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• W2023676009 abstract "The correct localization of integral membrane proteins to subcellular compartments is important for their functions. Synaptotagmin contains a single transmembrane domain that functions as a type I signal-anchor sequence in its N terminus and two calcium-binding domains (C2A and C2B) in its C terminus. Here, we demonstrate that the localization of an Arabidopsis synaptotagmin homolog, SYT1, to the plasma membrane (PM) is modulated by tandem C2 domains. An analysis of the roots of a transformant-expressing green fluorescent protein-tagged SYT1 driven by native SYT1 promoter suggested that SYT1 is synthesized in the endoplasmic reticulum, and then delivered to the PM via the exocytotic pathway. We transiently expressed a series of truncated proteins in protoplasts, and determined that tandem C2A-C2B domains were necessary for the localization of SYT1 to the PM. The PM localization of SYT1 was greatly reduced following mutation of the calcium-binding motifs of the C2B domain, based on sequence comparisons with other homologs, such as endomembrane-localized SYT5. The localization of SYT1 to the PM may have been required for the functional divergence that occurred in the molecular evolution of plant synaptotagmins. The correct localization of integral membrane proteins to subcellular compartments is important for their functions. Synaptotagmin contains a single transmembrane domain that functions as a type I signal-anchor sequence in its N terminus and two calcium-binding domains (C2A and C2B) in its C terminus. Here, we demonstrate that the localization of an Arabidopsis synaptotagmin homolog, SYT1, to the plasma membrane (PM) is modulated by tandem C2 domains. An analysis of the roots of a transformant-expressing green fluorescent protein-tagged SYT1 driven by native SYT1 promoter suggested that SYT1 is synthesized in the endoplasmic reticulum, and then delivered to the PM via the exocytotic pathway. We transiently expressed a series of truncated proteins in protoplasts, and determined that tandem C2A-C2B domains were necessary for the localization of SYT1 to the PM. The PM localization of SYT1 was greatly reduced following mutation of the calcium-binding motifs of the C2B domain, based on sequence comparisons with other homologs, such as endomembrane-localized SYT5. The localization of SYT1 to the PM may have been required for the functional divergence that occurred in the molecular evolution of plant synaptotagmins. IntroductionThe localization of integral membrane proteins to the appropriate intracellular compartment(s) is important for their functioning. Secretory proteins are transported to the apoplast through the default secretion pathway, in which the proteins move from the endoplasmic reticulum (ER) 4The abbreviations used are: ERendoplasmic reticulumSytsynaptotagminPMplasma membraneTGNtrans-Golgi networkEEearly endosomeSAsignal anchorBFAbrefeldin AGFPgreen fluorescent proteinEmGFPemerald GFPCFPcyan fluorescent proteinMES4-morpholineethanesulfonic acidSNAREsoluble N-ethylmaleimide-sensitive fusion protein attachment protein receptorsSMPplant synaptotagmin-like, mitochondria proteinPAQplasma-membrane-type aquaporin. to the Golgi and trans-Golgi network (TGN), and then finally to the plasma membrane (PM). However, the default pathway and sorting mechanism of integral membrane proteins are still unclear.Synaptotagmin (Syt) family proteins are calcium sensors that regulate exocytosis in mammalian cells (1.Südhof T.C. J. Biol. Chem. 2002; 277: 7629-7632Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). In the presynaptic membrane of neuron cells, Syt I is known to regulate the calcium-dependent exocytosis of synaptic vesicles containing neurotransmitter by detecting calcium influx via voltage-dependent calcium channels after activation of neuron cells (2.Bai J. Chapman E.R. Trends Biochem. Sci. 2004; 29: 143-151Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Some isoforms of Syt are also related to the other calcium-dependent secretions, such as insulin release in pancreatic cell and maintenance of bone mass in bone cells (3.Gustavsson N. Lao Y. Maximov A. Chuang J.C. Kostromina E. Repa J.J. Li C. Radda G.K. Südhof T.C. Han W. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3992-3997Crossref PubMed Scopus (144) Google Scholar, 4.Zhao H. Ito Y. Chappel J. Andrews N.W. Teitelbaum S.L. Ross F.P. Dev. Cell. 2008; 14: 914-925Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Syt homologs occur throughout the plant kingdom (5.Craxton M. BMC Genomics. 2004; 5: 43Crossref PubMed Scopus (109) Google Scholar). The Arabidopsis genome has five genes encoding Syt homologs (SYT1, -2, -4, and -5; full-length SYT3 cDNA has a stop codon at a functional region). Recently, we demonstrated that Arabidopsis SYT1 is involved in calcium-dependent freezing tolerance, which is related to the membrane-resealing system (6.Yamazaki T. Kawamura Y. Minami A. Uemura M. Plant Cell. 2008; 20: 3389-3404Crossref PubMed Scopus (118) Google Scholar). The membrane-resealing system was initially identified in animal cells as an emergency response system against disruption of the PM, and a mammalian Syt homolog, Syt VII, functions in membrane resealing as a sensor to detect the calcium influx from outside of the cells through the ruptured site of the PM (7.Bi G.Q. Alderton J.M. Steinhardt R.A. J. Cell Biol. 1995; 131: 1747-1758Crossref PubMed Scopus (236) Google Scholar, 8.Reddy A. Caler E.V. Andrews N.W. Cell. 2001; 106: 157-169Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar, 9.Steinhardt R.A. Bi G. Alderton J.M. Science. 1994; 263: 390-393Crossref PubMed Scopus (402) Google Scholar, 10.Terasaki M. Miyake K. McNeil P.L. J. Cell Biol. 1997; 139: 63-74Crossref PubMed Scopus (218) Google Scholar). In addition, Arabidopsis SYT1 is required for the maintenance of PM integrity and viability of root cells when exposed to severe salt stress, suggesting the involvement of the membrane-resealing system in such harsh conditions (11.Schapire A.L. Voigt B. Jasik J. Rosado A. Lopez-Cobollo R. Menzel D. Salinas J. Mancuso S. Valpuesta V. Baluska F. Botella M.A. Plant Cell. 2008; 20: 3374-3388Crossref PubMed Scopus (145) Google Scholar).The Syt family has one conserved transmembrane (TM) domain in the N terminus and two conserved calcium-binding domains (C2A and C2B) in the C terminus, and C2A and C2B of mammalian Syt I are actually bound to three and two calcium ions, respectively (1.Südhof T.C. J. Biol. Chem. 2002; 277: 7629-7632Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 2.Bai J. Chapman E.R. Trends Biochem. Sci. 2004; 29: 143-151Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 7.Bi G.Q. Alderton J.M. Steinhardt R.A. J. Cell Biol. 1995; 131: 1747-1758Crossref PubMed Scopus (236) Google Scholar, 12.Jahn R. Lang T. Südhof T.C. Cell. 2003; 112: 519-533Abstract Full Text Full Text PDF PubMed Scopus (1215) Google Scholar). Note that the membrane proteins with a single TM domain are classified according to protein structure and topogenesis: type I membrane proteins possess an N-terminal cleavable signal sequence and a subsequent stop-transfer sequence and are anchored with the Nexo/Ccyt topology; type II membrane proteins (also called SA-II proteins) possess an uncleaved type II signal-anchor (SA-II) sequence and are anchored with the Ncyt/Cexo topology; type I signal-anchor (SA-I) proteins (also called type III membrane proteins) possess an uncleaved SA-I sequence and are anchored with the Nexo/Ccyt topology; tail-anchored (TA) membrane proteins are anchored by a C-terminal TM domain with the Ncyt/Cexo topology (13.Goder V. Spiess M. FEBS Lett. 2001; 504: 87-93Crossref PubMed Scopus (132) Google Scholar, 14.High S. Flint N. Dobberstein B. J. Cell Biol. 1991; 113: 25-34Crossref PubMed Scopus (65) Google Scholar). Based on their structure and topology, the Syt family proteins are classified as SA-I proteins (15.Kida Y. Sakaguchi M. Fukuda M. Mikoshiba K. Mihara K. J. Cell Biol. 2000; 150: 719-730Crossref PubMed Scopus (45) Google Scholar).The structure of plant Syts differs from that of conventional mammalian Syts in several ways. The predicted TM domain of plant Syts consists of 23 or 22 amino acid residues, which is almost the same number of amino acid residues reported for mammalian Syts. However, plant Syts lack an N-terminal extension in the extracellular region, but have an SMP (plant synaptotagmin-like, mitochondria proteins) domain just beside the C-terminal side of the TM domain (5.Craxton M. BMC Genomics. 2004; 5: 43Crossref PubMed Scopus (109) Google Scholar, 16.Jiménez J.L. Davletov B. Proteins. 2007; 68: 770-778Crossref PubMed Scopus (8) Google Scholar). The SMP domain has also been identified in yeast Tcb and mammalian E-Syt families (mentioned later), although the function of the SMP domain in these proteins is unknown (5.Craxton M. BMC Genomics. 2004; 5: 43Crossref PubMed Scopus (109) Google Scholar, 16.Jiménez J.L. Davletov B. Proteins. 2007; 68: 770-778Crossref PubMed Scopus (8) Google Scholar). Our previous examination indicated that the topology of SYT1 is the same as that of mammalian Syt, i.e. the Nexo/Ccyt topology (6.Yamazaki T. Kawamura Y. Minami A. Uemura M. Plant Cell. 2008; 20: 3389-3404Crossref PubMed Scopus (118) Google Scholar). In addition, like mammalian Syts, the C2A domain of SYT1 has the conserved calcium-binding motif and exhibits the calcium-dependent interaction with lipid membranes; however, the C2B domain of SYT1 exhibits a calcium-independent interaction with the lipid membrane (11.Schapire A.L. Voigt B. Jasik J. Rosado A. Lopez-Cobollo R. Menzel D. Salinas J. Mancuso S. Valpuesta V. Baluska F. Botella M.A. Plant Cell. 2008; 20: 3374-3388Crossref PubMed Scopus (145) Google Scholar).Mammalian genomes contain thirteen Syt homologs, some of which are known to contain mRNA splicing variants. Mammalian Syt II, which is the most similar protein to Syt I, contains the SA-I sequence that integrates the translated proteins in the ER membrane and orientates C2A and C2B to the cytosol and the N-terminal extension to the extracellular space. It was established that the mammalian Syts examined in several cell types (for instance, neuron cells, endocrine β-cells, sperm cells, and mammalian cultured cells) are located in endomembrane systems (i.e. secretory vesicles, Golgi/TGN, lysosomes, acrosomes, etc.), but not in the PM (17.Berton F. Cornet V. Iborra C. Garrido J. Dargent B. Fukuda M. Seagar M. Marquèze B. Eur. J. Neurosci. 2000; 12: 1294-1302Crossref PubMed Scopus (45) Google Scholar, 18.Czibener C. Sherer N.M. Becker S.M. Pypaert M. Hui E. Chapman E.R. Mothes W. Andrews N.W. J. 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Benassy M.N. Hémar A. Lang J. Histochem. Cell Biol. 2007; 127: 625-632Crossref PubMed Scopus (22) Google Scholar, 27.Osborne S.L. Herreros J. Bastiaens P.I. Schiavo G. J. Biol. Chem. 1999; 274: 59-66Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 28.Perin M.S. Fried V.A. Mignery G.A. Jahn R. Südhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (648) Google Scholar, 29.Ting J.T. Kelley B.G. Sullivan J.M. J. Neurosci. 2006; 26: 372-380Crossref PubMed Scopus (31) Google Scholar). Interestingly, a novel mammalian Syt-related protein family, E-Syt (E-Syt1/KIAA0747/FAM62A, E-Syt2/FAM62B, and E-Syt3/FAM62C), contains five C2 domains (E-Syt1) or three C2 domains (E-Syt2 or E-Syt3) in the C terminus (5.Craxton M. BMC Genomics. 2004; 5: 43Crossref PubMed Scopus (109) Google Scholar, 30.Min S.W. Chang W.P. Südhof T.C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 3823-3828Crossref PubMed Scopus (136) Google Scholar, 31.Nagase T. Ishikawa K. Suyama M. Kikuno R. Miyajima N. Tanaka A. Kotani H. Nomura N. Ohara O. DNA Res. 1998; 5: 277-286Crossref PubMed Scopus (127) Google Scholar). E-Syt1 is localized to some endomembrane systems, but E-Syt2 and E-Syt3 are localized to the PM, indicating that the C2 domain is also involved in subcellular localization (30.Min S.W. Chang W.P. Südhof T.C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 3823-3828Crossref PubMed Scopus (136) Google Scholar). In addition, three yeast Syt homologs (Tcb1, Tcb2, and Tcb3) contain five or six C2 domains in their C termini, and Tcb2 seems to be localized to the PM (16.Jiménez J.L. Davletov B. Proteins. 2007; 68: 770-778Crossref PubMed Scopus (8) Google Scholar, 32.Creutz C.E. Snyder S.L. Schulz T.A. Cell Mol. Life Sci. 2004; 61: 1208-1220Crossref PubMed Scopus (43) Google Scholar). An Arabidopsis synaptotagmin, SYT1, is also localized to the PM (6.Yamazaki T. Kawamura Y. Minami A. Uemura M. Plant Cell. 2008; 20: 3389-3404Crossref PubMed Scopus (118) Google Scholar).In this study, we analyzed the mechanism by which plant SYT1 is delivered to the PM. Whereas mammalian Syts localize to endomembranes, including synaptic vesicles, the TGN, and endosomes and lysosomes, but not to the PM, Arabidopsis SYT1 localizes to the PM. We were thus interested in establishing whether there is a specific mechanism that facilitates the differential localization of mammalian and plant Syts. We found that SYT1 may be synthesized on rough ER and then delivered to the PM through the Golgi. Moreover, the tandem C2A-C2B domains were found to be necessary for the delivery of SYT1 to the PM. A motif in the C2B domain was found to be required for localization to the PM, which may have been modified during the course of evolution to permit a novel function of plant Syts. To our knowledge, this is the first report demonstrating the localization mechanism of a plant SA-I protein.DISCUSSIONSyt proteins are classified as SA-I proteins and occur throughout eukaryotes. In animal cells, Syt functions as a calcium sensor to promote the membrane fusion in calcium-dependent events, such as membrane resealing (8.Reddy A. Caler E.V. Andrews N.W. Cell. 2001; 106: 157-169Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar). In plant cells, we have recently demonstrated that the Arabidopsis Syt homolog, SYT1, is involved in calcium-dependent membrane resealing, which remarkably enhances freezing tolerance (6.Yamazaki T. Kawamura Y. Minami A. Uemura M. Plant Cell. 2008; 20: 3389-3404Crossref PubMed Scopus (118) Google Scholar). In addition, the C2A domain of SYT1 exhibits calcium-dependent binding to phospholipid membrane (11.Schapire A.L. Voigt B. Jasik J. Rosado A. Lopez-Cobollo R. Menzel D. Salinas J. Mancuso S. Valpuesta V. Baluska F. Botella M.A. Plant Cell. 2008; 20: 3374-3388Crossref PubMed Scopus (145) Google Scholar). Therefore, plant SYT1 may, like mammalian Syt proteins, act as a calcium sensor during membrane-membrane fusion events that are stimulated by calcium ions. However, the localization of plant SYT1 to the PM is different from that of mammalian Syts, because mammalian Syts have ubiquitously been found in endomembranes. Our current study suggests that the evolutionary modification of the C2B domain may be necessary for the delivery of SYT1 to the PM via the exocytotic pathway.Synthesis and Delivery of SYT1 to the Plasma MembraneOur biochemical analyses of SYT1-EmGFP driven by the SYT1 native promoter showed that GFP tagging at the C terminus of SYT1 has no effect on its localization and orientation (FIGURE 1, FIGURE 2). In addition, the biochemical localization of SYT1 to the PM in seedlings agreed with the localization based on GFP fluorescence in leaf epidermal and mesophyll cells (supplemental Fig. S1). In roots, the localization pattern of SYT1-EmGFP differed at the various developmental stages (Fig. 3). This is thought to result from the expression level of the SYT1 gene in root tissue, because the fluorescence pattern of EmGFP driven by the SYT1 promoter in roots corresponded to that of SYT1-EmGFP (supplemental Fig. S2).Furthermore, we observed the subcellular localization pattern of SYT1-EmGFP in different types of root cells expressing different levels of its transcription. In division and elongation zones, SYT1-EmGFP was localized not only to the PM, but also to the rough ER (Fig. 3, A and D). SYT1 may not be retained in the ER, because a chimeric GFP containing an ER retention signal, HEDL, is localized only to rough ER and cortical ER, but not to the PM (53.Ridge R.W. Uozumi Y. Plazinski J. Hurley U.A. Williamson R.E. Plant Cell Physiol. 1999; 40: 1253-1261Crossref PubMed Scopus (85) Google Scholar). In some dividing cells, SYT1-EmGFP was localized to the cell plate (Fig. 3A). The plant cell plate is generated not only by delivery of newly synthesized materials from the ER via the Golgi, but also by endocytic recycling, which provides materials from the PM (54.Dhonukshe P. Baluska F. Schlicht M. Hlavacka A. Šamaj J. Friml J. Gadella Jr., T.W. Dev. Cell. 2006; 10: 137-150Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Recent work based on short term labeling of the PM with FM4–64 shows that vesicles derived from the PM via the endocytotic pathway are localized to the EE and TGN but not to the Golgi (48.Bolte S. Talbot C. Boutte Y. Catrice O. Read N.D. Satiat-Jeunemaitre B. J. Microsc. 2004; 214: 159-173Crossref PubMed Scopus (442) Google Scholar). In our current study, vesicles containing SYT1-EmGFP did not colocalize completely with vesicles stained with FM4–64 (Fig. 3, A and B), suggesting that SYT1 is localized to the Golgi after synthesis on the ER, and less SYT1 is cycled from the PM back to the cytoplasm through the endocytotic pathway than typical materials incorporated in the PM. This is also supported by the incomplete localization of SYT1-EmGFP to BFA compartments (Fig. 3D), because the BFA compartment is speculated to be composed of TGN and EE membranes, but Golgi vesicles do not fuse to the BFA compartment (48.Bolte S. Talbot C. Boutte Y. Catrice O. Read N.D. Satiat-Jeunemaitre B. J. Microsc. 2004; 214: 159-173Crossref PubMed Scopus (442) Google Scholar, 50.Robinson D.G. Langhans M. Saint-Jore-Dupas C. Hawes C. Trends Plant Sci. 2008; 13: 405-408Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In addition, colocalization analysis of SYT1-EmGFP driven by the CaMV35S promoter and CFP-labeled Golgi marker showed that SYT1-EmGFP is also partially localized to Golgi vesicles (Fig. 5A). These results suggest that, after SYT1 is synthesized on the rough ER membrane, SYT1 is delivered to the PM by the exocytotic pathway via Golgi, and then tends to be retained in the PM. However, it is unclear whether SYT1 moves directly to the PM or via the TGN.Interestingly, SYT1-EmGFP was not uniformly localized to the PM and appeared to be located along some microfibers (Fig. 3C). We recently showed that SYT1 accumulates in a fraction of detergent-resistant plasma membrane regions, which are related to microdomains in the membrane, together with actins (55.Minami A. Fujiwara M. Furuto A. Fukao Y. Yamashita T. Kamo M. Kawamura Y. Uemura M. Plant Cell Physiol. 2009; 50: 341-359Crossref PubMed Scopus (117) Google Scholar). These results led us to speculate that, after the transport of SYT1 to the PM, SYT1 is redistributed in the PM via a specific mechanism. The localization of RHD2 NADPH oxidase, which is similar to that of SYT1, is affected by treatment with the microfilament-disrupting drug cytochalasin D, but not by the microtubule-disrupting drugs oryzalin or taxol (56.Takeda S. Gapper C. Kaya H. Bell E. Kuchitsu K. Dolan L. Science. 2008; 319: 1241-1244Crossref PubMed Scopus (383) Google Scholar). Although both microfilaments and microtubules are localized in the root hair tip, microtubules are aligned with the short axis of cortical cells, whereas microfilaments are not (57.Baluska F. Salaj J. Mathur J. Braun M. Jasper F. Samaj J. Chua N.H. Barlow P.W. Volkmann D. Dev. Biol. 2000; 227: 618-632Crossref PubMed Scopus (270) Google Scholar). It is possible that several factors facilitate the interaction between SYT1 and microfilaments.Localization of SYT1 to the PM Is Determined by Tandem C2A-C2B DomainsPlant Syts contain four domains (namely TM, transmembrane; SMP, synaptotagmin-like mitochondria membrane protein; and C2A and C2B, calcium-binding domains) (refer to Fig. 4A, row 1). The TM domain combined with a partial SMP domain was insufficient for anchoring of SYT1 to the membrane, but the TM domain combined with a complete SMP domain may be sufficient for localization to ER (Fig. 4B, rows 5 and 6; Fig. 5B, row 2), suggesting that the SMP domain is required for the integration of Syt into the rough ER membrane. This result also suggests that tandem C2A-C2B domains recruit SYT1 and then helps to transport it to the PM through the exocytotic pathway, because full-length SYT1 is localized to the PM. On the other hand, only tandem C2A-C2B domains are hard to be localized to the PM without SMP domain (Fig. 4B, row 2; Fig. 5A, row 2). In addition, less localization of tandem TM-SMP-C2A domains or of the C2B domain to the PM (Fig. 4B, rows 4 and 7) indicated that SMP-C2A domains or a single C2B domain may not be sufficient to localize SYT1 to the PM. Recently, transient expression analysis using a tobacco protoplast system showed that TM-SMP-C2A of SYT1 is localized to the ER (58.Lewis J.D. Lazarowitz S.G. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 2491-2496Crossref PubMed Scopus (169) Google Scholar). We also confirmed that the TM-SMP-C2A of SYT1 was partially localized to ER (supplemental Fig. S5). This result suggests that tandem C2 domains require the SMP domain to interact with the PM.We clearly showed that, like mammalian Syts, SYT1 had the Nexo/Ccyt topology (Fig. 2), demonstrating that SYT1 is a SA-I protein. Based on animal and fungal models, the localization mechanism of plant integral membrane proteins has been described in terms of a membrane trafficking pathway. In tobacco cells, modification of the TM domain of human lysosome-associated protein LAMP1 or pea vacuolar sorting receptor VSR/BP80, which are type I membrane proteins with the Nexo/Ccyt topology, shows that the length of TM domain determines the localization of the proteins, with a short TM domain (17 amino acid residues) resulting in the membrane proteins being transported to the ER, and a long TM domain (22 or 23 amino acid residues) resulting in them being transported to the PM (59.Brandizzi F. Frangne N. Marc-Martin S. Hawes C. Neuhaus J.M. Paris N. Plant Cell. 2002; 14: 1077-1092Crossref PubMed Scopus (184) Google Scholar). In mammalian Syts, the TM length also affects the localization of tail-anchored proteins that possess a single TM domain at the C terminus with an Ncyt/Cexo topology, such as cytochrome b5 or syntaxin (60.Pedrazzini E. Villa A. Borgese N. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 4207-4212Crossref PubMed Scopus (89) Google Scholar, 61.Watson R.T. Pessin J.E. Am. J. Physiol. Cell Physiol. 2001; 281: C215-C223Crossref PubMed Google Scholar). For example, the localization of syntaxin, a component of membrane-membrane fusion, to the PM is determined by the long length of its TM domain (≥23 amino acid residues). A prediction program for the presence of TM domain, SOSUI (bp.nuap.nagoya-u.ac.jp/sosui), calculated that the length of the putative TM domain of SYT1 is 23 amino acid residues long, and that the corresponding regions in the other Arabidopsis SYT1 homologous proteins are of the same length. In the case of SYT1, however, the long TM domain does not determine localization to the PM, because truncated SYT1 without tandem C2A-C2B domains cannot localize to the PM (Fig. 4B, lane 6).Evolutionary Modulation of Plant Synaptotagmin LocalizationThe phylogenetic tree in Fig. 6 showed that plant Syt genes, which contain two calcium-binding domains in their C termini, were clearly separated into three clades (the SYT1/2, SYT3, and SYT4/5). Each clade contained Syt genes of monocotyledonous and eudicotyledonous plants, suggesting that the three clades diverged before the speciation of monocots and eudicots in the early stages of the angiosperm evolutionary process (Fig. 6A). In addition, the conservation of exon-intron structures among AtSyt1, AtSyt2, AtSyt4, and AtSyt5 implies that the angiosperm Syt genes share a common ancestral gene of SYT1/2, SYT3, and SYT4/5 (Fig. 6B). The most significant difference between the SYT1/2 and SYT3 clades and the SYT4/5 clade is the lack of the calcium-binding motifs in Loop 1 and Loop 3 of the C2B domain (supplemental Fig. S4). It is unclear whether genes in the SYT1/2 and SYT3 clades lost their calcium-binding motifs after duplication of the genes of SYT1/2/3 and SYT4/5 clades. However, a common ancestor of the Syt genes may have retained the calcium-binding motifs both in the C2A domain and in the C2B domain, because the calcium-binding motifs were conserved in both C2 domains in mammalian Syts.The loss of the calcium-binding motifs in the C2B domain during evolution may result in alterations in the localization of plant Syts in the SYT1/2/3 clades. In fact, SYT1 is localized mainly to the PM and partially to endomembranes, such as Golgi, but SYT5 is localized only to endomembranes (Fig. 7C). Moreover, the SYT1 mutant, which contains the calcium-binding motif in Loop 1 (but not in Loop 3) of the C2B domain by referring SYT5 sequences, was localized mainly to endomembranes. Loop 1 of the C2A domain of mammalian Syt1 bound to calcium was shown to penetrate the lipid bilayer, and NMR data show that Loop 2 and Loop 3 of the C2A domain also bind to phospholipids with calcium (62.Chae Y.K. Abildgaard F. Chapman E.R. Markley J.L. J. Biol. Chem. 1998; 273: 25659-25663Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 63.Chapman E.R. Davis A.F. J. Biol. Chem. 1998; 273: 13995-14001Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Because the C2B domain of SYT1 interacts with liposomes in a calcium-independent manner in vitro (11.Schapire A.L. Voigt B. Jasik J. Rosado A. Lopez-Cobollo R. Menzel D. Salinas J. Mancuso S. Valpuesta V. Baluska F. Botella M.A. Plant Cell. 2008; 20: 3374-3388Crossref PubMed Scopus (145) Google Scholar), the lack of calcium-binding motifs may permit the calcium-independent interaction between SYT1 and the membrane. In addition, the evolutionarily modified amino acid residues of the C2 domains in SYT1, except for arginine and lysine, seem to be more bulky and hydrophobic than those of the SYT4/5 clade (Fig. 7B), raising the possibility that the calcium-independent penetration of Loop 1 into the lipid bilayer occurs and imparts some degree of specificity for the binding to the PM. On the other hand, as described above, the C2B domain alone may not contribute to the localization of SYT1 to the PM, for which tandem C2A-C2B domains are needed.Reconstructed fragments of the C2A or C2B domain in mammalian Syt I demonstrate that the C2B domain, but not the C2A domain, triggers the membrane-membrane fusion mediated by the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex in a calcium-dependent manner (64.Gaffaney J.D. Dunning F.M. Wang Z. Hui E. Chapman E.R. J. Biol. Chem. 2008; 283: 31763-31775Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In addition, the C2B domain interacts in a calcium-independent manner with a complex of SNAP-25/syntaxin known to target SNARE proteins to the PM of neuron cells, and the calcium-independent interaction is imparted by a polybasic region of the C2B domain (65.Rickman C. Archer D.A. Meunier F.A. Craxton M. Fukuda M. Burgoyne R.D. Davletov B. J. Biol. Chem. 2004; 279: 12574-12579Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). This calcium-independent interaction of the C2B domain with lipid membrane accelerates the calcium-triggered SNARE-mediated fusion (66.Loewen C.A. Lee S.M. Shin Y.K. Reist N.E. Mol. Biol. Cell. 2006; 17: 5211-5226Crossref PubMed Scopus (47) Google Scholar). Because the polybasic region is conserved in the C2B domain of plant Syts (see supplemental Fig. S4), plant Syts may interact in a calcium-independent manner with the SNAP-25-syntaxin complex. Therefore, to further understand the evolutionary effect of the absence of calcium-binding motifs in the C2B domain on plant Syts of SYT1/2/3 clades, it is necessary to investigate the interaction between plant SNARE proteins and Arabidopsis SYT1, and to assess their contribution to the localization of SYT1.Default Pathway and Sorting Mechanism of Plant SytsMechanisms or rules that determine the sorting of plant integral membrane proteins to the appropriate compartments after synthesis on rough ER have been proposed, such as the “length rule” described above. However, the length rule is not sufficient to interpret the sorting mechanism of the type I membrane proteins, because signals in the cytoplasmic tail also affect the export from the ER via the Golgi to the prevacuole compartment (67.daSilva L.L. Foresti O. Denecke J. Plant Cell." @default.
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• W2023676009 title "Arabidopsis Synaptotagmin SYT1, a Type I Signal-anchor Protein, Requires Tandem C2 Domains for Delivery to the Plasma Membrane" @default.
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