions as a
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cytosolic facing receptor and transfers precursor proteins to the Tom40 channel.
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It can be phosphorylated by Casein Kinase 2 (CK2) and the mitochondrial bound
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Casein Kinase 1 (CK1), to stimulate activity and assembly of the TOM complex
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(Harbauer et al., 2014). Protein kinase A (PKA) can phosphorylate Tom22,
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impairing its import rate. Thus, PKA and CK1 and CK2 act antagonistically. It has
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also been demonstrated that the cyclin-dependent kinase Cdk1 stimulated
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assembly of the TOM complex by phosphorylation of Tom6, an accessory
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subunit of the TOM complex, enhancing its import into mitochondria (Gerbeth et
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al., 2013). Thus, in yeast mitochondria, the phosphorylation of the protein import
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machinery itself appears to play a role in import.
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While the overall theme of mitochondrial protein targeting, machinery and
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pathways utilized is conserved between different systems, significant variations
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have been observed in plants. Firstly, the plant outer mitochondrial protein
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receptor, Tom20 is not an ortholog to the yeast or mammalian receptors (Perry et
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al., 2006). Structural studies on the plant Tom20 import receptor suggest a
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“discontinuous bidentate hydrophobic binding” mechanism, somewhat different to
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that observed in other systems (Rimmer et al., 2011). Furthermore, plant
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mitochondria are required to distinguish between chloroplast and mitochondrial
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proteins. Transit peptides and presequences display some similarities in that
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both are enriched in positively charged residues. However, while mitochondrial
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presequences are proposed to form α-amphilphilic structures, chloroplast transit
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peptides do not, and are instead predicted to form β-sheet secondary structures
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(Zhang and Glaser, 2002). The identification of a plant specific outer membrane
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receptor, Toc64 involved in the import of specific precursor proteins (Chew et al.,
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2004) highlights the possibility of additional and varied mechanisms between
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plant mitochondrial protein targeting and other systems.
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Previously, we have identified an outer mitochondrial membrane protein,
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called purple acid phosphatase 2 (AtPAP2) that is also dual targeted to the outer
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envelope in chloroplasts (Sun et al., 2012; Sun et al., 2012). Similar to Toc33/34
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and Tom20, AtPAP2 is anchored on the outer membranes by a C-terminal
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transmembrane motif. Overexpression of AtPAP2 resulted in an altered growth
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phenotype with elevated ATP levels (Sun et al., 2012) and thus the function of
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this protein was investigated. Here we show that the phosphorylation and
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dephosphorylation of the presequence of the precursor MORF3 (pMORF3) alters
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the kinetics of its import. Furthermore it was shown that the phosphorylation of
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pMORF3 when translated in wheat germ lysate resulted in relatively slow import
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kinetics and the inhibition of phosphorylation by site directed mutagenesis
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resulted in faster import kinetics. Together these results show that the
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phosphorylation and dephosphorylation of a mitochondrial precursor protein does
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play a role in its rate of import. The role of the outer membrane AtPAP2 protein
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and STY kinases was also investigated, supporting a role for phosphorylation
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and dephosphorylation in the import of some precursor proteins into plant
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mitochondria.
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RESULTS
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AtPAP2 Interacts with the Presequences of MORF Proteins
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Using AtPAP2 as the bait in a yeast two-hybrid screen, four MORF family
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proteins, including MORF2/3/5/6, were identified (Fig. 1A). When the predicted
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mitochondrial
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MORF3/5/6 (∆50aaMORF3/5/6) were removed, their interactions with AtPAP2
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were completely abolished (Fig. 1B). Previously a large-scale protein
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phosphorylation study in Arabidopsis revealed three experimentally identified
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phosphorylation sites (Thr17, Ser20 and Thr35) on the presequence of MORF3
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(Wang et al., 2013). Mutations were introduced within the predicted
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phosphorylation sites of the MORF3 presequence, pMORF3-T17/S20/T35A, and
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it was found that the mutated prey construct did not interact with AtPAP2 (Fig.
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1B).
targeting
presequences
of
MORF2
(∆40aaMORF2)
and
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To confirm these interaction assays, the localization of MORF2/3/5/6 was
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tested by biolistic transformation with GFP fusion constructs in combination with
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specific organelle markers AOX-RFP (mitochondria) and SSU-RFP (chloroplasts).
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The MORF2-green fluorescent protein (GFP) fusion protein was observed to be
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targeted to plastids, the MORF3-GFP and MORF5-GFP fusion proteins were
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observed to be targeted to mitochondria, and the MORF6-GFP fusion protein
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was observed to be targeted to both plastid and mitochondria (Fig. 2 and
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Supplemental Fig. S1). A bimolecular fluorescence complementation assay
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(BiFC) was used to verify in vivo interactions between AtPAP2 and MORF2/3/5/6.
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In the BiFC assay, the co-expression of NYFP-AtPAP2 and MORF2-CYFP
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reconstituted a functional YFP signal in the chloroplasts; the co-expression of
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NYFP-AtPAP2 and MORF3/5-CYFP reconstituted a functional YFP signal in the
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mitochondria; the co-expression of NYFP-AtPAP2 and MORF6-CYFP generated
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functional YFP signals in both the chloroplasts and mitochondria (Fig. 1C).
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STY8 and STY17 Phosphorylates the Presequences of Nuclear-encoded
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Mitochondrial Proteins
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It was found that rabbit reticulocyte lysate (RRL), wheat germ lysate (WGL)
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and leaf extract (LE) contain kinases that phosphorylate pMORF3, but the
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pMORF3 phosphorylating kinase activity of RRL is much weaker than that of
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WGL and LE (Fig. 3A). The specific kinase inhibitor, JNJ-101198409 (JNJ),
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inhibits
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phosphorylation, and the non-specific phosphatase inhibitor, NaF, inhibits
STY
kinase
autophosphorylation
and
chloroplast
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substrate
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chloroplast substrate dephosphorylation (Waegemann and Soll, 1996; Lamberti
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et al., 2011). An in vitro phosphorylation assay indicated that JNJ can specifically
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inhibit the phosphorylation of pMORF3 by an intrinsic kinase in WGL, but the
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phosphorylation of intrinsic proteins in WGL was not affected (Fig. 3A, lane 3 vs.
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lane 4). However, the phosphorylation of pMORF3 by an intrinsic kinase in LE
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was not inhibited by JNJ (Fig. 3A, lane 12 vs. lane 11), suggesting that the
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pMORF3 phosphorylating kinases in WGL and LE have different properties. The
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weak pMORF3 phosphorylating activity in RRL was not affected by JNJ (Fig. 3A,
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lane 8 vs. lane 7). Compared with WGL (lane 1) and LE (lane 9), RRL had very
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weak kinase activity, not only to pMORF3 (lane 7), but also toward intrinsic
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proteins (lane 5). The results of the in vitro kinase assay reflect a balance of
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intrinsic kinase and phosphatase activities. Therefore, the activities of
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phosphatases in WGL, RRL and LE were determined (Fig. 3B) by including a
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phosphatase inhibitor (NaF) in the kinase assays. Our results show that WGL not
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only has rich kinase activities, but is also rich in phosphatase activities that could
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counteract the kinase activities in the WGL (Fig. 3B, lane 6 vs. lane 5). LE also
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contained substantial phosphatase activities (Fig. 3B, lane 2 vs. lane 1). For RRL,
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NaF had little effect on its kinase activities suggesting that the lack of
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phosphorylation of pMORF3 is because RRL does not have rich intrinsic kinase
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activities, and not due to the presence of high intrinsic phosphatase activities.
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The STY8 and STY17 kinases phosphorylate transit peptides for some
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nuclear-encoded chloroplast proteins (Martin et al., 2006; Lamberti et al., 2011).
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The precursor protein (pMORF3) but not the ∆50aaMORF3, was phosphorylated
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in the in vitro phosphorylation assay with STY8 or STY17 (Fig. 3C). The
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phosphorylation
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phosphorylated by STY8 or STY17, indicating that one or all of Thr17, Ser20 and
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Thr35 are the target phosphorylation sites of STY8 and STY17 (Fig. 3C).
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Additionally pMORF2/5/6 were all phosphorylated by STY8 and STY17 but upon
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removal of the presequences (∆40aaMORF2 and ∆50aaMORF3/5/6) no
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phosphorylation by STY8 or STY17 could be observed (Fig. 3C).
deficient
mutant
of
pMORF3-T17/S20/T35A
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was
not
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Dephosphorylation of the Presequence of pMORF3 is Required for Import
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into Mitochondria
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RRL and WGL have both been used to transcribe and translate precursor
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proteins for in vitro chloroplast and mitochondria protein import studies, though it
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has been observed that WGL-synthesized precursors are generally import-
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incompetent into mitochondria (Dessi et al., 2003; Biswas and Getz, 2004). Here,
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we examined whether pMORF3 synthesized in RRL (RRL-pMORF3) and WGL
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(WGL-pMORF3) are import competent into isolated Arabidopsis thaliana
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mitochondria. In the mitochondrial import assay, RRL-pMORF3 was imported
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and protease protected when incubated with mitochondria as evidenced by the
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generation of the mature form of MORF3 (mMORF3) (Fig. 4A lanes 1-3). As the
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generation of mMORF3 was sensitive to the addition of valinomycin, an
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ionophore that dissipates the membrane potential (Fig. 4A, lanes 4 and 5), the
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generation of mMORF3 is thus a direct result of correct import and processing.
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Interestingly, the transcription and translation of WGL-pMORF3 results in a
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mature-like product of the same apparent molecular weight as mMORF3 prior
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addition of mitochondria (Fig. 4A, lane 6). This has been previously reported for
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other mitochondrial precursor proteins synthesized in WGL and is likely
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generated by a mitochondrial peptidase/protease within the WGL, as wheat germ
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is a rich source of mitochondrial proteins (Peiffer et al., 1990). Never the less, the
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import and processing of WGL-pMORF3, as determined by the generation of a
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protease protected band (Fig. 4A, lane 8) was very weak compared to RRL-
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pMORF3 (Fig. 4A, lane 3). Furthermore, the truncated precursor protein of
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pMORF3 translated in RRL (RRL-∆50aaMORF3) could not be imported into
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mitochondria, indicating that the presequence of pMORF3 is essential for import
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(Supplemental Fig. S2).
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We have shown that WGL and LE contain kinases that could
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phosphorylate pMORF3 in the in vitro kinase assay (Fig. 3A) and as it had been
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previously shown that the addition of WGL to RRL-synthesized precursor
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proteins reduce the rate of import into mitochondria (Dessi et al., 2003), we
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further tested if this was also the case for pMORF3. The import rates of both LE-
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treated and WGL-treated RRL-pMORF3 were reduced when treated prior to
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import into isolated mitochondria, suggesting that LE and WGL both contain
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unknown inhibitor(s) that can reduce the import ability of RRL-pMORF3 into
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mitochondria (Fig. 4B). As pMORF3 can be phosphorylated by intrinsic kinases
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in WGL and LE, but not RRL (Fig. 3A), it was tested if the phosphorylation of
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pMORF3 in WGL and LE affected the rate of import uptake into isolated
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mitochondria. Although RRL contains an unknown inhibitor of the STY-
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dependent phosphorylation of pMORF3 (Supplemental Fig. S3), the treatment of
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RRL-pMORF3
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phosphorylated RRL-pMORF3 (Supplemental Fig. S3). RRL-pMORF3 pre-
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incubated with STY8 kinase to increase the amount of the phosphorylated
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pMORF3 prior to mitochondrial import displayed reduced amounts of protein
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import into mitochondria compared to untreated RRL-pMORF3 (Fig. 4C). In the
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presence of phosphatase inhibitor NaF, the import rate of STY8-treated RRL-
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pMORF3 (lane 5) exhibited additive inhibitory effect (Fig. 4C). These results
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suggest that STY8 phosphorylation of the presequence of pMORF3 impedes its
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import into mitochondria and that its dephosphorylation is required prior to
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translocation.
by
recombinant
STY8
could
increase
the
amount
of
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Next, we examined whether the phosphorylation of the presequence is
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necessary for the import of pMORF3 into mitochondria. As shown in Fig. 4D, the
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RRL-synthesized phosphorylation deficient mutant pMORF3-T17/S20/T35A was
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imported normally, similar to wild type RRL-pMORF3, indicating that the
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phosphorylation of the presequence of pMORF3 is not required for import. Taken
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together, unlike in the import of pSSU into chloroplasts, the phosphorylation of
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pMORF3 by STY kinases does not enhance, but rather impedes import into
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mitochondria. Furthermore, addition of NaF reduced the import rate of RRL-
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pMORF3-T17/S20/T35A. Therefore, the slower import rate of pMORF3 (Fig. 4D,
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lane 3) and pMORF3-T17/S20/T35A (Fig. 4D, lane 7) in the presence of the
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phosphatase inhibitor NaF may be due to its effects on the phosphorylation
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status of the TOM and TIM complexes, in addition to the phosphorylation status
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of the presequence of pMORF3 (Sugiyama et al., 2008; Nakagami et al., 2010;
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Schmidt et al., 2011; Havelund et al., 2013).
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HSP70 and 14-3-3 Interact with pMORF3 but not with pMORF3-
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T17/S20/T35A
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HSP70 interacts with precursor proteins during translocation into mitochondria,
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chloroplasts and endoplasmic reticulum (Deshaies et al., 1988; Zimmermann et
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al., 1988; Beckmann et al., 1990), whereas 14-3-3 proteins recognize transit
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peptides of chloroplast precursors containing a phosphopeptide binding motif and
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then form a guidance complex together with HSP70 (Muslin et al., 1996). It was
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investigated if pMORF3 also has the ability to interact with 14-3-3 and HSP70
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using a co-immunoprecipitation assay. STY8-treated RRL-pMORF3 interacted
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with
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T17/S20/T35A mutant could not interact with HSP70 or 14-3-3 (Fig. 5A and 5B).
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This result suggests that the phosphorylation of the presequence of pMORF3 by
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STY8 kinase enables it to bind to both HSP70 and 14-3-3 proteins in vitro.
HSP70
and
14-3-3;
whereas
the
STY8-treated
RRL-pMORF3-
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Import Rate of the WGL-pMORF3 into pap2 T-DNA Mitochondria is Slower
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The C-terminal transmembrane domain of PAP2 anchors AtPAP2 to the
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outer membrane of chloroplasts and mitochondria (Sun et al., 2012). AtPAP2
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was highly expressed in mitochondria of Arabidopsis thaliana overexpressing
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AtPAP2 line (OE7) but was not expressed in pap2 T-DNA line (Supplemental Fig.
346
S4). We compared the import rates of RRL-pMORF3 and WGL-pMORF3 into WT
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(Col-0), OE7 and pap2 mitochondria. The import rates of largely non-
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phosphorylated RRL-pMORF3 were significantly faster than the import rates of
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more extensively phosphorylated WGL-pMORF3 into the mitochondria of all
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three lines (Fig. 5C and 5D). As the presequence of pMORF3 translated in WGL
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is phosphorylated (Fig. 3A), the presequence must be dephosphorylated before
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import. By contrast, the import rate of WGL-pMORF3 into mitochondria isolated
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from pap2 was lower when compared to WT (Col-0) (Fig. 5D; lanes 5-7), this
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decrease in import was not observed for mitochondria isolated from OE7. The
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import rate of WGL-pMORF3-T17/S20/T35A was also tested into mitochondria
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isolated from WT (Col-0), OE7 and pap2 and no disparity in import ability was
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observed (Fig. 5E). In addition, the import rate of WGL-pMORF3 and WGL-
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pMORF3-T17/S20/T35A was tested into mitochondria isolated from into WT (Col-
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0) and it was determined that the import of WGL-pMORF3-T17/S20/T35A
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reached maximum ability within the first 2 minutes of the import assay unlike
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WGL-pMORF3 which reached maximum import uptake at 15 minutes suggesting
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the import kinetics between the two precursors are altered (Fig. 5F).
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DISCUSSION
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The outer mitochondrial membrane preprotein receptors of plants are not
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orthologous to those in fungal or mammalian systems (Chew et al., 2004; Perry
368
et al., 2006). This is proposed to be due to the unique cellular environments of
369
plant cells, which contain plastids, and thus the requirement to sort proteins
370
between these organelles, a situation that does not exist in animals or fungi. The
371
in vivo and in vitro import of precursor proteins into mitochondria from plants
372
(Boutry et al., 1987; Whelan et al., 1988) has been known for some time. While in
373
vivo methods are generally considered to reflect the in planta situation with
374
respect to targeting specificity, in vitro approaches allow the dissection of the
375
molecular mechanisms of protein import, from synthesis of the precursor protein
376
in the cytosol to assembly into a functional unit in mitochondria (Murcha et al.,
377
2014). For in vitro studies, precursor proteins are synthesized in cell free
378
translation lysates, most typically the rabbit reticulocyte lysate. These
379
approaches have been very successful in elucidating the molecular mechanisms
380
required to achieve mitochondrial protein import.
381
A noticeable and puzzling feature of in vitro protein import into plant
382
mitochondria is that while it is readily supported by import from rabbit reticulocyte
383
lysates, wheat germ translation lysates do not generally support mitochondrial
384
protein import (Dessi et al., 2003). This is puzzling given the efficient import of
385
chloroplast precursor protein from wheat germ translation lysates (Waegemann
386
and Soll, 1996; May and Soll, 2000), although some inhibitory effects on protein
387
import into chloroplasts can also be attributed to proteinaceous factors in wheat
388
germ translation lysates (Schleiff et al., 2002). Detailed studies with wheat germ
389
translation lysates show that not only can mitochondrial import not be supported,
390
but in fact, addition of wheat germ lysate to otherwise mitochondrial import
391
competent precursor protein inhibits import (Dessi et al., 2003). Thus it has been
392
proposed that there are inhibitory factors present in the wheat germ lysate,
393
although the identity of these factors remains unidentified.
20
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
394
Here it is presented that phosphorylation of at least one mitochondrial
395
precursor protein, pMORF3, results in a large decrease in the amount of import
396
into mitochondria. Several lines of evidence show this decrease associated with
397
phosphorylation. Firstly, mutation of threonine as well as serine residues to
398
alanine, that cannot be phosphorylated, results in much greater import from a
399
wheat germ translation lysate. Secondly, addition of WGL or LE shown to
400
phosphorylate pMORF3 reduces the amount of import into mitochondria, and that
401
addition a NaF, a general inhibitor of phosphatases, results in a reduction in the
402
amount of protein import into mitochondria. Thirdly, Arabidopsis line pap2 which
403
has the outer membrane phosphatase inactivated, imports WGL-pMORF3 slower
404
than wild type or lines that have it over-expressed. Finally it can be shown that
405
the presequence was the site of action, as it was required to interact with AtPAP2,
406
and that purified STY7 and STY8 can phosphorylate the presequence of
407
pMORF3.
408
dephosphorylation in the import of pMORF3 into mitochondria on the
409
presequence of pMORF3.
Combined
these
shown
a
role
for
phosphorylation
and
410
STY kinases are present in the cytosol, and AtPAP2 is present on the
411
outer membranes of both organelles (Martin et al., 2006; Sun et al., 2012). In
412
vivo BiFC assays showed that pMORF3/5 interacted with AtPAP2 on
413
mitochondria but not with AtPAP2 on chloroplasts (Fig.1C). In contrast, in vivo
414
BiFC assays showed that chloroplast targeted precursor protein pMORF2
415
interacted with AtPAP2 on chloroplasts but not with AtPAP2 on the mitochondria
416
(Fig. 1C). These data indicate that the interactions of these nuclear-encoded
417
proteins with AtPAP2 are not the sole determining factors of their targeting to
418
these two organelles. Instead, other receptors on TOC (Toc34, Toc64, Toc159)
419
and TOM (Tom20, OM64) complexes are responsible for cargo specificity (Ye et
420
al., 2012; Ye et al., 2015).
421
What is the purpose of the phosphorylation and dephosphorylation of
422
transit peptides and presequences of precursor proteins when both processes
423
are energy-consuming? The localizations of at least 751 mitochondrial proteins
21
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
424
(Duncan et al., 2011) and 1323 chloroplastic proteins (Smith et al., 2004) have
425
been confirmed. Both mitochondria- and chloroplast-targeting signals are located
426
at their N-termini. Although these sequences are enriched in basic and
427
hydroxylated amino acids and are deficient in acidic residues (Perry et al., 2008),
428
their sequences are versatile. The process of STY kinase phosphorylation, 14-3-
429
3 binding, and interaction with TOC/TOM can greatly reduce the variety of
430
sequences of transit peptides. Complex formation also enhances the import
431
efficiency of the chloroplast precursor proteins into chloroplasts, and impedes the
432
import efficiency of mitochondrial precursor proteins into mitochondria.
433
This design may serve to coordinate protein imports into chloroplasts and
434
mitochondria and have evolved early in the green lineage. A single STY-like
435
gene and a single AtPAP2-like gene are present in the genome of Ostreococcus
436
tauri (Supplemental Fig. S5 and Supplemental Table S1), a unicellular
437
photosynthetic alga that contains a single chloroplast and a single mitochondrion
438
per cell. Both STY-like kinases and AtPAP2-like phosphatases can be found in
439
green plants (Supplemental Fig. S5, Supplemental Table 1), suggesting that the
440
genes may have co-evolved in the plant kingdom (Martin et al., 2006; Lamberti et
441
al., 2011; Sun et al., 2012). Conversely, MORF family members only evolved in
442
flowering plants (Supplemental Fig. S5) and work together with RNA sequence-
443
specific pentatricopeptide repeat (PPR) family members in RNA editing in
444
mitochondria and chloroplasts (Takenaka et al., 2012). Bioinformatics analysis of
445
the first 66 amino acids of 334 MORFs from flowering plants showed that serine
446
is the dominant residue at positions 17 – 29 (Supplemental Fig. S6), implying that
447
phosphorylation at the serine residues at the presequences of MORFs may be of
448
biological significance.
449
Modulation of mitochondrial import process or import apparatus were
450
shown to have various effects on the physiology of plants. For example, the
451
limiting step for the import of precursor protein appears to be the amount of the
452
inner membrane translocase TIM17:23 (Wang et al., 2012). Increasing the
453
amount of AtTim23 resulted in increased protein import but significantly reduced
22
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
454
growth, while significantly reducing the rate of protein import into mitochondria
455
had no detectable effect of growth rate in Arabidopsis (Wang et al., 2012). By
456
contrast, Arabidopsis plants with all three Tom20 receptor isoforms inactivated
457
show only some reduced growth rate (Lister et al., 2007). In the case of AtPAP2,
458
while overexpression of AtPAP2 results in enhanced growth and higher seed
459
yield (Sun et al., 2012; Sun et al., 2012), sole targeting of AtPAP2 to
460
mitochondria results in early senescence and lower seed yield (Supplemental Fig.
461
S7). This shows the in vivo biological effects of the overexpression of AtPAP2 in
462
mitochondria on the physiology of plants.
463
In conclusion the observation that at least some mitochondrial precursor
464
protein can be phosphorylated, and that this impedes import into mitochondria,
465
uncovers a novel step in the import of precursor protein into mitochondria and
466
may have implication for protein sorting and specificity of import.
467
468
23
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
469
METHODS
470
Plant Materials and Growth Conditions
471
Wild-type Arabidopsis thaliana ecotype Columbia-0 (WT), Arabidopsis thaliana
472
overexpressing AtPAP2 line (OE7), and a T-DNA insertion mutant (pap2,
473
Salk_013567) (Sun et al., 2012) were used in this study. All of the plants were
474
grown under long-day conditions (16 h light/8 h dark) in a controlled-environment
475
chamber. Arabidopsis seeds were sterilized with 20% (v/v) bleach and grown for
476
14 days on Murashige and Skoog (MS) medium that was supplemented with 3%
477
(w /v) sucrose.
478
479
Plasmid Construction
480
First strand cDNA was reverse-transcribed by Reverse Transcription System
481
(Promega, USA) according to the manufacturer’s instructions. Full length coding
482
sequences of MORF2/3/5/6, AtPAP2, pF1β, STY8 and STY17, coding sequence
483
of MORF2 with deletion of the first 120 nucleotides following the start codon
484
(∆40aaMORF2), and coding sequences of MORF3/5/6 with deletion of the first
485
150 nucleotides following the start codon (∆50aaMORF3/5/6) were amplified by
486
Platinum® Pfx DNA Polymerase (Life Technologies, USA) using primers listed in
487
Supplemental Table S2. For site-directed mutagenesis, phosphorylation deficient
488
mutant MORF3-T17/S20/T35A was amplified by primers containing mutated
489
bases as described by Heckman and Pease (2007).
490
To produce transgenic lines that only overexpressed AtPAP2 on
491
mitochondria, cDNA encoding a.a. 1-588 of AtPAP2 and a.a.163-202 of Tom20-3
492
was amplified separately and then both purified PCR products were combined
493
and a chimeric PCR product was amplified by forward primer of AtPAP2 and
494
reverse primer of Tom20-3. The PCR product was cloned into the pCXSN vector
495
(Chen et al., 2009) to generate pCXSN-P2Tom.
496
For the yeast two-hybrid study, the PCR product of AtPAP2 was digested
497
with NdeI and SalI, and cloned into pGBKT7 vector (Clontech, USA). The PCR
24
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
498
products of MORF2/3/5/6, ∆40aaMORF2, ∆50aaMORF3/5/6 and MORF3-
499
T17/S20/T35A were digested with NdeI and XhoI, and the PCR product of STY8
500
was digested with NdeI and SacI and ligated into the pGADT7 vector (Clontech,
501
USA). For the bimolecular fluorescence complementation (BiFC) assay, the
502
cDNA of AtPAP2 was amplified and digested with SpeI, and cloned into the
503
pSPYNE vector, while the cDNAs of MORF2/3/5/6 were amplified and cut by
504
XbaI and SalI, and cloned into the pSPYCE vector (Walter et al., 2004). For
505
overproduction of recombinant proteins, the PCR products of MORF2/3/5/6,
506
∆40aaMORF2, ∆50aaMORF3/5/6, MORF3-T17/S20/T35A and pF1β were
507
digested with NdeI and SalI and cloned into the pET21a vector (Novagen,
508
Germany), the PCR products of STY8 and STY17 were digested with NcoI and
509
SacI and cloned into pET28a vector (Novagen, Germany). For GFP analysis, the
510
PCR products of MORF2/3/5/6 containing attB sites were transferred into the
511
pDONR201 vector (Life Technologies, USA) by Gateway BP reaction (Life
512
Technologies,
513
pDONR201 vector containing the fragment of interest was transferred into the C-
514
terminal GFP vector (Life Technologies) by Gateway LR reaction (Life
515
Technologies, USA) according to the manufacturer’s instructions.
USA)
according
to
the
manufacturer’s
instructions.
The
516
517
Expression of Recombinant Proteins
518
The coding sequences of STY8 and STY17 were subcloned into the pET28a
519
vector
520
mMORF2/3/5/6 and pMORF3-T17/S20/T35A were subcloned into the pET21a
521
vector (Novagen, Germany). The overexpression and purification of STY8 and
522
STY17 were performed as described by Lamberti et al. (2011). The other
523
recombinant proteins were purified as described by Inoue and Akita (2008).
(Novagen,
Germany);
the
coding
sequences
of
pMORF2/3/5/6,
524
525
Mitochondria Isolation
526
14-day-old seedlings that were grown on MS agar were used for mitochondria
527
isolation as described by Day et al. (1985); Lister et al. (2007). For
25
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
528
immunoblotting analysis, wash buffer (300 mM sucrose and 10 mM TES) without
529
bovine serum albumin (BSA) was used for the final mitochondria wash.
530
531
In Vitro Mitochondrial Protein Import
532
Rabbit reticulocyte TNT in vitro transcription/translation lysate (RRL; Promega,
533
USA) and wheat germ TNT in vitro transcription/translation lysate (WGL;
534
Promega, USA) were used to synthesize [35S]-Met–precursor proteins as
535
previously described (Dessi et al., 2003). The in vitro mitochondrial protein import
536
assay was performed as described by Lister et al. (2007).
537
538
In Vitro Phosphorylation Assay
539
This assay was performed as described (Martin et al., 2006). The recombinant
540
substrate proteins and recombinant STY8 and STY17 kinases were expressed in
541
the E. coli BL21 (PLysS) strain and purified. Two micrograms of recombinant
542
substrate proteins were incubated with 2 µg of recombinant STY8/17, 35 µg of
543
WGL, 200 µg of RRL and 10 µg of LE in the reaction buffer (20 mM Tris-HCl, pH
544
7.5, 5 mM MgCl2, 0.5 mM MnCl2, 2.5 µM ATP) containing 5 µCi of [γ-32P] ATP.
545
The reaction was incubated at 30 °C for 15 minutes and terminated by adding 20
546
µl of 5 × sodium dodecyl sulfate (SDS)-loading buffer. The reaction products
547
were analyzed in a 15% (v/v) sodium dodecyl sulfate-polyacrylamide gel
548
electrophoresis (SDS-PAGE) gel and exposed to Hyperfilm MP (GE Healthcare,
549
USA).
550
551
Yeast Two-hybrid Interaction and BiFC Assays
552
The Clontech Matchmaker two-hybrid system was used according to the
553
manufacturer’s instructions. Positive interactions were verified on triple dropout
554
medium without Trp, Leu or His (-TLH). BiFC assays were performed as
555
described in (Walter et al., 2004). The pSPYNE and pSPYCE vectors containing
556
genes of interest were co-transformed into the leaf cells of tobacco (Nicotiana
557
benthamiana) by Agrobacterium infiltration. Biofluorescence was detected with
558
an LSM710 confocal laser scanning microscope (Zeiss, Germany).
26
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
559
560
Immunoblot Assay
561
Fourteen-day-old wild type Arabidopsis thaliana ecotype Columbia-0 (WT),
562
Arabidopsis thaliana overexpressing AtPAP2 line (OE7), and pap2 T-DNA
563
insertion
564
Mitochondrial proteins were separated on a 10% (w/v) SDS-PAGE gel and
565
transferred to Hybond-C nitrocellulose membranes (GE Healthcare, USA). The
566
membrane containing mitochondrial proteins were immunodetected by anti-
567
AtPAP2 antibodies as described by Sun et al. (2012).
mutant
(Salk_013567)
were
used
for
mitochondrial
isolation.
568
569
Co-immunoprecipitation Assay
570
[35S]-Met–labeled pMORF3 and [35S]-Met–labeled pMORF3-T17/S20/T35A
571
synthesized by RRL (Promega, USA), with or without 15 min treatment of STY8,
572
were incubated with 2 μM JNJ and 10 µg WGL (Promega, USA), which was used
573
as a source of 14-3-3 and HSP70 protein. The mixture were then mixed with anti-
574
14-3-3 (gift from Prof. Carol MacKintosh) and anti-HSP70 (Agrisera, Sweden)
575
antibodies for one hour, and the bound proteins were pulled down using
576
Sepharose-Protein A (Amersham, USA) and detected by autoradiography (Dessi
577
et al., 2003).
578
579
GFP Subcellular Localization Studies
580
Full length MORF2/3/5/6 was cloned into the gateway vector pDONRTM201 and
581
then transferred into a vector containing GFP according to the manufacturer’s
582
instructions (Invitrogen, USA). MORF2/3/5/6 fused to GFP was co-transformed
583
with a positive control containing red fluorescent protein (RFP) into Arabidopsis
584
cell suspensions by the PDS-1000/He biolistic transformation system (Bio-Rad,
585
USA) as described by Xu et al. (2013).
586
587
Plant Transformation
588
The pCXSN-P2Tom vector was transformed into Agrobacterium tumefaciens
589
strain GV3101 by freeze and thaw method as described by Weigel and
27
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
590
Glazebrook (2006). After that, Agrobacterium tumefaciens containing pCXSN-
591
P2Tom was introduced into WT by floral dip method as previously described
592
(Clough and Bent, 1998). Homozygous CaMV35S:P2-Tom overexpression lines
593
were selected on MS plates containing antibiotics as previously described by
594
Chen et al. (2009). Multiple independent expression lines were verified by
595
immunobloting assay using anti-AtPAP2 antibody.
596
597
Phylogenetic Analysis
598
For phylogenetic analysis of PAP2, two steps were conducted to identify PAPs
599
including AtPAP2/9-like proteins across 64 species ranging from algae to higher
600
plants to human with genome sequence available. Firstly, Blastp (e-value ≤ 1e-5)
601
was used to search homologous protein using 42 seed PAP proteins including
602
PAPs with a unique C-terminal hydrophobic motif (C-tail) against non-redundant
603
protein sequences of plants (taxid: 3193), in which all PAP candidate sequences
604
with signals of the five conserved motifs (DxG, GDISY, GNHE, QGHR, and
605
GHVH) within the proteins were identified; secondly, HMM matrix was built for
606
the C-terminal hydrophobic motif of PAP proteins using HMM build, and the
607
candidate PAP sequences in step 1 were used to identify PAP proteins with a C-
608
tail using HMM search, in which the AtPAP2/9-like proteins were identified. For
609
phylogenetic analysis of STY proteins, a similar strategy was implemented. First,
610
Blastp (e-value ≤ 1e-5) was used to search homologous proteins using 20 seed
611
STYs (each with an ACT motif and a kinase activate segment motif) against the
612
64 species protein database, in which STY candidate sequences were identified.
613
Second, to make sure each candidate STY contain the ACT and activate
614
segment motifs, HMM matrix was built for ACT and kinase activate segment
615
motif, respectively, and search the candidate STYs identified in step 1 using
616
HMM search, in which the STY protein set for each species was determined. For
617
phylogenetic analysis of MORF, a similar strategy to that used for PAP and STY
618
was implemented. First, Blastp (e-value ≤ 1e-5) was used to search homologous
619
proteins using 9 seed MORF proteins from Arabidopsis thaliana against the 64
620
species protein database in which the MORF candidate sequences were
28
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2015 American Society of Plant Biologists. All rights reserved.
621
identified. Second, a HMM matrix was used to screen the identified orthologs for
622
a conserved GCT motif.
623
624
Accession Numbers
625
Sequence data from this work can be found in the Arabidopsis Genome Initiative
626
or GenBank/EMBL databases under the following accession numbers:
627
AT1G13900
(AtPAP2),
628
AT1G32580
(MORF5),
629
AT4G35780 (STY17).
AT2G33430
AT2G35240
(MORF2),
(MORF6),
AT3G06790
AT2G17700
(MORF3),
(STY8)
and
630
631
ACKNOWLEDGMENTS
632
YSL, RZ, SC and XG are grateful for the award of a University of Hong Kong
633
PhD scholarship. We thank Dr. Clive Lo (University of Hong Kong) for generous
634
gifts of BiFC vectors.
635
636
637
AUTHOR CONTRIBUTIONS
638
BLL designed the study. RZ generated the P2Tom lines. SF generated the OE
639
lines. SC carried out phylogenetic analysis. XG produced recombinant STY
640
kinases. YSL wrote the manuscript and carried out all the other experiments. OD
641
and MWM developed the mitochondrial import assays. JW provided guidance on
642
this study, and all authors were involved in writing and/or revising the manuscript.
643
644
29
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
645
Figure Legends
646
Figure 1. Interactions of AtPAP2 with MORF proteins as demonstrated by yeast
647
two-hybrid and BiFC assays.
648
A, Yeast two-hybrid assay using MORF2/3/5/6 proteins and AtPAP2 in the
649
double drop-out medium without tryptophan or leucine (-TL) and in the triple
650
drop-out medium without tryptophan, leucine or histidine (-TLH). MORF2 also
651
shows interactions with quadruple drop out medium (-TLHA). B, The interaction
652
is abolished when the presequences were removed (∆40aaMORF2 and
653
∆50aaMORF3/5/6), or when three threonine residues on the presequence were
654
changed to alanine (AD-pMORF3-T17/S20/T35A mutant). The pGBKT7 and
655
pGADT7 vectors were used as negative controls. C, BiFC visualization of
656
interactions between AtPAP2 and MORFs in Agrobacterium-infiltrated tobacco (N.
657
benthamiana) leaves. The co-expression of NYFP-AtPAP2 and MORF2-CYFP
658
reconstituted a functional YFP signal in the chloroplasts. The co-expression of
659
NYFP-AtPAP2 and MORF3/5-CYFP reconstituted a functional YFP signal in the
660
mitochondria.
661
reconstituted functional YFP signals in both mitochondria and chloroplasts. The
662
scale bar indicates 10 µm. The yellow arrow represents a chloroplast; the white
663
arrow represents a mitochondrion.
The
co-expression
of
NYFP-AtPAP2
and
MORF6-CYFP
664
665
Figure 2. Subcellular localizations of MORFs in Arabidopsis cell suspension.
666
GFP was fused to the C-termini of Arabidopsis thaliana MORF2/3/5/6
667
(MORF2/3/5/6-GFP), while RFP was fused to the C-termini of Glycine max
668
mitochondrial alternative oxidase (AOX-RFP) (Carrie et al., 2009) and Pisum
669
sativum small subunit of Rubisco (SSU-RFP) (Carrie et al., 2009). The scale bar
670
indicates 10 µm.
671
672
Figure 3. Phosphorylation of recombinant pMORF3 protein by various kinase
673
sources and the effects of inhibitors.
30
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
674
A, The ability of RRL (200 µg), WGL (35 µg) and LE (10 µg) to phosphorylate
675
recombinant pMORF3 protein in the presence or absence of the kinase inhibitor
676
JNJ was examined.
677
B, The ability of RRL, WGL and LE to dephosphorylate the phosphorylated
678
recombinant pMORF3 protein was analyzed in the presence or absence of the
679
phosphatase inhibitor NaF.
680
C, Recombinant pMORFs, the corresponding mature proteins (mMORFs) and a
681
substitution mutant of pMORF3 (pMORF3-T17/S20/T35A) were produced in E.
682
coli and purified. The recombinant proteins were phosphorylated by recombinant
683
STY8 and STY17 kinases. pF1β was used as a control.
684
685
Figure 4. Mitochondrial import efficiency of RRL-pMORF3 and WGL-pMORF3
686
under various conditions.
687
A, RRL-pMORF3 (lanes 2–3) and WGL-pMORF3 (lanes 7–8) were successfully
688
imported into mitochondria. In the presence of valinomycin, the import of RRL-
689
pMORF3 (lanes 4–5) and WGL-pMORF3 (lanes 9–10) was inhibited. Proteinase
690
K was used to digest non-imported pMORF3 (lanes 3, 5, 8, and 10).
691
B, Pre-incubation of RRL-pMORF3 with WGL (lanes 4–5) and LE (lane 7)
692
reduced the mitochondrial import efficiency. Import experiments were repeated
693
three times and statistical differences were based on one-way ANOVA analysis
694
followed by Tukey Honestly Significant Differences test. The amount of imported
695
mMORF3 without addition of WGL or LE (lane 2) was taken as 1.0. The value
696
marked with different letters (a, b) are significantly different (P < 0.01).
697
C, Pre-incubation of RRL-pMORF3 with NaF (lane 3), STY8 (lane 4) or both
698
(lane 5) reduced the mitochondrial import efficiency. * represents an additional 10
699
minutes of incubation with kinase assay buffer before the in vitro kinase assay.
700
Import experiments were repeated three times and statistical differences were
701
based on one-way ANOVA analysis followed by Tukey Honestly Significant
702
Differences test. The amount of imported mMORF3 without STY8 or NaF
703
treatment (lane 2) was taken as 1.0. The value marked with different letters (a, b,
704
c) are significantly different (P < 0.01).
31
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
705
D, RRL-pMORF3-T17/S20/T35A (lanes 4–5) was imported normally into
706
mitochondria. The amount of imported mMORF3 without NaF treatment (lanes 2
707
and 5) was taken as 1.0. In the presence of NaF, the import rate was reduced.
708
Statistical differences with P < 0.01 (**) were based on student's t test .
709
710
Figure 5. Comparative co-immunoprecipitation and mitochondrial import assays
711
between pMORF3 and pMORF3-T17/S20/T35A.
712
A-B, RRL-pMORF3 and pMORF3-T17/S20/T35A, with or without 15 min
713
treatment with STY8 kinase, were immunoprecipitated with anti-HSP70 (A) and
714
anti-14-3-3 (B) antibodies. Without STY8 treatment, pMORF3 showed a weak
715
interaction with HSP70. In contrast, STY8 treatment of RRL-pMORF3 enhanced
716
pMORF3’s interaction with 14-3-3 and HSP70 but had no effect on pMORF3-
717
T17/S20/T35A. * represents an additional 10 minutes of incubation in kinase
718
assay buffer before the in vitro kinase assay.
719
C, The import efficiencies of RRL-pMORF3 into WT (lanes 2–4), OE7 (lanes 5–7)
720
and pap2 (lanes 8–10) mitochondria. Import experiments were repeated three
721
times and the import rates were not significantly different between mitochondria.
722
D-E, The import efficiencies of WGL-pMORF3 (D) and WGL-pMORF3-
723
T17/S20/T35A (E) into WT, pap2 and OE7 mitochondria. Import experiments
724
were repeated three times. The import rate of WGL-pMORF3 into pap2 (lanes 5–
725
7) mitochondria was significantly reduced, with a P value < 0.05 (*) and 0.01 (**)
726
using student's t test, but the import rates of WGL-pMORF3-T17/S20/T35A into
727
different mitochondria were not significantly different.
728
F, The import rate of WGL-pMORF3-T17/S20/T35A into WT mitochondria was
729
significantly faster than that of WGL-pMORF3, with a P value < 0.01 (**) using
730
student's t test. Import experiments were repeated three times.
731
732
733
734
735
32
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
736
737
Supplementary Data Files:
738
Supplementary Figure S1. Negative controls of subcellular localizations of
739
MORFs in Arabidopsis cell suspension. The scale bar indicates 10 µm.
740
Supplementary Figure S2. Mitochondrial import efficiency of RRL-pMORF3 and
741
RRL-∆50aaMORF3. RRL-pMORF3 (lanes 2-3), but not RRL-∆50aaMORF3 (lane
742
11), was imported into mitochondria. In the presence of valinomycin, import of
743
RRL-pMORF3 (lanes 4-5, 8-9) was inhibited. Mitochondrial outer membrane was
744
ruptured after import (lanes 6-9) and the bands between precursor and mature
745
proteins disappeared in lane 7 (vs. lane 3), indicating that these bands are
746
partially imported pMORF3 that had yet to be translocated into matrix. *
747
represents additional band formed after PK digestion.
748
Supplementary Figure S3. The effects of RRL and WGL on the activities of
749
STY8 kinase. Addition of recombinant STY8 increased the amounts of
750
phosphorylated pMORF3 in presence of RRL (200 µg, lanes 6-7) and WGL (35
751
µg, lanes 10-11), respectively. However, in the presence of RRL, the pMORF3-
752
phosphorylating ability of STY8 is lower (lane 6 vs. lane 1). * represents
753
additional 10 minutes incubation with kinase assay buffer before in vitro kinase
754
assay.
755
Supplementary Figure S4. The expression level of AtPAP2 in mitochondrial
756
protein of WT, OE7 and pap2 T-DNA line.
757
A, Phenotype of 28-d-old plants of WT, OE7 and pap2 T-DNA line.
758
B, Immunodetection of AtPAP2 in different concentration of mitochondrial protein
759
of WT, OE7 and pap2, and separated by SDS-PAGE (C).
760
Supplementary Figure S5. Distribution of STY, PAP and MORF genes.
761
Supplementary Figure S6. Serine is the dominant amino acid at the
762
presequences of MORF family proteins. Multiple alignment of first 66 a.a. of 334
33
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
763
high-confident
MORF
family
proteins
were
analyzed
764
(http://weblogo.berkeley.edu/logo.cgi) (Crooks et al., 2004).
by
WebLogo
765
766
Supplementary Figure S7. Growth phenotypes of P2Tom OE lines.
767
A, Phenotypes of 45-d-old WT, P2Tom3-8 and P2Tom4-4 lines (right). The two
768
P2Tom lines showed an early senescence phenotype (left).
769
B, Seed yield of the WT and P2Tom lines upon maturity. Top panel: Average
770
silique numbers and seed yield per plant (n = 15). Bottom panel: Statistics
771
analysis of seed length and width (n = 15) and 100-seed weight.
772
C, Seed morphology of WT, P2Tom3-8 and P2Tom4-4. ** indicates P < 0.01.
773
774
Supplementary Table S1. Summary of STYs, MORFs and AtPAP2 in different
775
plant sources.
776
Supplementary Table S2. List of primer sequences used for overproduction of
777
recombinant proteins, site-directed mutagenesis, yeast-two hybrid, GFP and
778
BiFC studies.
34
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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