Phosphorylation and Dephosphorylation of the Presequence of Precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during Import into Mitochondria from Arabidopsis
Abstract:The nucleus-encoded mitochondria-targeted proteins, multiple organellar RNA editing factors (MORF3, MORF5, and MORF6), interact with Arabidopsis (Arabidopsis thaliana) PURPLE ACID PHOSPHATASE2 (AtPAP2) located on the chloroplast and mitochondrial outer membranes in a presequence-dependent manner. Phosphorylation of the presequence of the precursor MORF3 (pMORF3) by endogenous kinases in wheat germ translation lysate, leaf extracts, or STY kinases, but not in rabbit reticulocyte translation lysate, resulted in … Show more
“…1B, AtPAP2 interacted with pSSU and mSSU but not the transit peptide in chloroplasts. AtPAP2 was shown to interact with pMORF3 in mitochondria, 3 but no signal in the mitochondria was seen for pSSU/mSSU, indicating that the interactions of AtPAP2 with pSSU/mSSU are specific to chloroplasts. Figure 1.AtPAP2 on outer chloroplast membrane binds pSSU.…”
Section: Atpap2 On the Outer Membrane Of Chloroplasts Recognizes Pssumentioning
confidence: 90%
“…AtPAP2 was shown to interact with the full-length sequence of pMORF3 but not with the mature protein of MORF (mMORF3), indicating that it recognizes the presequence of pMORF3 3 . To examine whether AtPAP2 interacts with AtSSU1B (AT5G38430), yeast 2-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were carried out.…”
Section: Atpap2 On the Outer Membrane Of Chloroplasts Recognizes Pssumentioning
confidence: 99%
“…Both of these complexes contain channel proteins (Toc75/Tom40) and distinct receptors (Toc33/34, Toc159, and Toc64 on chloroplasts, and Tom20 and OM64 on mitochondria). A number of preproteins targeted to chloroplasts and mitochondria can be phosphorylated at their transit peptides/presequences 3-5 . To import pSSU and pMORF3 in their complex forms through the TOC and TOM, respectively, 2 additional steps are required: First pSSU/pMORF3 must be dissociated from 14-3-3 and expose their transit peptide/presequence, followed by the dephosphorylation of the transit peptide/presequence.…”
Section: Conclusion and Perspectivementioning
confidence: 99%
“…Multiple layers of control, including reversible phosphorylation at the transit peptides/presequenes of preproteins, are involved in fine tuning the translocation processes 2,3 . Cytosolic STY kinases were shown to phosphorylate the Ser34 residue of the transit peptide of tobacco small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, SSU) 4,5 and the Thr20/Ser27/Thr35 residues of the presequence of Multiple Organellar RNA Editing Factor 3 (pMORF3), 3 which are then bound by 14-3-3 3,6 . NaF, a broad-range phosphatase inhibitor, inhibits the importing of pSSU and pMORF3 into chloroplasts and mitochondria, respectively.…”
mentioning
confidence: 99%
“…AtPAP2 was shown to interact with the presequences of a number of pMORF proteins, and the import of STY8-treated, presequence-phosphorylated pMORF3 into mitochondria isolated from AtPAP2 T-DNA line was slower than in mitochondria isolated from the wild-type plants 3 . The goal of this study was to examine if AtPAP2 also interacts with pSSU and modulates its import into chloroplasts.…”
Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) is the only phosphatase that is dual-targeted to both chloroplasts and mitochondria. Like Toc33/34 of the TOC and Tom 20 of the TOM, AtPAP2 is anchored to the outer membranes of chloroplasts and mitochondria via a hydrophobic C-terminal motif. AtPAP2 on the mitochondria was previously shown to recognize the presequences of several nuclear-encoded mitochondrial proteins and modulate the import of pMORF3 into the mitochondria. Here we show that AtPAP2 binds to the small subunit of Rubisco (pSSU) and that chloroplast import experiments demonstrated that pSSU was imported less efficiently into pap2 chloroplasts than into wild-type chloroplasts. We propose that AtPAP2 is an outer membrane-bound phosphatase receptor that facilitates the import of selected proteins into chloroplasts.
“…1B, AtPAP2 interacted with pSSU and mSSU but not the transit peptide in chloroplasts. AtPAP2 was shown to interact with pMORF3 in mitochondria, 3 but no signal in the mitochondria was seen for pSSU/mSSU, indicating that the interactions of AtPAP2 with pSSU/mSSU are specific to chloroplasts. Figure 1.AtPAP2 on outer chloroplast membrane binds pSSU.…”
Section: Atpap2 On the Outer Membrane Of Chloroplasts Recognizes Pssumentioning
confidence: 90%
“…AtPAP2 was shown to interact with the full-length sequence of pMORF3 but not with the mature protein of MORF (mMORF3), indicating that it recognizes the presequence of pMORF3 3 . To examine whether AtPAP2 interacts with AtSSU1B (AT5G38430), yeast 2-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were carried out.…”
Section: Atpap2 On the Outer Membrane Of Chloroplasts Recognizes Pssumentioning
confidence: 99%
“…Both of these complexes contain channel proteins (Toc75/Tom40) and distinct receptors (Toc33/34, Toc159, and Toc64 on chloroplasts, and Tom20 and OM64 on mitochondria). A number of preproteins targeted to chloroplasts and mitochondria can be phosphorylated at their transit peptides/presequences 3-5 . To import pSSU and pMORF3 in their complex forms through the TOC and TOM, respectively, 2 additional steps are required: First pSSU/pMORF3 must be dissociated from 14-3-3 and expose their transit peptide/presequence, followed by the dephosphorylation of the transit peptide/presequence.…”
Section: Conclusion and Perspectivementioning
confidence: 99%
“…Multiple layers of control, including reversible phosphorylation at the transit peptides/presequenes of preproteins, are involved in fine tuning the translocation processes 2,3 . Cytosolic STY kinases were shown to phosphorylate the Ser34 residue of the transit peptide of tobacco small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, SSU) 4,5 and the Thr20/Ser27/Thr35 residues of the presequence of Multiple Organellar RNA Editing Factor 3 (pMORF3), 3 which are then bound by 14-3-3 3,6 . NaF, a broad-range phosphatase inhibitor, inhibits the importing of pSSU and pMORF3 into chloroplasts and mitochondria, respectively.…”
mentioning
confidence: 99%
“…AtPAP2 was shown to interact with the presequences of a number of pMORF proteins, and the import of STY8-treated, presequence-phosphorylated pMORF3 into mitochondria isolated from AtPAP2 T-DNA line was slower than in mitochondria isolated from the wild-type plants 3 . The goal of this study was to examine if AtPAP2 also interacts with pSSU and modulates its import into chloroplasts.…”
Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) is the only phosphatase that is dual-targeted to both chloroplasts and mitochondria. Like Toc33/34 of the TOC and Tom 20 of the TOM, AtPAP2 is anchored to the outer membranes of chloroplasts and mitochondria via a hydrophobic C-terminal motif. AtPAP2 on the mitochondria was previously shown to recognize the presequences of several nuclear-encoded mitochondrial proteins and modulate the import of pMORF3 into the mitochondria. Here we show that AtPAP2 binds to the small subunit of Rubisco (pSSU) and that chloroplast import experiments demonstrated that pSSU was imported less efficiently into pap2 chloroplasts than into wild-type chloroplasts. We propose that AtPAP2 is an outer membrane-bound phosphatase receptor that facilitates the import of selected proteins into chloroplasts.
Key message
P-subfamily PPR protein OsPPR939, which can be phosphorylated by OsS6K1, regulates plant growth and pollen development by involving in the splicing of mitochondrial nad5 introns 1, 2, and 3.
Abstract
In land plants, pentatricopeptide repeat (PPR) proteins play key roles in mitochondrial group II intron splicing, but how these nucleus-encoded proteins are imported into mitochondria is unknown. To date, a few PPR proteins have been characterized in rice (Oryza sativa). Here, we demonstrate that the mitochondrion-localized P-subfamily PPR protein OsPPR939 is required for the splicing of nad5 introns 1, 2, and 3 in rice. Complete knockout or partial disruption of OsPPR939 function resulted in different degrees of growth retardation and pollen sterility. The dramatically reduced splicing efficiency of these introns in osppr939-4 and osppr939-5 led to reduced mitochondrial complex I abundance and activity and enhanced expression of alternative respiratory pathway genes. Complementation with OsPPR939 rescued the defective plant morphology of osppr939-4 and restored its decreased splicing efficiency of nad5 introns 1, 2, and 3. Therefore, OsPPR939 plays crucial roles in plant growth and pollen development by splicing mitochondrial nad5 introns 1, 2, and 3. More importantly, the 12th amino acid Ser in the N-terminal targeting sequence of OsPPR939 is phosphorylated by OsS6K1, and truncated OsPPR939 with a non-phosphorylatable S12A mutation in its presequence could not be imported into mitochondria, suggesting that phosphorylation of this amino acid plays an important role in the mitochondrial import of OsPPR939. To our knowledge, the 12th residue Ser on OsPPR939 is the first experimentally proven phosphorylation site in PPR proteins. Our results provide a basis for investigating the regulatory mechanism of PPR proteins at the post-translational level.
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