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ScienceDirect Plant vacuolar trafficking driven by RAB and SNARE proteins Tomohiro Uemura1 and Takashi Ueda1,2 Membrane-bounded organelles are connected to each other by membrane trafficking, which is accomplished by membrane fusion between transport vesicles and target organelles mediated by RAB GTPases and SNARE proteins. Of those trafficking pathways networking plant organelles, the vacuolar trafficking pathway has recently been shown to be uniquely diversified from non-plant systems, most likely reflecting unique functions of plant vacuoles such as the storage of proteins and other organic compounds, generation of turgor pressure, and space-filling to enlarge plant bodies. Plantunique trafficking machineries in addition to evolutionarily conserved molecular components are allocated to this trafficking pathway in distinctive ways. In this review, we summarize recent findings on SNARE proteins and RAB GTPases mediating vacuolar transport in plants, especially focusing on the functions and regulation of two distinct transSNARE complexes and RAB5 and RAB7 in multiple vacuolar trafficking pathways. Addresses 1 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan 2 Japan Science and Technology Agency (JST), PRESTO, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan Corresponding author: Ueda, Takashi ([email protected])
Current Opinion in Plant Biology 2014, 22:116–121 This review comes from a themed issue on Cell biology Edited by Shaul Yalovsky and Viktor Zˇa´rsky´ For a complete overview see the Issue and the Editorial Available online 23rd October 2014 http://dx.doi.org/10.1016/j.pbi.2014.10.002 1369-5266/# 2014 Elsevier Ltd. All rights reserved.
Introduction Membrane-bounded organelles such as the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), endosomes, and vacuoles are connected by the membrane trafficking system, which play pivotal roles in not only basic cellular activities but also higher-order physiological functions such as development and environmental responses. In a trafficking pathway, transport vesicles loaded with cargo molecules bud from the donor organelle by the action of the coat protein complex, whereas it is uncertain whether secretory vesicles possess a coat protein complex or not [1]. The transport vesicles Current Opinion in Plant Biology 2014, 22:116–121
are delivered to the destination organelle and then tethered and fused to the target membrane to discharge the cargo molecules [2,3]. The specificity of the tethering and fusion between two membranes is determined by several evolutionarily conserved components, which include RAB GTPase, a member of the Ras super family, and the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) protein [4–6]. These proteins exhibit restricted and specific subcellular localizations and are thus also deemed to be determinants of the organelle identity. The membrane trafficking system in plant cells comprises three major trafficking pathways: the biosyntheticsecretory pathway, the endocytic pathway, and the vacuolar transport pathway. In the biosynthetic-secretory pathway, newly synthesized proteins in the ER are delivered to the plasma membrane or cell exterior via the Golgi apparatus and the TGN [7]. In the endocytic pathway, extracellular and plasma membrane components are taken up into the cell, with some being recycled back to the plasma membrane and others delivered to the vacuole for degradation through early and late endosomes (EE and LE, respectively) [8]. The plant endocytic pathway harbors distinctive features compared with non-plant systems, which are exemplified by the early endosomal function of the TGN [9]. The third pathway, the vacuolar transport pathway, is also fulfilled by unique and complex mechanisms, most likely reflecting the elaborate functions of the plant vacuole, which include the storage of proteins and sugars, space filling to increase the volume of the cell, and defense responses in addition to the lytic function shared with other organisms [10,11]. Several proteins synthesized at the ER are shown to be transported to the vacuole through the Golgi apparatus, TGN, and multivesicular endosome (MVE) [12]. In addition to this pathway, some vacuolar storage proteins are also transported to the vacuole directly from the ER [13]. The Golgi-independent vacuolar transport is also proposed for some membrane proteins including vacuolar H(+)-pyrophosphatase and vacuolar H(+)-adenosinetriphosphatase, although molecular machineries mediating this pathway have not been identified yet [14,15]. Some populations of LE/MVE have been reported to be generated by direct maturation from the TGN [16]. To develop such a complex vacuolar trafficking system, plants should have produced complex and unique mechanisms during evolution. In this review, we summarize the recent findings on unique aspects of plant vacuolar trafficking, especially highlighting the functions and regulation of RAB GTPases and SNARE proteins. www.sciencedirect.com
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SNAREs regulating vacuolar transport pathway SNARE proteins are classified into five subgroups, Qa-SNAREs, Qb-SNAREs, Qc-SNAREs, R-SNAREs, and Qb/Qc-SNAREs, based on the sequence similarity in the conserved SNARE motif (Figure 1). The assembly of distinctive combinations of three Q-SNARE domains, which are provided by the Qa-SNAREs, QbSNAREs, and Qc-SNAREs or the Qa-SNAREs and Qb/ Qc-SNAREs, and one R-SNARE domain into a tight complex leads to membrane fusion [17]. In the vacuolar trafficking pathway, two SYP2 members, SYP21/PEP12 and SYP22/VAM3, localize to the LE/MVE and vacuolar membrane and play major roles as Qa-SNAREs [18]. Although the syp21 mutant does not exhibit an abnormal phenotype, the syp22 mutant exhibits pleiotropic phenotypes such as semi-dwarfism, late flowering, and a poorly developed vascular network in leaves [19–22]. The double mutation syp21 syp22 results in gametophytic lethality [23,24], and vacuolar transport of 12S globulin and 2S albumin is impaired in the syp22 mutant with knocked down SYP21 [23]. These results indicate redundant functions of SYP2 members in vacuolar transport. However, the overexpression of SYP21 has also been reported to cause homotypic fusion of the prevacuolar compartment (PVC) and to trap vacuolar cargoes in the PVC in tobacco cells, whereas the overexpression of SYP22 does not exert such effects [25]. Thus, SYP21 and SYP22 may also possess some specific functions in the vacuolar trafficking pathway.
Qb-VTI11 is a binding partner of SYP22 and SYP21, and its loss of function by the zig-1 mutation results in zigzagshaped inflorescences, defective shoot gravitropic responses, and abnormal vacuolar morphology [26–29]. The crucial role of VTI11 in PI3P-mediated vacuolar biogenesis was also demonstrated recently by the characterization of another mutant allele of VTI11, itt3 [30]. The Qc-SNARE that partners with SYP22 is SYP5; two closely related SYP5 members (SYP51 and SYP52) are encoded in the Arabidopsis genome. Although both of the SYP5 members are localized to the vacuolar membrane and TGN and complement the yeast vam7 mutation, the functions of these proteins in vacuolar transport seem to be different; SYP51 is essential for transporting GFP-chitinase, whereas SYP52 plays a role in the transport of Aleurain-GFP. It is also reported that SYP51 and SYP52 behave as inhibitory or interfering SNAREs (i-SNAREs) when overexpressed, accumulating on the vacuolar membrane [31]. VAMP713 (and most likely also VAMP711 and VAMP712), the R-SNARE residing on the vacuolar membrane [32], is also a member of the SYP22 complex [33]. Physiological studies indicated that this complex is involved in the salt stress response; the syp22 mutant is tolerant to salt stress [34], and impairment in VAMP711 results in inhibition of the fusion of H2O2-containing vesicles with the vacuolar membrane under salt stress [35]. In addition to the R-VAMP713 described above, another R-SNARE present on the LE/MVE, VAMP727, has been shown to form a complex with SYP22, VTI11, and SYP5
Figure 1
SYP21/PEP12 (SYP22/VAM3)
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Subcellular localization of SNARE proteins regulating transport around the MVE/LE and the vacuole. The R-SNARE present on the MVE/LE, VAMP727, mediates the heterotypic fusion between the MVE/LE and vacuole by forming a complex with Qa-SYP2, Qb-VTI1, and Qc-SYP5. Given the vacuolar localization of VAMP71, homotypic fusion between vacuoles is most likely mediated by the SNARE complex consisting of Qa-SYP2, Qb-VTI1, Qc-SYP5, and R-VAMP71. www.sciencedirect.com
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[36]. VAMP727 is characterized by the insertion of an acidic sequence in its N-terminal longin domain. R-SNARE proteins with a similar acidic insertion are well conserved in seed plants, whereas the lycophyte and moss that have had their genomes sequenced do not harbor this type of R-SNARE. The distinctive distribution of VAMP727 homologs may indicate that vacuolar trafficking pathways specifically diversified in seed plant lineages, and further studies of the R-SNARE in basal plant lineages are required to unveil the origin of this R-SNARE.
RAB GTPases mediating vacuolar trafficking in plants RAB5 and RAB7 are major regulators of endosomal/ vacuolar trafficking in animal and yeast cells and are also present in plants. In addition to orthologous genes for these RAB GTPases, the Arabidopsis genome contains a plant-specific RAB5 member, ARA6/RABF1 [37]. ARA6 and conventional RAB5, RHA1/RABF2a and ARA7/ RABF2b localize to distinct populations of the MVE with considerable overlap [33,38,39] and regulate distinct trafficking pathways; ARA7 and RHA1 play a critical role in the vacuolar trafficking pathway [33,40,41,42], and ARA6 mediates transport from the MVE to the plasma membrane (PM) in Arabidopsis [33]. ARA6 has also been shown to act in the vacuolar transport of soluble cargo and recycling of vacuolar sorting receptors from the MVE to the TGN in tobacco cells [40,43]. These results imply the multifunctionality of ARA6, with diversified functions of this protein in different plants or tissues. RAB7 is also involved in trafficking to the vacuole. There are eight RAB7-related GTPases in Arabidopsis, which are further divided into three subgroups (RABG1, G2, and G3a-f) [37]. Some of these members have been localized to the vacuolar membrane and the MVE [8,44]. This group has been implicated in abiotic and biotic stress responses. The overexpression of RABG3e results in the accumulation of a high concentration of sodium in vacuole and confers tolerance to salt and osmotic stresses [45]. The expression of RABG3e has been shown to be upregulated by treatment with superoxide or salicylic acid and by infection with pathogens [45]. The RABG group also plays a critical role in TE (tracheary element) differentiation through its function in autophagy, which is shown by experiments using RABG3b [46–48]. The function of RAB7 in vacuolar trafficking has been precisely demonstrated recently. Induction of the dominant negative mutant of RABG3f (RABG3fT22N) caused dilation of the MVE and deformation of the vacuole [44]. In this plant, soluble vacuolar cargos are mistargeted to the apoplast, and vacuolar storage proteins are not properly degraded during the germination process. In a consistent manner, multiple mutations of RABG3 members resulted in fragmented (or unfused) protein storage vacuoles (PSVs) in mature embryos, and several mutants Current Opinion in Plant Biology 2014, 22:116–121
including a quintuple mutant of RABG3 (rabg3,b,c,d,e,f) accumulated unprocessed precursors of 12S globulin and missecreted the artificial cargo GFP-CT24 into the extracellular space [49]. These lines of evidence indicated that RABG3 plays a critical role in the vacuolar transport of soluble cargo proteins in Arabidopsis. The comparable effect of dominant negative RAB7 on soluble cargo is also observed in tobacco protoplast cells [40]. The Arabidopsis sextuple rabg3a,b,c,d,e,f mutant exhibited semi-dwarfism in its early developmental stages [49], and the induction of RABG3fT22N caused severe growth defects in seedlings [44]. Thus, RAB7 activity is required for the normal development of Arabidopsis, which is also supported by analyses of the guanine nucleotide exchange factor (GEF) of RAB7 as described later [44,50]. In addition to RAB7, RAB5 is required for the vacuolar transport of soluble cargo [40,41,49,51]. The similar effects of impairment in RAB5 and RAB7 suggest that these proteins act in the same trafficking pathway, as proposed in animal and plant systems [43,52]. However, the genetic analyses of RAB5-related and RAB7-related mutants indicated that vacuolar morphogenesis is more sensitive to mutations in RAB7-related components, although defects in RAB5-related machineries primarily affect the transport of soluble cargo to the PSV [49]. Furthermore, rab5 and rab7 mutations exert counteracting effects in the vti11/zig-1 mutant. These results strongly suggest that RAB5 and RAB7 regulate genetically separable membrane trafficking events. The overexpression of dominant-negative mutants of RAB5 and RAB7 has also been reported to distinctly affect the vacuolar transport of membrane proteins in tobacco [40]. Thus, RAB5 and RAB7 seem to be recruited to plant vacuolar trafficking pathways in distinct ways from non-plant systems.
RAB conversion in the vacuolar trafficking pathway In animal cells, the GEF for RAB7, consisting of SAND1 and CCZ1, mediates the endosomal maturation from the RAB5-positive EE to the RAB7-positive LE [53–55]. However, whether the RAB conversion also occurs in the vacuolar trafficking pathway in plants remained unclear. Recently, the Arabidopsis SAND–CCZ1 complex was characterized, which outlined conserved and unique aspects of the RAB conversion in plant cells [44,49,50]. One SAND1 homolog (SAND, At2g28390) and two CCZ1 homologs (CCZ1a, At1g16020 and CCZ1b, At1g80910) exist in Arabidopsis. SAND and CCZ1 form a complex that specifically interacts with canonical RAB5/ RABF2 in the GTP-bound active state, thus acting as the effector for RAB5. The SAND–CCZ1 complex also binds to GDP-bound RAB7 and serves as the GEF for RAB7. These results clearly indicate that the molecular function of the SAND–CCZ1 complex is conserved www.sciencedirect.com
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Figure 2
RAB7
SAND CCZ1
RAB5
RAB5 GDP
GTP
GDP
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VPS9a RAB5
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(a) aleulein phaseolin 12S globlin VAMP71
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Schematic model of vacuolar trafficking pathways in plant cells. Vacuolar trafficking in Arabidopsis comprises at least four trafficking routes. (a) The route depending on RAB5-to-RAB7 conversion, where RAB5 is activated by VPS9a (GEF) and targeted to the MVE/LE, leads to the recruitment of the SAND/CCZ1 complex. The SAND/CCZ1 complex activates RAB7, which is required for transport from the MVE/LE to the vacuole. (b) VAMP71 is transported by the AP-3-dependent but RAB5-independent and RAB7-independent pathway. (c) SYP22 is a cargo of the RAB5-dependent and AP-3-independent pathway. (d) V-PPase and V-ATPase are targeted to the vacuole directly from the ER.
between plant and non-plant systems, mediating RAB conversion and being responsible for the trafficking pathway dependent on both RAB5 and RAB7. However, the timing of the SAND–CCZ1 action appears to be different in animals and plants. In animal cells, RAB conversion is associated with early-to-late endosomal maturation [55,56]. In contrast, the SAND–CCZ1 complex is not required for late endosomal MVE formation in plants but does seem to be crucial for MVE-to-vacuole trafficking [44,50]. This difference in the timing of the RAB conversion could be linked to the timing of the RAB5 action, which is responsible for early endosomal events in animal cells but regulates late endosomal trafficking in plant cells. The diversified endosomal trafficking is also highlighted by the RAB7-independent trafficking pathways to the vacuole in plants. Two vacuolar membrane proteins, SYP22 and VAMP713, are distinctly affected by ccz1 and vps9a-2 mutations, which impair RAB7-dependent and RAB5-dependent trafficking, respectively. The vps9a-2 mutation, but not the ccz1 mutation, hampers the vacuolar targeting of SYP22, whereas the vacuolar localization of VAMP713 is not affected by either of these mutations [49]. Interestingly, impairments in the adaptor protein complex 3 (AP-3) specifically delocalize www.sciencedirect.com
VAMP713. Thus, at least four vacuolar trafficking pathways occur in Arabidopsis, as summarized in Figure 2.
Conclusion and perspectives The transport of soluble vacuolar cargo, such as 12S globulin, phaseolin, aleurain, and CT24, requires both RAB5 and RAB7 [49,50], with the RAB5-to-RAB7 conversion on the MVE mediated by the SAND–CCZ1 complex playing a crucial role. In addition to this pathway, at least two trafficking pathways distinctly depending on RAB5 and RAB7 operate in plants, which are responsible for transporting distinctive cargos [40,49]. These findings suggest that plants have evolved unique and complex vacuolar trafficking pathways compared with non-plant systems by recruiting the conserved molecular machineries in a distinct way from other organisms. Autophagy-related and Golgi-independent transport from the ER to the vacuole is another example of such trafficking pathway [57]. This pathway also involves an exocyst subcomplex, although Rab and SNARE molecules associated with this pathway have not been identified thus far. Future studies on the molecular mechanisms of these plant-specific vacuolar trafficking pathways will reveal how plants have utilized unique vacuolar trafficking routes during evolution. The significance of the Current Opinion in Plant Biology 2014, 22:116–121
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secondary loss of conserved machinery components in endosomal trafficking would also be an interesting future research target. Recent genomic analyses revealed that ancient molecular machineries such as RAB21, which is predicted to function on the endosome, are secondarily lost in most angiosperms, including Arabidopsis, whereas basal species such as Physcomitrella patens and Selaginella moellendorffii still harbor RAB21 [58,59]. Comparative analysis between moss RAB21 and Arabidopsis RAB5/7 could reveal how and why plants have secondarily lost this ancient RAB member. The acquisition of novel RAB and SNARE molecules such as ARA6 and VAMP727 should be another way to expand endosomal/vacuolar trafficking pathways [60]. Thus, there were several strategic options for the diversification of vacuolar trafficking pathways. It would be extremely interesting to ask how plants have developed their unique vacuolar trafficking pathways during evolution and which plant-unique vacuolar function is associated with each plantunique vacuolar trafficking pathway.
Acknowledgements
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This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and JST, PRESTO.
20. Ohtomo I, Ueda H, Shimada T, Nishiyama C, Komoto Y, HaraNishimura I, Takahashi T: Identification of an allele of VAM3/ SYP22 that confers a semi-dwarf phenotype in Arabidopsis thaliana. Plant Cell Physiol 2005, 46:1358-1365.
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The authors showed that the MON1–CCZ1 complex serves as the Rab5 effector and mediates RAB5-to-RAB7 conversion on the PVC. 45. Mazel A, Leshem Y, Tiwari BS, Levine A: Induction of salt and osmotic stress tolerance by overexpression of an intracellular vesicle trafficking protein AtRab7 (AtRabG3e). Plant Physiol 2004, 134:118-128. 46. Kwon SI, Cho HJ, Jung JH, Yoshimoto K, Shirasu K, Park OK: The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis. Plant J: Cell Mol Biol 2010, 64:151-164. 47. Kwon SI, Cho HJ, Kim SR, Park OK: The Rab GTPase RabG3b positively regulates autophagy and immunity-associated hypersensitive cell death in Arabidopsis. Plant Physiol 2013, 161:1722-1736. 48. Kwon SI, Cho HJ, Lee JS, Jin H, Shin SJ, Kwon M, Noh EW, Park OK: Overexpression of constitutively active Arabidopsis RabG3b promotes xylem development in transgenic poplars. Plant Cell Environ 2011, 34:2212-2224. 49. Ebine K, Inoue T, Ito J, Ito E, Uemura T, Goh T, Abe H, Sato K, Nakano A, Ueda T: Plant vacuolar trafficking occurs through distinctly regulated pathways. Curr Biol 2014, 24:1375-1382. The authors demonstrated that the SAND–CCZ1 complex serves as a RAB7 GEF and that RAB5 and RAB7 distinctly regulate vacuolar trafficking pathways in Arabidopsis. 50. Singh MK, Kru¨ger F, Beckmann H, Brumm S, Vermeer JEM, Munnik T, Mayer U, Stierhof Y-D, Grefen C, Schumacher K et al.: Protein delivery to vacuole requires SAND protein-dependent RabGTPase conversion. Curr Biol 2014, 24:1383-1389. The authors showed that SAND is a RAB5 effector and that SANDdependent activation of RAB7 is necessary for MVB-vacuole fusion but not for multivesicularization of the endosome. 51. Sohn EJ, Kim ES, Zhao M, Kim SJ, Kim H, Kim YW, Lee YJ, Hillmer S, Sohn U, Jiang L et al.: Rha1, an Arabidopsis Rab5 homolog, plays a critical role in the vacuolar trafficking of soluble cargo proteins. Plant Cell 2003, 15:1057-1070. 52. Balderhaar HJ, Ungermann C: CORVET and HOPS tethering complexes — coordinators of endosome and lysosome fusion. J Cell Sci 2013, 126:1307-1316. 53. Cabrera M, Nordmann M, Perz A, Schmedt D, Gerondopoulos A, Barr F, Piehler J, Engelbrecht-Vandre S, Ungermann C: The Mon1-Ccz1 GEF activates the Rab7 GTPase Ypt7 via a longinfold-Rab interface and association with PI3P-positive membranes. J Cell Sci 2014, 127:1043-1051. 54. Nordmann M, Cabrera M, Perz A, Brocker C, Ostrowicz C, Engelbrecht-Vandre S, Ungermann C: The Mon1–Ccz1 complex is the GEF of the late endosomal Rab7 homolog Ypt7. Curr Biol: CB 2010, 20:1654-1659. 55. Poteryaev D, Datta S, Ackema K, Zerial M, Spang A: Identification of the switch in early-to-late endosome transition. Cell 2010, 141:497-508. 56. Yousefian J, Troost T, Grawe F, Sasamura T, Fortini M, Klein T: Dmon1 controls recruitment of Rab7 to maturing endosomes in Drosophila. J Cell Sci 2013, 126:1583-1594. 57. Kulich I, Pecˇenkova´ T, Sekeresˇ J, Smetana O, Fendrych M, Foissner I, Ho¨ftberger M, Za´rsky´ V: Arabidopsis exocyst subcomplex containing subunit EXO70B1 is involved in autophagy-related transport to the vacuole. Traffic 2013, 11:1155-1165. 58. Banks JA, Nishiyama T, Hasebe M, Bowman JL, Gribskov M, dePamphilis C, Albert VA, Aono N, Aoyama T, Ambrose BA et al.: The Selaginella genome identifies genetic changes associated with the evolution of vascular plants. Science 2011, 332:960-963. 59. Rensing SA, Lang D, Zimmer AD, Terry A, Salamov A, Shapiro H, Nishiyama T, Perroud PF, Lindquist EA, Kamisugi Y et al.: The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants. Science 2008, 319:64-69. 60. Fujimoto M, Ueda T: Conserved and plant-unique mechanisms regulating plant post-Golgi traffic. Front Plant Sci 2012, 3:197. A recent review of plant-unique aspects of post-Golgi trafficking. Current Opinion in Plant Biology 2014, 22:116–121
ScienceDirect Plant vacuolar trafficking driven by RAB and SNARE proteins Tomohiro Uemura1 and Takashi Ueda1,2 Membrane-bounded organelles are connected to each other by membrane trafficking, which is accomplished by membrane fusion between transport vesicles and target organelles mediated by RAB GTPases and SNARE proteins. Of those trafficking pathways networking plant organelles, the vacuolar trafficking pathway has recently been shown to be uniquely diversified from non-plant systems, most likely reflecting unique functions of plant vacuoles such as the storage of proteins and other organic compounds, generation of turgor pressure, and space-filling to enlarge plant bodies. Plantunique trafficking machineries in addition to evolutionarily conserved molecular components are allocated to this trafficking pathway in distinctive ways. In this review, we summarize recent findings on SNARE proteins and RAB GTPases mediating vacuolar transport in plants, especially focusing on the functions and regulation of two distinct transSNARE complexes and RAB5 and RAB7 in multiple vacuolar trafficking pathways. Addresses 1 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan 2 Japan Science and Technology Agency (JST), PRESTO, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan Corresponding author: Ueda, Takashi ([email protected])
Current Opinion in Plant Biology 2014, 22:116–121 This review comes from a themed issue on Cell biology Edited by Shaul Yalovsky and Viktor Zˇa´rsky´ For a complete overview see the Issue and the Editorial Available online 23rd October 2014 http://dx.doi.org/10.1016/j.pbi.2014.10.002 1369-5266/# 2014 Elsevier Ltd. All rights reserved.
Introduction Membrane-bounded organelles such as the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), endosomes, and vacuoles are connected by the membrane trafficking system, which play pivotal roles in not only basic cellular activities but also higher-order physiological functions such as development and environmental responses. In a trafficking pathway, transport vesicles loaded with cargo molecules bud from the donor organelle by the action of the coat protein complex, whereas it is uncertain whether secretory vesicles possess a coat protein complex or not [1]. The transport vesicles Current Opinion in Plant Biology 2014, 22:116–121
are delivered to the destination organelle and then tethered and fused to the target membrane to discharge the cargo molecules [2,3]. The specificity of the tethering and fusion between two membranes is determined by several evolutionarily conserved components, which include RAB GTPase, a member of the Ras super family, and the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) protein [4–6]. These proteins exhibit restricted and specific subcellular localizations and are thus also deemed to be determinants of the organelle identity. The membrane trafficking system in plant cells comprises three major trafficking pathways: the biosyntheticsecretory pathway, the endocytic pathway, and the vacuolar transport pathway. In the biosynthetic-secretory pathway, newly synthesized proteins in the ER are delivered to the plasma membrane or cell exterior via the Golgi apparatus and the TGN [7]. In the endocytic pathway, extracellular and plasma membrane components are taken up into the cell, with some being recycled back to the plasma membrane and others delivered to the vacuole for degradation through early and late endosomes (EE and LE, respectively) [8]. The plant endocytic pathway harbors distinctive features compared with non-plant systems, which are exemplified by the early endosomal function of the TGN [9]. The third pathway, the vacuolar transport pathway, is also fulfilled by unique and complex mechanisms, most likely reflecting the elaborate functions of the plant vacuole, which include the storage of proteins and sugars, space filling to increase the volume of the cell, and defense responses in addition to the lytic function shared with other organisms [10,11]. Several proteins synthesized at the ER are shown to be transported to the vacuole through the Golgi apparatus, TGN, and multivesicular endosome (MVE) [12]. In addition to this pathway, some vacuolar storage proteins are also transported to the vacuole directly from the ER [13]. The Golgi-independent vacuolar transport is also proposed for some membrane proteins including vacuolar H(+)-pyrophosphatase and vacuolar H(+)-adenosinetriphosphatase, although molecular machineries mediating this pathway have not been identified yet [14,15]. Some populations of LE/MVE have been reported to be generated by direct maturation from the TGN [16]. To develop such a complex vacuolar trafficking system, plants should have produced complex and unique mechanisms during evolution. In this review, we summarize the recent findings on unique aspects of plant vacuolar trafficking, especially highlighting the functions and regulation of RAB GTPases and SNARE proteins. www.sciencedirect.com
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SNAREs regulating vacuolar transport pathway SNARE proteins are classified into five subgroups, Qa-SNAREs, Qb-SNAREs, Qc-SNAREs, R-SNAREs, and Qb/Qc-SNAREs, based on the sequence similarity in the conserved SNARE motif (Figure 1). The assembly of distinctive combinations of three Q-SNARE domains, which are provided by the Qa-SNAREs, QbSNAREs, and Qc-SNAREs or the Qa-SNAREs and Qb/ Qc-SNAREs, and one R-SNARE domain into a tight complex leads to membrane fusion [17]. In the vacuolar trafficking pathway, two SYP2 members, SYP21/PEP12 and SYP22/VAM3, localize to the LE/MVE and vacuolar membrane and play major roles as Qa-SNAREs [18]. Although the syp21 mutant does not exhibit an abnormal phenotype, the syp22 mutant exhibits pleiotropic phenotypes such as semi-dwarfism, late flowering, and a poorly developed vascular network in leaves [19–22]. The double mutation syp21 syp22 results in gametophytic lethality [23,24], and vacuolar transport of 12S globulin and 2S albumin is impaired in the syp22 mutant with knocked down SYP21 [23]. These results indicate redundant functions of SYP2 members in vacuolar transport. However, the overexpression of SYP21 has also been reported to cause homotypic fusion of the prevacuolar compartment (PVC) and to trap vacuolar cargoes in the PVC in tobacco cells, whereas the overexpression of SYP22 does not exert such effects [25]. Thus, SYP21 and SYP22 may also possess some specific functions in the vacuolar trafficking pathway.
Qb-VTI11 is a binding partner of SYP22 and SYP21, and its loss of function by the zig-1 mutation results in zigzagshaped inflorescences, defective shoot gravitropic responses, and abnormal vacuolar morphology [26–29]. The crucial role of VTI11 in PI3P-mediated vacuolar biogenesis was also demonstrated recently by the characterization of another mutant allele of VTI11, itt3 [30]. The Qc-SNARE that partners with SYP22 is SYP5; two closely related SYP5 members (SYP51 and SYP52) are encoded in the Arabidopsis genome. Although both of the SYP5 members are localized to the vacuolar membrane and TGN and complement the yeast vam7 mutation, the functions of these proteins in vacuolar transport seem to be different; SYP51 is essential for transporting GFP-chitinase, whereas SYP52 plays a role in the transport of Aleurain-GFP. It is also reported that SYP51 and SYP52 behave as inhibitory or interfering SNAREs (i-SNAREs) when overexpressed, accumulating on the vacuolar membrane [31]. VAMP713 (and most likely also VAMP711 and VAMP712), the R-SNARE residing on the vacuolar membrane [32], is also a member of the SYP22 complex [33]. Physiological studies indicated that this complex is involved in the salt stress response; the syp22 mutant is tolerant to salt stress [34], and impairment in VAMP711 results in inhibition of the fusion of H2O2-containing vesicles with the vacuolar membrane under salt stress [35]. In addition to the R-VAMP713 described above, another R-SNARE present on the LE/MVE, VAMP727, has been shown to form a complex with SYP22, VTI11, and SYP5
Figure 1
SYP21/PEP12 (SYP22/VAM3)
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Subcellular localization of SNARE proteins regulating transport around the MVE/LE and the vacuole. The R-SNARE present on the MVE/LE, VAMP727, mediates the heterotypic fusion between the MVE/LE and vacuole by forming a complex with Qa-SYP2, Qb-VTI1, and Qc-SYP5. Given the vacuolar localization of VAMP71, homotypic fusion between vacuoles is most likely mediated by the SNARE complex consisting of Qa-SYP2, Qb-VTI1, Qc-SYP5, and R-VAMP71. www.sciencedirect.com
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[36]. VAMP727 is characterized by the insertion of an acidic sequence in its N-terminal longin domain. R-SNARE proteins with a similar acidic insertion are well conserved in seed plants, whereas the lycophyte and moss that have had their genomes sequenced do not harbor this type of R-SNARE. The distinctive distribution of VAMP727 homologs may indicate that vacuolar trafficking pathways specifically diversified in seed plant lineages, and further studies of the R-SNARE in basal plant lineages are required to unveil the origin of this R-SNARE.
RAB GTPases mediating vacuolar trafficking in plants RAB5 and RAB7 are major regulators of endosomal/ vacuolar trafficking in animal and yeast cells and are also present in plants. In addition to orthologous genes for these RAB GTPases, the Arabidopsis genome contains a plant-specific RAB5 member, ARA6/RABF1 [37]. ARA6 and conventional RAB5, RHA1/RABF2a and ARA7/ RABF2b localize to distinct populations of the MVE with considerable overlap [33,38,39] and regulate distinct trafficking pathways; ARA7 and RHA1 play a critical role in the vacuolar trafficking pathway [33,40,41,42], and ARA6 mediates transport from the MVE to the plasma membrane (PM) in Arabidopsis [33]. ARA6 has also been shown to act in the vacuolar transport of soluble cargo and recycling of vacuolar sorting receptors from the MVE to the TGN in tobacco cells [40,43]. These results imply the multifunctionality of ARA6, with diversified functions of this protein in different plants or tissues. RAB7 is also involved in trafficking to the vacuole. There are eight RAB7-related GTPases in Arabidopsis, which are further divided into three subgroups (RABG1, G2, and G3a-f) [37]. Some of these members have been localized to the vacuolar membrane and the MVE [8,44]. This group has been implicated in abiotic and biotic stress responses. The overexpression of RABG3e results in the accumulation of a high concentration of sodium in vacuole and confers tolerance to salt and osmotic stresses [45]. The expression of RABG3e has been shown to be upregulated by treatment with superoxide or salicylic acid and by infection with pathogens [45]. The RABG group also plays a critical role in TE (tracheary element) differentiation through its function in autophagy, which is shown by experiments using RABG3b [46–48]. The function of RAB7 in vacuolar trafficking has been precisely demonstrated recently. Induction of the dominant negative mutant of RABG3f (RABG3fT22N) caused dilation of the MVE and deformation of the vacuole [44]. In this plant, soluble vacuolar cargos are mistargeted to the apoplast, and vacuolar storage proteins are not properly degraded during the germination process. In a consistent manner, multiple mutations of RABG3 members resulted in fragmented (or unfused) protein storage vacuoles (PSVs) in mature embryos, and several mutants Current Opinion in Plant Biology 2014, 22:116–121
including a quintuple mutant of RABG3 (rabg3,b,c,d,e,f) accumulated unprocessed precursors of 12S globulin and missecreted the artificial cargo GFP-CT24 into the extracellular space [49]. These lines of evidence indicated that RABG3 plays a critical role in the vacuolar transport of soluble cargo proteins in Arabidopsis. The comparable effect of dominant negative RAB7 on soluble cargo is also observed in tobacco protoplast cells [40]. The Arabidopsis sextuple rabg3a,b,c,d,e,f mutant exhibited semi-dwarfism in its early developmental stages [49], and the induction of RABG3fT22N caused severe growth defects in seedlings [44]. Thus, RAB7 activity is required for the normal development of Arabidopsis, which is also supported by analyses of the guanine nucleotide exchange factor (GEF) of RAB7 as described later [44,50]. In addition to RAB7, RAB5 is required for the vacuolar transport of soluble cargo [40,41,49,51]. The similar effects of impairment in RAB5 and RAB7 suggest that these proteins act in the same trafficking pathway, as proposed in animal and plant systems [43,52]. However, the genetic analyses of RAB5-related and RAB7-related mutants indicated that vacuolar morphogenesis is more sensitive to mutations in RAB7-related components, although defects in RAB5-related machineries primarily affect the transport of soluble cargo to the PSV [49]. Furthermore, rab5 and rab7 mutations exert counteracting effects in the vti11/zig-1 mutant. These results strongly suggest that RAB5 and RAB7 regulate genetically separable membrane trafficking events. The overexpression of dominant-negative mutants of RAB5 and RAB7 has also been reported to distinctly affect the vacuolar transport of membrane proteins in tobacco [40]. Thus, RAB5 and RAB7 seem to be recruited to plant vacuolar trafficking pathways in distinct ways from non-plant systems.
RAB conversion in the vacuolar trafficking pathway In animal cells, the GEF for RAB7, consisting of SAND1 and CCZ1, mediates the endosomal maturation from the RAB5-positive EE to the RAB7-positive LE [53–55]. However, whether the RAB conversion also occurs in the vacuolar trafficking pathway in plants remained unclear. Recently, the Arabidopsis SAND–CCZ1 complex was characterized, which outlined conserved and unique aspects of the RAB conversion in plant cells [44,49,50]. One SAND1 homolog (SAND, At2g28390) and two CCZ1 homologs (CCZ1a, At1g16020 and CCZ1b, At1g80910) exist in Arabidopsis. SAND and CCZ1 form a complex that specifically interacts with canonical RAB5/ RABF2 in the GTP-bound active state, thus acting as the effector for RAB5. The SAND–CCZ1 complex also binds to GDP-bound RAB7 and serves as the GEF for RAB7. These results clearly indicate that the molecular function of the SAND–CCZ1 complex is conserved www.sciencedirect.com
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Figure 2
RAB7
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GTP
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(a) aleulein phaseolin 12S globlin VAMP71
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Schematic model of vacuolar trafficking pathways in plant cells. Vacuolar trafficking in Arabidopsis comprises at least four trafficking routes. (a) The route depending on RAB5-to-RAB7 conversion, where RAB5 is activated by VPS9a (GEF) and targeted to the MVE/LE, leads to the recruitment of the SAND/CCZ1 complex. The SAND/CCZ1 complex activates RAB7, which is required for transport from the MVE/LE to the vacuole. (b) VAMP71 is transported by the AP-3-dependent but RAB5-independent and RAB7-independent pathway. (c) SYP22 is a cargo of the RAB5-dependent and AP-3-independent pathway. (d) V-PPase and V-ATPase are targeted to the vacuole directly from the ER.
between plant and non-plant systems, mediating RAB conversion and being responsible for the trafficking pathway dependent on both RAB5 and RAB7. However, the timing of the SAND–CCZ1 action appears to be different in animals and plants. In animal cells, RAB conversion is associated with early-to-late endosomal maturation [55,56]. In contrast, the SAND–CCZ1 complex is not required for late endosomal MVE formation in plants but does seem to be crucial for MVE-to-vacuole trafficking [44,50]. This difference in the timing of the RAB conversion could be linked to the timing of the RAB5 action, which is responsible for early endosomal events in animal cells but regulates late endosomal trafficking in plant cells. The diversified endosomal trafficking is also highlighted by the RAB7-independent trafficking pathways to the vacuole in plants. Two vacuolar membrane proteins, SYP22 and VAMP713, are distinctly affected by ccz1 and vps9a-2 mutations, which impair RAB7-dependent and RAB5-dependent trafficking, respectively. The vps9a-2 mutation, but not the ccz1 mutation, hampers the vacuolar targeting of SYP22, whereas the vacuolar localization of VAMP713 is not affected by either of these mutations [49]. Interestingly, impairments in the adaptor protein complex 3 (AP-3) specifically delocalize www.sciencedirect.com
VAMP713. Thus, at least four vacuolar trafficking pathways occur in Arabidopsis, as summarized in Figure 2.
Conclusion and perspectives The transport of soluble vacuolar cargo, such as 12S globulin, phaseolin, aleurain, and CT24, requires both RAB5 and RAB7 [49,50], with the RAB5-to-RAB7 conversion on the MVE mediated by the SAND–CCZ1 complex playing a crucial role. In addition to this pathway, at least two trafficking pathways distinctly depending on RAB5 and RAB7 operate in plants, which are responsible for transporting distinctive cargos [40,49]. These findings suggest that plants have evolved unique and complex vacuolar trafficking pathways compared with non-plant systems by recruiting the conserved molecular machineries in a distinct way from other organisms. Autophagy-related and Golgi-independent transport from the ER to the vacuole is another example of such trafficking pathway [57]. This pathway also involves an exocyst subcomplex, although Rab and SNARE molecules associated with this pathway have not been identified thus far. Future studies on the molecular mechanisms of these plant-specific vacuolar trafficking pathways will reveal how plants have utilized unique vacuolar trafficking routes during evolution. The significance of the Current Opinion in Plant Biology 2014, 22:116–121
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secondary loss of conserved machinery components in endosomal trafficking would also be an interesting future research target. Recent genomic analyses revealed that ancient molecular machineries such as RAB21, which is predicted to function on the endosome, are secondarily lost in most angiosperms, including Arabidopsis, whereas basal species such as Physcomitrella patens and Selaginella moellendorffii still harbor RAB21 [58,59]. Comparative analysis between moss RAB21 and Arabidopsis RAB5/7 could reveal how and why plants have secondarily lost this ancient RAB member. The acquisition of novel RAB and SNARE molecules such as ARA6 and VAMP727 should be another way to expand endosomal/vacuolar trafficking pathways [60]. Thus, there were several strategic options for the diversification of vacuolar trafficking pathways. It would be extremely interesting to ask how plants have developed their unique vacuolar trafficking pathways during evolution and which plant-unique vacuolar function is associated with each plantunique vacuolar trafficking pathway.
Acknowledgements
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This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and JST, PRESTO.
20. Ohtomo I, Ueda H, Shimada T, Nishiyama C, Komoto Y, HaraNishimura I, Takahashi T: Identification of an allele of VAM3/ SYP22 that confers a semi-dwarf phenotype in Arabidopsis thaliana. Plant Cell Physiol 2005, 46:1358-1365.
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