PLANT SIGNALING & BEHAVIOR 2016, VOL. 11, NO. 9, e1221558 (5 pages) http://dx.doi.org/10.1080/15592324.2016.1221558
SHORT COMMUNICATION
Golgi-to-plastid trafficking of proteins through secretory pathway: Insights into vesicle-mediated import toward the plastids Marouane Baslama,b, Kazusato Oikawaa, Aya Kitajima-Kogaa, Kentaro Kanekob, and Toshiaki Mitsuia,b a Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Niigata, Japan; bGraduate School of Science and Technology, Niigata University, Ikarashi, Niigata, Japan
ABSTRACT
ARTICLE HISTORY
The diversity of protein targeting pathways to plastids and their regulation in response to developmental and metabolic status is a key issue in the regulation of cellular function in plants. The general import pathways that target proteins into and across the plastid envelope with changes in gene expression are critical for plant development by regulating the response to physiological and metabolic changes within the cell. Glycoprotein targeting to complex plastids involves routing through the secretory pathway, among others. However, the mechanisms of trafficking via this system remain poorly understood. The present article discusses our results in site-specific N-glycosylation of nucleotide pyrophosphatase/ phosphodiesterases (NPPs) glycoproteins and highlights protein delivery in Golgi/plastid pathway via the secretory pathway. Furthermore, we outline the hypotheses that explain the mechanism for importing vesicles trafficking with nucleus-encoded proteins into plastids.
Received 20 June 2016 Revised 31 July 2016 Accepted 1 August 2016
Abbreviations: Amy, amylase; CAH, carbonic anhydrase; COP, coat protein complex; ER, endoplasmic reticulum;
ES, endoplasmic system; GFP, green fluorescent protein; MITS1, mitochondrial-targeting signal 1; NPPs, nucleotide pyrophosphatase/phosphodiesterases; TOC, translocons in the outer envelope membrane of chloroplasts; TIC, translocons in the inner envelope membrane of chloroplasts
Organisms are exposed to a wide range of environmental perturbations, including both short-term and longer-term changes. Plants have developed capabilities to regulate their physiological and developmental states through genome-wide gene expression programs which act in response to environmental fluctuations by relaying signals that adjust the accumulation of proteins in different cellular compartments. In vesicle trafficking, newly synthesized proteins are delivered to the appropriate intracellular compartments, or are secreted extracellularly. More than 95% of plastid and mitochondrial proteins are nuclear genome encoded, synthesized on cytosolic ribosomes as precursor proteins and post-translationally imported into the target organelle. The targeting of proteins to specific subcellular organelles is directed by N-terminal presequences, such as chloroplast transit and mitochondrial targeting peptides. Precursors are imported by translocases of the outer and inner envelope membranes of chloroplasts, TOC and TIC complexes, at the expense of internal ATP. Finally, after import, processing and folding of the precursor protein takes place inside the target organelle.1 Previously, TOC/TICindependent pathways for chloroplast glycoprotein targeting have been identified. However, proteins have been reported to use alternative translocation pathways, such examples indicating that the TOC/TIC machinery is not exclusively responsible for transport of proteins into plastids. These proteins are most likely transported via vesicles from the CONTACT Toshiaki Mitsui Japan
[email protected]
KEYWORDS
Glycoprotein; golgi-vesicle budding; invagination; nucleotide pyrophosphatase/ phosphodiesterases; N-glycoproteomes; pass-through model; signal peptide; trans-Golgi compartments; vesiclemediated pathway
ER to the Golgi and further to the plastid envelope bypassing the TOC/TIC system,2 which becomes N-glycosylated in the ER.3 Recently, we reported that the trans-Golgi compartments participate in the Golgi-to-plastid trafficking and targeting mechanism of nucleotide pyrophosphatase/phosphodiesterases (NPPs). One other possibility that we raised in our discussion of the results characterizing NPP 1, 2 and 6, is further reinforced here (Fig. 1). As shown in Fig. 1A, the fluorescence distribution patterns of NPP1-GFP, NPP2-GFP and NPP6-GFP largely overlaps with those of chlorophyll autofluorescence. Fluorescence spectroscopy measurements emitted by each NPP-GFP form showed that ca. 73–82% was localized in the chloroplasts of transformed cells.4 This localization pattern was further supported using onion epidermal cells transiently expressing NPP-GFP.4 It is worth noting that the remaining (18–27%) background signal was distributed among the ER, Golgi apparatus and/or the other endomembrane systems. These results strongly suggest that NPP glycoproteins localize in the plastid. N-linked glycosylation is an important, prominent and abundant posttranslational modification in the cell. However to date, little is known about the N-glycoproteomes of plants.5 The N-glycans that are covalently linked to proteins are involved in numerous biological processes. The details of N-glycosylation modification on the plastidial NPP proteins were investigated. As shown by the data presented in Fig. 1B
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Niigata 9502181,
Published with license by Taylor & Francis Group, LLC © Marouane Baslam, Kazusato Oikawa, Aya Kitajima-Koga, Kentaro Kaneko, and Toshiaki Mitsui This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
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Figure 1. (A) Fluorescence images of chloroplasts with small vesicles in rice cells expressing NPP1-GFP, NPP2-GFP and NPP6-GFP. The stable transformant cells were sectioned with a vibratome to a thickness of 25 mm, and observed by confocal laser scanning microscopy. (a) to (c), (d) to (f) and (g) to (i) represent rice cells transformed with NPP1-GFP, NPP2-GFP and NPP6-GFP, respectively. [a], [d] and [g] GFP fluorescence; [b], [e] and [h] chlorophyll autofluorescence; [c], [f] and [i] GFP and chlorophyll autofluorescence merged. Bars D 5 mm. Non-merged vesicle-like fluorescence of NPP-GFP (arrows) can be seen in both the surface and the interior of plastids. (B) Determination of N-glycosylation sites in NPP1 by Mass Spectrometry. The trypsin-digested NPP1 was subjected to the MS2/MS3 analyses. The three glycopeptides conjugated with HexNAc (m/z 1038.93, 1144.96 and 936.93) were detected in the MS2spectra. Furthermore, the MS3 analyses showed the N-glycosylation sites, those are 124Asn, 326 Asn and 579Asn. (C) MS3 spectrum of a glycopeptide consisting of glycan attached to LTAFNHSSLLFEYK (parent mass m/z 1348.12, z D 2) derived from NPP1. y, y ion; b, b ion; yo, y ion that has lost water; bo, b ion that has lost water. C1 and C2 display singly and doubly charged fragment ions, respectively.
PLANT SIGNALING & BEHAVIOR
and 1C, it was found that 3 glycopeptides conjugated with HexNAc were detected in rice cells. MS3 spectrometric analyses revealed 3 N-glycosylation sites at positions 124Asn, 326Asn and 579 Asn of NPP1. These N-glycans could act upon protein structure and dynamics on the folded glycoproteins. The SignalP and PSORT algorithms showed that all the deduced amino acid sequences of NPP 1, 2, and 6 have cleavable hydrophobic N-terminal extensions that act as potential signal peptides to the ER and putative N-glycosylation sites. The glycosylation site of 579Asn was conserved in all NPP polypeptides, although the other sites, 124Asn and 326Asn, were characteristic for NPP1 and 6 polypeptides. The N-glycome analyses of NPPs revealed differential a(1,3)-fucosylation of glycan chains. The contents of the oligosaccharide chains with fucose residues in NPP 1, 2 and 6 were calculated to be approximately 55, 44 and 73%, respectively. These results show the occurrence of differential and selective glycosylation in each NPP isozyme. In plant cells, fucose transfer to oligosaccharides by fucosyltransferases has been shown to be located in the late compartments of the Golgi apparatus. Our previous study revealed that the NPP1 protein synthesized in the wheat germ cell-free translation system had no enzymatic activity, probably indicating that the conjugation of glycan chains is required for the expression of enzyme function.6 Furthermore, Buren et al. (2011),7 who performed a series of in vivo analyses of mutant CAH1 a-type carbonic anhydrase with disrupted glycosylation sites, demonstrated that Arabidopsis unglycosylated CAH1 misfolded and lost its enzyme activity. However, the details of terminal sugar characters of plant Nglycans related to their functions remain to be elucidated. Scanning for signal peptides of 916 nuclear-encoded plastid proteins in Arabidopsis suggested that part of total proteins are targeted via the secretory system.8 The glycoproteins targeted from the Golgi apparatus through the secretory pathway to the chloroplast of higher plants have been described: a-amylase I-1 (AmyI-1),2,9 manganese superoxide dismutase 1 (MSD1),10 NPP16 and more recently, NPP 2 and 64, and an a-type carbonic anhydrase 1 (CAH1).7 The N-glycosylated protein AmyI-1, MSD1 and NPP 1, 2 and 6 were localized in rice plastids, while CAH1 was identified in Arabidopsis chloroplasts. The passage of these proteins through the secretory pathway en route to plastids was discovered using a combination of cellbiological techniques, including the analysis of trafficking of green-fluorescent-protein (GFP) fusion proteins in the presence of pharmacological agents that disrupt Golgi trafficking and high-pressure frozen/freeze-substituted techniques. Trafficking these proteins through the secretory pathway before entering the chloroplast might be related to protein folding11,12 and the need for post-translational modifications, such as Nglycosylation, for proper folding, and to enhance the stability and/or function of these proteins.7 Given the functional role of N-glycosylation for these proteins, it seems likely that their glycosylation status serves as a signal of the overall energy status of the cell and thus contributes to the regulation of carbon metabolism.13 Moreover, recent studies have provided information concerning protein N-glycan structures and their pathways in different phyla.14-19 Phylogenetic analyses of higher plant proteins directed to plastids via the endomembrane system (ES); CAH1, NPP1, aAmy7, and protein disulfide isomerase (RB60) from the green alga Chlamydomonas reinhardtii – used
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for its peculiar capacity for dual post- and co-translational targeting to both the plastid and endomembrane proteins revealed that vesicular trafficking of these proteins to plastids evolved long after cyanobacterial endosymbiosis to permit their glycosylation and/or transport to more than one cellular compartment.20 Interestingly, some glycoproteins are transported to the mitochondria from the ER-Golgi system through the secretory pathway. The mechanism for targeting of a plant protein, mitochondrial-targeting signal 1 (MITS1), to mitochondria has been reported.21 The successful targeting of Arabidopsis thaliana MITS1 is influenced by an N-terminal extension serving as a targeting peptide as well as by domains in the full-length protein. Functional dissection of the MITS1 N-terminal extension has shown the existence of 3 regions that coordinate the mitochondrial targeting signal, including a cryptic signal for protein targeting to the secretory pathway.21 Furthermore, the N-termini of the NADPH: protochlorophylide oxidoreductase A (PORA) from barley etioplasts carries a shorter transit peptidelike presequence22 but is translocated across the outer plastid envelope through the OEP16-1 pore.23 Such proteins do not carry a transit peptide but a signal peptide instead and are transported via ER and Golgi in vesicle-mediated pathways. Transport between the ER and the Golgi apparatus is bidirectional and believed to be mediated by different coated vesicles. Coat protein complex I (COPI) and COPII vesicles are respectively thought to function in the retrograde and anterograde, membrane traffic between the ER and the Golgi apparatus.24-26 These proteins are presumably transported across the remaining membranes in a TOC/TICindependent manner. Flores-Perez and Jarvis reported that TOC/TIC machinery is not believed to be able to translocate such bulky molecules.27 As in primary plastids, there is no need for members of the TOC machinery to transport glycoproteins because the addition of glycans occurs exclusively in the ER and Golgi and stromal glycoproteins necessarily use a vesiclemediated route. Given this lack of specific information about glycoprotein import into plastids, Fig. 2 depicts the 3 possibilities of vesicle-reltaed protein transport from Golgi to chloroplast. In the fusing/budding model (Fig 2A), the translocation across the outer envelope membrane of chloroplasts could be achieved by fusion of post-Golgi vesicles with the outer chloroplast membrane, thereby releasing the glycosylated protein into the intermembrane space. Subsequent targeting of cargo across the inner membrane could involve the TIC complex, an unknown transporter in the inner envelope membrane or a second vesicle budding from the inner membrane itself.28 The complication with this hypothesis is that the Golgi vesicle membrane is likely inserted within the outer envelope membrane (OM) of the plastid. In contrast, no signal of GFP-tagged proteins was observed at the chloroplast periphery. Another hypothesis suggests that Golgi-vesicles may target plastid through an invagination model (Fig 2B). However, if microautophagic vacuole invagination were involved in the uptake of membrane vesicles, vesicles encircled by the plastid envelope membranes in addition to small invagination would be frequently detected in the plastid stroma. However, distinctive vesicles such as these were scarce, suggesting that an as yet undetermined vesicle uptake mechanism might operate in
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Figure 2. Hypotheses for vesicle-mediated import into plastids in plants. (A) Fusing/budding model, (B) Invagination model and (C) Pass-through model. See text for details concerning the 3 models (A, B and C) on glycoprotein import into plastid stroma. GV, Golgi vesicle; IM, inner envelope membrane; OM, outer envelope membrane.
protein importation into plastids. The imported vesicles are perhaps subsequently broken up in the organelle. The moment of plastid entry of membrane vesicles was seen in EM observations using quick-frozen cells,2 suggesting that the vesiclemediated manner might pass through the plastid envelope membranes by a mechanism other than by a budding model or microautophagic vacuole invagination into the organelle (Fig 2C). Consistent with this pass-through model, plastid-targeted protein can freely diffuse through pores or through a non-specific protein transporter. Recently, Sugano et al.29 speculated that OsVAMP714, an intracellular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), can play a role in membrane fusing between the Golgi apparatus and chloroplasts since SNARE protein functions are closely linked to subcellular localizations of the imported glycoprotein cargo into plastids. Additional studies are needed to fully understand how nuclear-encoded proteins are imported into the plastids. In plants, the sorting of proteins in the cell is more complex due to the presence of different organelles, and hence, different types of membranes. Herein, we showed that not all nuclear-encoded plastid proteins are targeted to the canonical TOC/TIC complex. Proteins presenting an N-terminal signal peptide are often directed to the secretory pathway,
translocated into the ER, and sorted for transport to external space including the cell wall or vacuole, or retained in the ER, Golgi, and plasma membranes. Proteins without an ER signal sequence are translated into the cytosol, and if equipped with appropriate targeting signals, can be transported into particular organelles such as the plastids or mitochondria. The secretory pathway is a further notable example of the diverse array of routes that have evolved to mediate protein trafficking to plastids and how plant cells have evolved several chloroplast import pathways working in parallel. An inventory of proteins involved in vesicular trafficking via the secretory pathway and models involved in the entry of vesicles into plastids have been herein compiled. The routes that mediate plastid–nuclear communication, the characterization of cross-signaling pathways and the elucidation of vesiclemediated import into plastids will provide important clues as to the overall nature of how plastids provide essential metabolic and signaling functions within all plant cells in response to constantly changing environmental conditions.
Disclosure of potential conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
PLANT SIGNALING & BEHAVIOR
Acknowledgment We apologize to those colleagues whose work is not cited due to space limitations. This research was supported by KAKENHI Grants-in-Aid for Scientific Research (A) (15H02486) from Japan Society for the Promotion of Sciences and Grant for Promotion of KAAB Projects (Niigata University) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
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13. Lehtim€aki N, Koskela MM, Mulo P. Posttranslational modifications of chloroplast proteins: An emerging field. Plant Physiol 2015; 168:76875; http://dx.doi.org/10.1104/pp.15.00117 14. Decker EL, Reski R. Glycoprotein production in moss bioreactors. Plant Cell Rep 2011; 31:453-60; PMID:21960098; http://dx.doi.org/ 10.1007/s00299-011-1152-5 15. Mathieu-Rivet E, Kiefer-Meyer MC, Vanier G, Ovide C, Burel C, Lerouge P, Bardor M. Protein N-glycosylation in eukaryotic microalgae and its impact on the production of nuclear expressed biopharmaceuticals. Front Plant Sci 2014; 5:359; PMID:25183966; http://dx.doi. org/10.3389/fpls.2014.00359 16. Ruiz-May E, Hucko S, Howe KJ, Zhang S, Sherwood RW, Thannhauser TW, Rose JK. A comparative study of lectin affinity based plant N-glycoproteome profiling using tomato fruit as a model. Mol Cell Proteomics 2014; 13:566-79; PMID:24198434; http://dx.doi.org/ 10.1074/mcp.M113.028969 17. Lerouge P, Cabanes-Macheteau M, Rayon C, Fischette-Laine AC, Gomord V, Faye L. N-Glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol Biol 1998; 38:31-48; PMID:9738959; http://dx.doi.org/10.1023/A:1006012005654 18. Zielinska DF, Gnad F, Wisniewski JR, Mann M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell 2010; 141:897-907; PMID:20510933; http://dx.doi. org/10.1016/j.cell.2010.04.012 19. Mathieu-Rivet E, Scholz M, Arias C, Dardelle F, Schulze S, Le Mauff F, Teo G, Hochmal AK, Blanco-Rivero A, Loutelier-Bourhis C, et al. Exploring the N-glycosylation pathway in Chlamydomonas reinhardtii unravels novel complex structures. Mol Cell Proteomics 2013; 12:3160-83; PMID:23912651; http://dx.doi.org/ 10.1074/mcp.M113.028191 20. Gagat P, Body» A, Mackiewicz P. How protein targeting to primary plastids via the endomembrane system could have evolved? A new hypothesis based on phylogenetic studies. Biol Direct 2013; 8:18; PMID:23845039; http://dx.doi.org/10.1186/1745-6150-8-18 21. Chatre L, Matheson LA, Jack AS, Hanton SL, Brandizzi F. Efficient mitochondrial targeting relies on co-operation of multiple protein signals in plants. J Exp Bot 2009; 60:741-9; PMID:19112171; http://dx. doi.org/10.1093/jxb/ern319 22. Pl€ oscher M, Reisinger V, Eichacker LA. Proteomic comparison of etioplast and chloroplast protein complexes. J Proteomics 2011; 74:1256-65; PMID:21440687; http://dx.doi.org/10.1016/j.jprot.2011. 03.020 23. Samol I, Rossig C, Buhr F, Springer A, Pollmann S, Lahroussi A, von Wettstein D, Reinbothe C, Reinbothe S. The outer chloroplast envelope protein OEP16-1 for plastid import of NADPH:protochlorophyllide oxidoreductase A in Arabidopsis thaliana. Plant Cell Physiol 2011; 52:96-111; PMID:21098556; http://dx.doi.org/10.1093/pcp/ pcq177 24. Hawes C, Satiat-Jeunemaitre B. The plant Golgi apparatus–going with the flow. Biochim Biophys Acta 2005; 1744:93-107; PMID:15922463; http://dx.doi.org/10.1016/j.bbamcr.2005.03.009 25. Faso C, Boulaflous A, Brandizzi F. The plant Golgi apparatus: last 10 years of answered and open questions. FEBS Lett 2009; 583:3752-7; PMID:19800330; http://dx.doi.org/10.1016/j.febslet.2009.09.046 26. Brandizzi F, Barlowe C. Organization of the ER-Golgi interface for membrane traffic control. Nat Rev Mol Cell Biol 2013; 14:382-92; PMID:23698585; http://dx.doi.org/10.1038/nrm3588 Jarvis P. Molecular chaperone involvement in chloro27. Flores-Perez U, plast protein import. Biochim Biophys Acta 2013; 1833:332-40; PMID: 22521451; http://dx.doi.org/10.1016/j.bbamcr.2012.03.019 28. Radhamony RN, Theg SM. Evidence for an ER to Golgi to chloroplast protein transport pathway. Trends Cell Biol 2006; 16:385-7; PMID:16815014; http://dx.doi.org/10.1016/j.tcb.2006.06.003 29. Sugano S, Hayashi N, Kawagoe Y, Mochizuki S, Inoue H, Mori M, Nishizawa Y, Jiang CJ, Matsui M, Takatsuji H. Rice OsVAMP714, a membrane-trafficking protein localized to the chloroplast and vacuolar membrane, is involved in resistance to rice blast disease. Plant Mol Biol 2016; 91:81-95; PMID:26879413; http://dx.doi.org/10.1007/ s11103-016-0444-0
SHORT COMMUNICATION
Golgi-to-plastid trafficking of proteins through secretory pathway: Insights into vesicle-mediated import toward the plastids Marouane Baslama,b, Kazusato Oikawaa, Aya Kitajima-Kogaa, Kentaro Kanekob, and Toshiaki Mitsuia,b a Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Niigata, Japan; bGraduate School of Science and Technology, Niigata University, Ikarashi, Niigata, Japan
ABSTRACT
ARTICLE HISTORY
The diversity of protein targeting pathways to plastids and their regulation in response to developmental and metabolic status is a key issue in the regulation of cellular function in plants. The general import pathways that target proteins into and across the plastid envelope with changes in gene expression are critical for plant development by regulating the response to physiological and metabolic changes within the cell. Glycoprotein targeting to complex plastids involves routing through the secretory pathway, among others. However, the mechanisms of trafficking via this system remain poorly understood. The present article discusses our results in site-specific N-glycosylation of nucleotide pyrophosphatase/ phosphodiesterases (NPPs) glycoproteins and highlights protein delivery in Golgi/plastid pathway via the secretory pathway. Furthermore, we outline the hypotheses that explain the mechanism for importing vesicles trafficking with nucleus-encoded proteins into plastids.
Received 20 June 2016 Revised 31 July 2016 Accepted 1 August 2016
Abbreviations: Amy, amylase; CAH, carbonic anhydrase; COP, coat protein complex; ER, endoplasmic reticulum;
ES, endoplasmic system; GFP, green fluorescent protein; MITS1, mitochondrial-targeting signal 1; NPPs, nucleotide pyrophosphatase/phosphodiesterases; TOC, translocons in the outer envelope membrane of chloroplasts; TIC, translocons in the inner envelope membrane of chloroplasts
Organisms are exposed to a wide range of environmental perturbations, including both short-term and longer-term changes. Plants have developed capabilities to regulate their physiological and developmental states through genome-wide gene expression programs which act in response to environmental fluctuations by relaying signals that adjust the accumulation of proteins in different cellular compartments. In vesicle trafficking, newly synthesized proteins are delivered to the appropriate intracellular compartments, or are secreted extracellularly. More than 95% of plastid and mitochondrial proteins are nuclear genome encoded, synthesized on cytosolic ribosomes as precursor proteins and post-translationally imported into the target organelle. The targeting of proteins to specific subcellular organelles is directed by N-terminal presequences, such as chloroplast transit and mitochondrial targeting peptides. Precursors are imported by translocases of the outer and inner envelope membranes of chloroplasts, TOC and TIC complexes, at the expense of internal ATP. Finally, after import, processing and folding of the precursor protein takes place inside the target organelle.1 Previously, TOC/TICindependent pathways for chloroplast glycoprotein targeting have been identified. However, proteins have been reported to use alternative translocation pathways, such examples indicating that the TOC/TIC machinery is not exclusively responsible for transport of proteins into plastids. These proteins are most likely transported via vesicles from the CONTACT Toshiaki Mitsui Japan
[email protected]
KEYWORDS
Glycoprotein; golgi-vesicle budding; invagination; nucleotide pyrophosphatase/ phosphodiesterases; N-glycoproteomes; pass-through model; signal peptide; trans-Golgi compartments; vesiclemediated pathway
ER to the Golgi and further to the plastid envelope bypassing the TOC/TIC system,2 which becomes N-glycosylated in the ER.3 Recently, we reported that the trans-Golgi compartments participate in the Golgi-to-plastid trafficking and targeting mechanism of nucleotide pyrophosphatase/phosphodiesterases (NPPs). One other possibility that we raised in our discussion of the results characterizing NPP 1, 2 and 6, is further reinforced here (Fig. 1). As shown in Fig. 1A, the fluorescence distribution patterns of NPP1-GFP, NPP2-GFP and NPP6-GFP largely overlaps with those of chlorophyll autofluorescence. Fluorescence spectroscopy measurements emitted by each NPP-GFP form showed that ca. 73–82% was localized in the chloroplasts of transformed cells.4 This localization pattern was further supported using onion epidermal cells transiently expressing NPP-GFP.4 It is worth noting that the remaining (18–27%) background signal was distributed among the ER, Golgi apparatus and/or the other endomembrane systems. These results strongly suggest that NPP glycoproteins localize in the plastid. N-linked glycosylation is an important, prominent and abundant posttranslational modification in the cell. However to date, little is known about the N-glycoproteomes of plants.5 The N-glycans that are covalently linked to proteins are involved in numerous biological processes. The details of N-glycosylation modification on the plastidial NPP proteins were investigated. As shown by the data presented in Fig. 1B
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Niigata 9502181,
Published with license by Taylor & Francis Group, LLC © Marouane Baslam, Kazusato Oikawa, Aya Kitajima-Koga, Kentaro Kaneko, and Toshiaki Mitsui This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.
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Figure 1. (A) Fluorescence images of chloroplasts with small vesicles in rice cells expressing NPP1-GFP, NPP2-GFP and NPP6-GFP. The stable transformant cells were sectioned with a vibratome to a thickness of 25 mm, and observed by confocal laser scanning microscopy. (a) to (c), (d) to (f) and (g) to (i) represent rice cells transformed with NPP1-GFP, NPP2-GFP and NPP6-GFP, respectively. [a], [d] and [g] GFP fluorescence; [b], [e] and [h] chlorophyll autofluorescence; [c], [f] and [i] GFP and chlorophyll autofluorescence merged. Bars D 5 mm. Non-merged vesicle-like fluorescence of NPP-GFP (arrows) can be seen in both the surface and the interior of plastids. (B) Determination of N-glycosylation sites in NPP1 by Mass Spectrometry. The trypsin-digested NPP1 was subjected to the MS2/MS3 analyses. The three glycopeptides conjugated with HexNAc (m/z 1038.93, 1144.96 and 936.93) were detected in the MS2spectra. Furthermore, the MS3 analyses showed the N-glycosylation sites, those are 124Asn, 326 Asn and 579Asn. (C) MS3 spectrum of a glycopeptide consisting of glycan attached to LTAFNHSSLLFEYK (parent mass m/z 1348.12, z D 2) derived from NPP1. y, y ion; b, b ion; yo, y ion that has lost water; bo, b ion that has lost water. C1 and C2 display singly and doubly charged fragment ions, respectively.
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and 1C, it was found that 3 glycopeptides conjugated with HexNAc were detected in rice cells. MS3 spectrometric analyses revealed 3 N-glycosylation sites at positions 124Asn, 326Asn and 579 Asn of NPP1. These N-glycans could act upon protein structure and dynamics on the folded glycoproteins. The SignalP and PSORT algorithms showed that all the deduced amino acid sequences of NPP 1, 2, and 6 have cleavable hydrophobic N-terminal extensions that act as potential signal peptides to the ER and putative N-glycosylation sites. The glycosylation site of 579Asn was conserved in all NPP polypeptides, although the other sites, 124Asn and 326Asn, were characteristic for NPP1 and 6 polypeptides. The N-glycome analyses of NPPs revealed differential a(1,3)-fucosylation of glycan chains. The contents of the oligosaccharide chains with fucose residues in NPP 1, 2 and 6 were calculated to be approximately 55, 44 and 73%, respectively. These results show the occurrence of differential and selective glycosylation in each NPP isozyme. In plant cells, fucose transfer to oligosaccharides by fucosyltransferases has been shown to be located in the late compartments of the Golgi apparatus. Our previous study revealed that the NPP1 protein synthesized in the wheat germ cell-free translation system had no enzymatic activity, probably indicating that the conjugation of glycan chains is required for the expression of enzyme function.6 Furthermore, Buren et al. (2011),7 who performed a series of in vivo analyses of mutant CAH1 a-type carbonic anhydrase with disrupted glycosylation sites, demonstrated that Arabidopsis unglycosylated CAH1 misfolded and lost its enzyme activity. However, the details of terminal sugar characters of plant Nglycans related to their functions remain to be elucidated. Scanning for signal peptides of 916 nuclear-encoded plastid proteins in Arabidopsis suggested that part of total proteins are targeted via the secretory system.8 The glycoproteins targeted from the Golgi apparatus through the secretory pathway to the chloroplast of higher plants have been described: a-amylase I-1 (AmyI-1),2,9 manganese superoxide dismutase 1 (MSD1),10 NPP16 and more recently, NPP 2 and 64, and an a-type carbonic anhydrase 1 (CAH1).7 The N-glycosylated protein AmyI-1, MSD1 and NPP 1, 2 and 6 were localized in rice plastids, while CAH1 was identified in Arabidopsis chloroplasts. The passage of these proteins through the secretory pathway en route to plastids was discovered using a combination of cellbiological techniques, including the analysis of trafficking of green-fluorescent-protein (GFP) fusion proteins in the presence of pharmacological agents that disrupt Golgi trafficking and high-pressure frozen/freeze-substituted techniques. Trafficking these proteins through the secretory pathway before entering the chloroplast might be related to protein folding11,12 and the need for post-translational modifications, such as Nglycosylation, for proper folding, and to enhance the stability and/or function of these proteins.7 Given the functional role of N-glycosylation for these proteins, it seems likely that their glycosylation status serves as a signal of the overall energy status of the cell and thus contributes to the regulation of carbon metabolism.13 Moreover, recent studies have provided information concerning protein N-glycan structures and their pathways in different phyla.14-19 Phylogenetic analyses of higher plant proteins directed to plastids via the endomembrane system (ES); CAH1, NPP1, aAmy7, and protein disulfide isomerase (RB60) from the green alga Chlamydomonas reinhardtii – used
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for its peculiar capacity for dual post- and co-translational targeting to both the plastid and endomembrane proteins revealed that vesicular trafficking of these proteins to plastids evolved long after cyanobacterial endosymbiosis to permit their glycosylation and/or transport to more than one cellular compartment.20 Interestingly, some glycoproteins are transported to the mitochondria from the ER-Golgi system through the secretory pathway. The mechanism for targeting of a plant protein, mitochondrial-targeting signal 1 (MITS1), to mitochondria has been reported.21 The successful targeting of Arabidopsis thaliana MITS1 is influenced by an N-terminal extension serving as a targeting peptide as well as by domains in the full-length protein. Functional dissection of the MITS1 N-terminal extension has shown the existence of 3 regions that coordinate the mitochondrial targeting signal, including a cryptic signal for protein targeting to the secretory pathway.21 Furthermore, the N-termini of the NADPH: protochlorophylide oxidoreductase A (PORA) from barley etioplasts carries a shorter transit peptidelike presequence22 but is translocated across the outer plastid envelope through the OEP16-1 pore.23 Such proteins do not carry a transit peptide but a signal peptide instead and are transported via ER and Golgi in vesicle-mediated pathways. Transport between the ER and the Golgi apparatus is bidirectional and believed to be mediated by different coated vesicles. Coat protein complex I (COPI) and COPII vesicles are respectively thought to function in the retrograde and anterograde, membrane traffic between the ER and the Golgi apparatus.24-26 These proteins are presumably transported across the remaining membranes in a TOC/TICindependent manner. Flores-Perez and Jarvis reported that TOC/TIC machinery is not believed to be able to translocate such bulky molecules.27 As in primary plastids, there is no need for members of the TOC machinery to transport glycoproteins because the addition of glycans occurs exclusively in the ER and Golgi and stromal glycoproteins necessarily use a vesiclemediated route. Given this lack of specific information about glycoprotein import into plastids, Fig. 2 depicts the 3 possibilities of vesicle-reltaed protein transport from Golgi to chloroplast. In the fusing/budding model (Fig 2A), the translocation across the outer envelope membrane of chloroplasts could be achieved by fusion of post-Golgi vesicles with the outer chloroplast membrane, thereby releasing the glycosylated protein into the intermembrane space. Subsequent targeting of cargo across the inner membrane could involve the TIC complex, an unknown transporter in the inner envelope membrane or a second vesicle budding from the inner membrane itself.28 The complication with this hypothesis is that the Golgi vesicle membrane is likely inserted within the outer envelope membrane (OM) of the plastid. In contrast, no signal of GFP-tagged proteins was observed at the chloroplast periphery. Another hypothesis suggests that Golgi-vesicles may target plastid through an invagination model (Fig 2B). However, if microautophagic vacuole invagination were involved in the uptake of membrane vesicles, vesicles encircled by the plastid envelope membranes in addition to small invagination would be frequently detected in the plastid stroma. However, distinctive vesicles such as these were scarce, suggesting that an as yet undetermined vesicle uptake mechanism might operate in
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Figure 2. Hypotheses for vesicle-mediated import into plastids in plants. (A) Fusing/budding model, (B) Invagination model and (C) Pass-through model. See text for details concerning the 3 models (A, B and C) on glycoprotein import into plastid stroma. GV, Golgi vesicle; IM, inner envelope membrane; OM, outer envelope membrane.
protein importation into plastids. The imported vesicles are perhaps subsequently broken up in the organelle. The moment of plastid entry of membrane vesicles was seen in EM observations using quick-frozen cells,2 suggesting that the vesiclemediated manner might pass through the plastid envelope membranes by a mechanism other than by a budding model or microautophagic vacuole invagination into the organelle (Fig 2C). Consistent with this pass-through model, plastid-targeted protein can freely diffuse through pores or through a non-specific protein transporter. Recently, Sugano et al.29 speculated that OsVAMP714, an intracellular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), can play a role in membrane fusing between the Golgi apparatus and chloroplasts since SNARE protein functions are closely linked to subcellular localizations of the imported glycoprotein cargo into plastids. Additional studies are needed to fully understand how nuclear-encoded proteins are imported into the plastids. In plants, the sorting of proteins in the cell is more complex due to the presence of different organelles, and hence, different types of membranes. Herein, we showed that not all nuclear-encoded plastid proteins are targeted to the canonical TOC/TIC complex. Proteins presenting an N-terminal signal peptide are often directed to the secretory pathway,
translocated into the ER, and sorted for transport to external space including the cell wall or vacuole, or retained in the ER, Golgi, and plasma membranes. Proteins without an ER signal sequence are translated into the cytosol, and if equipped with appropriate targeting signals, can be transported into particular organelles such as the plastids or mitochondria. The secretory pathway is a further notable example of the diverse array of routes that have evolved to mediate protein trafficking to plastids and how plant cells have evolved several chloroplast import pathways working in parallel. An inventory of proteins involved in vesicular trafficking via the secretory pathway and models involved in the entry of vesicles into plastids have been herein compiled. The routes that mediate plastid–nuclear communication, the characterization of cross-signaling pathways and the elucidation of vesiclemediated import into plastids will provide important clues as to the overall nature of how plastids provide essential metabolic and signaling functions within all plant cells in response to constantly changing environmental conditions.
Disclosure of potential conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Acknowledgment We apologize to those colleagues whose work is not cited due to space limitations. This research was supported by KAKENHI Grants-in-Aid for Scientific Research (A) (15H02486) from Japan Society for the Promotion of Sciences and Grant for Promotion of KAAB Projects (Niigata University) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
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