- Email: [email protected]
The longin domain regulates subcellular targeting of VAMP7 in Arabidopsis thaliana
FEBS Letters 579 (2005) 2842–2846
FEBS 29558
The longin domain regulates subcellular targeting of VAMP7 in Arabidopsis thaliana Tomohiro Uemuraa,*, Masa H. Satob,1, Kunio Takeyasua b
a Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan Graduate School of Human and Environmental Studies, Kyoto University, Yoshidanihonmatsu, Sakyo-ku, Kyoto 606-8501, Japan
Received 1 March 2005; revised 14 April 2005; accepted 14 April 2005 Available online 26 April 2005 Edited by Ulf-Ingo Flu¨gge
Abstract SNAREs (soluble N-ethyl-maleimide sensitive factor attachment protein receptors) which locate on the specific organelle membrane assure the correct vesicular transport by mediating specific membrane fusions. SNAREs are referred to as R- or Q-SNAREs on the basis of the amino acid sequence similarities and specific conserved residues. All of the Arabidopsis RSNAREs have a N-terminal domain, called the longin domain (LD). In this study, we investigated the vacuolar targeting mechanism of Arabidopsis R-SNAREs. The vacuolar localized AtVAMP711 was used as the mother protein of GFP-tagged chimeric proteins joined to several domains such as the LD, the SNARE motif (SNM) and the transmembrane domain (TMD) of other organelle-localized R-SNAREs. The results showed that, whereas the TMD is not relevant for the vacuolar targeting, a complete LD is essential for the vacuolar and subcellular targeting. Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Membrane traffic; Vesicle fusion; R-SNARE; Longin domain; Arabidopsis
1. Introduction In eukaryotic cells, the specific delivery of soluble and membrane proteins to the distinct subcellular compartments is accomplished by means of the trafficking of the transport vesicles through the endomembrane systems. The transport vesicles budding from one compartment fuse with the membrane of another compartment and discharge the cargo molecules. The spe-
*
Corresponding author. Present address: Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Fax: +81 3 3814 1408. E-mail addresses: [email protected], [email protected] (T. Uemura).
cific membrane fusions are mediated by a family of SNAREs [1–4]. The SNARE proteins share a coiled-coil domain built upon the SNARE motif that is composed of a hydrophobic heptad repeat [5]. Based on the amino acid sequence similarity of the SNARE motifs (SNMs), the SNARE proteins have been divided into four subgroups: Qa-SNARE, Qb-SNARE, Qc-SNARE and R-SNARE [6]. A complex consisting of a cross-subfamily four-helix bundle (i.e., one R-SNAREs and three Q SNAREs (Qa, Qb and Qc)) is called the trans-SNARE complex, and governs a specific membrane fusion [7,8]. R-SNAREs are divided into two subclasses; ÔlonginsÕ that containing the N-terminal longin domain (LD) of 120–140 amino acids preceding the SNM, and ÔbrevinsÕ that lack the LD [9]. Interestingly, plants lack the brevins. The longins are classified into three groups, Sec22- [10,11], YKT6- [12,13] and VAMP7like groups [14]. The Sec22- and YKT6-like groups occur in all eukaryotes, whereas the VAMP7-like group showed a restricted distribution. Arabidopsis possess eleven genes encoding the VAMP7-like group, but yeast lacks VAMP7-like genes [9,15,16]. In order to assure the correct intra-cellular transport, each SNARE molecule must be localized to a specific subcellular compartment. We have determined the specific subcellular localization of Arabidopsis eleven VAMP7 proteins (AtVAMP711AtVAMP714, AtVAMP721-AtVAM727) [17]. In spite of the high similarities among the VAMP7 proteins, they showed different subcellular localizations. AtVAMP711, AtVAMP712 and AtVAMP713 were localized to the vacuolar membrane, AtVAMP714, AtVAMP723 and AtVAMP727 on the Golgi apparatus, the ER and the endosome, respectively. AtVAMP721, AtVAMP722, AtVAMP724, AtVAMP725 and AtVAMP726 were mainly localized to the plasma membrane and the endosome. Thus, the Arabidopsis VAMP7 proteins will be useful markers to elucidate molecular mechanisms for the specific organelle localization of the R-SNARE proteins. In this study, to identify domains determining specific targeting of VAMP7 SNAREs, a vacuolar-localized VAMP7 protein, AtVAMP711, was chosen. After chimeric recombination with other SNAREs, the domain determining the vacuolar targeting was identified by monitoring the changes of the subcellular localization.
1 Present address: Faculty of Human Environmental Sciences, Kyoto Prefectural University, Shimogamo-nakaragi-cho, Sakyo-ku, Kyoto 606-8522, Japan
2. Materials and methods
Abbreviations: SNAREs, soluble N-ethyl-maleimide sensitive factor attachment protein receptors; LD, longin domain; SNM, SNARE motif; TMD, transmembrane region; PM, plasma membrane; TGN, trans-Golgi network; GFP, green fluorescent protein
2.1. Plasmids construction of chimeric cDNAs The cDNAs for Arabidopsis VAMP7 proteins have been isolated by RT-PCR [17]. By using these cDNAs as templates, the cDNA fragment necessary for the chimeric recombination was amplified by using
0014-5793/$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.04.022
T. Uemura et al. / FEBS Letters 579 (2005) 2842–2846 sequential PCR methods [18]. Open reading frames (ORFs) of the amplified chimeric cDNA were fused to the ORFs of sGFP (synthetic GFP) of the CaMV35S-sGFP(S65T)-NOS3 0 vector (provided by Y. Niwa). For the C-terminal replacement study, cDNAs that corresponded to residues 176–219 (CT-l) and 189–219 (CT-s) of AtVAMP711 or residues 180–219 (CT-l) and 193–219 (CT-s) of AtVAMP721 were used. For the N-terminal deletion or replacement studies, cDNAs that corresponded to residues 1–111 (NT-s) and 1– 126 (NT-l) of AtVAMP711, residues 1–111 (NT-s) of AtVAMP712, residues 1–112 (NT-s) of AtVAMP713, residues 1–127 (NT-l) of AtVAMP714, residues 1- 130 (NT-l) of AtVAMP721 or residues 1–149 (NT-s) of AtVAMP727 were used. 2.2. Transient expression of the GFP-tagged protein in protoplast cells The transient expression of the GFP-fused proteins in Arabidopsis suspension cultured cells was performed as described previously [19,20]. Briefly, 2 g of cultured cells was incubated in 25 ml of enzyme solution (0.4 M mannitol, 5 mM EGTA, 1% cellulase R-10 and 0.05% pectolyase Y-23) for 1–2 h at 30 °C and filtered with a nylon mesh (125 lm pore). The resultant protoplasts were washed twice with 25 ml of solution A (0.4 M mannitol, 70 mM CaCl2, and 5 mM MES-KOH, pH 5.7) and resuspended in 1 ml of the MaMg solution (0.4 M mannitol, 15 mM MgCl2, and 5 mM MES-KOH, pH 5.7). After the addition of 2 lg of plasmid and 50 lg of carrier DNA to 100 ll of the protoplast solution, 400 ll of DNA uptake solution (0.4 M mannnitol, 40% polyethylene glycol (PEG) 6000 and 0.1 M Ca(NO3)2) was added. The protoplasts were incubated on ice for 20 min and then diluted with 10 ml of a dilution solution (0.4 M mannitol, 125 mM CaCl2, 5 mM KCl, 5 mM glucose and 1.5 mM MESKOH, pH 5.7). The collected protoplasts were resuspended in 4 ml of MS medium containing 0.4 M mannitol and incubated with gentle agitation at 23 °C for 16 h in the dark. 2.3. Confocal laser scanning microscopy Observations were done using a confocal laser scanning microscope, Zeiss LSM LSM5 PASCAL equipped with green HeNe and argon lasers.
3. Results VAMP7 proteins consist of three distinct domains, the LD, SNM and the transmembrane domain (TMD). Amino acid se-
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quence analysis showed that vacuolar localized VAMP711, AtVAMP712 and AtVAMP713 share over 90% similarities with each other, and 80% and 60% similarities with Golgi-localized VAMP714 and plasma membrane/endosome localized VAMP7 proteins, respectively. In spite of the high homology of the VAMP7s proteins, they showed different subcellular localizations [17]. To determine the roles played by the three different domains (LD, SNM and TMD) of VAMP7 proteins in vacuolar sorting, AtVAMP711 was used as a model of vacuolar localized protein (Fig. 1A). AtVAMP721, AtVAMP714 and AtVAMP727 were used as the PM-, Golgi-, and endosome-localized proteins, respectively (Fig. 1B, C and D). We have already shown that the dot structures of GFP-AtVAMP714 and GFP-VAMP727 were merged with the fluorescence of SYP31 (Golgi marker) and endocytic dye FM4-64 (endosome marker), respectively [17,21]. Chimeric proteins were generated by replacing the corresponding domains of these proteins with that of AtVAMP711. 3.1. TMD is not the determinant for vacuolar localization To examine whether the TMD is the determinant for the vacuolar targeting or not, the GFP tagged chimeric AtVAMP711 proteins replacing the C-terminal (CT) region with those of PM-localized AtVAMP721, were transiently expressed in the protoplast of Arabidopsis suspension cells. Two different CT fragment were used in the experiments: CT-s (short), including TMD and intravesicular tail, and CT-l (long), including further 13 residues from the coiled coil domain. GFP fluorescence of GFP-AtVAMP711/PM-typeCT-s and GFP-AtVAMP711/PM-type-CT-l was observed on the vacuolar membrane like wild type AtVAMP711 (Fig. 2A and B, compared with Fig. 1A). GFP-AtVAMP721/ Vac-type-CT-s and GFP-AtVAMP721/Vac-type-CT-l which contain the vacuolar-type of CT-s and CT-l, respectively, were appeared on the plasma membrane, but not on the vacuolar membrane (Fig. 2C and D). These results suggest that the CT containing TMD is not a vacuolar targeting determinant.
Fig. 1. Schematic drawings and subcellular localizations of the VAMP7 proteins used in this study. The GFP-tagged VAMP7 proteins were transiently expressed in the protoplasts prepared from Arabidopsis suspension cultured cells. Fluorescence and Nomarski images are shown for GFPAtVAMP711 (A), GFP-AtVAMP721 (B), GFP-AtVAMP714 (C) and GFP-AtVAMP727 (D). Arrowheads in (C) show the dot-like structures of Golgi apparatus. Arrowheads in (D) show the dot-like structures of endosomes as confirmed in [15]. Scale bars = 10 lm.
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T. Uemura et al. / FEBS Letters 579 (2005) 2842–2846
Fig. 2. Schematic drawings and subcellular localizations of chimeric proteins. The GFP-tagged chimeric proteins, of which the TMDs were replaced for with those of other VAMP7 proteins, were transiently expressed in the protoplasts prepared from Arabidopsis suspension cultured cells. Fluorescence and Nomarski images are shown for GFP-AtVAMP711/PM-type-CT-s (A) and GFP-AtVAMP711/PM-type-CT-l (B), GFPAtVAMP721/Vac-type-CT-s (C) and GFP-AtVAMP721/Vac-type-CT-l (D). Scale bars = 10 lm.
3.2. The LD is critical for the vacuolar localization GFP-tagged truncated AtVAMP711(GFP-AtVAMP7 11DNTs), lacking the N-terminal short fragment (NT-s) which is corresponded to the truncated LD, lost the vacuolar-targeting ability and appeared on the plasma membrane and the small dot structures which are considered to be trans-Golgi network (TGN), or endosome (Fig. 3A, compared with Fig. 1A). GFP-AtVAMP712DNT-s and GFP-AtVAMP713DNT-s, which were the NT-s-deleted proteins of other vacuolar-localized VAMP7 proteins, also lost their vacuolar-targeting properties and were localized on the plasma membrane and the TGN/endosome (Fig. 3B and C). These results imply that the LD may be important for the vacuolar targeting of the vacuolar-localized VAMPs (AtVAMP711, AtVAMP712 and AtVAMP713). However, a substitution of the NT-s of AtVAMP721 (PM) with that of AtVAMP711 (vacuolar NT-s) (GFP-tagged vacuolar-type-NTs/VAMP721) did not affect the PM and the TGN/endosome localization, resulting in no vacuolar localization (Fig. 4A, compared with Fig. 1A and B). Further addition of 15 amino acids to the NT-s (which is corresponded to the complete LD [10,13] in an additional chimera), GFP-vacuolar-type-LD/VAMP721 (containing of the LD of the vacuolar type and the SNM and the TMD of the PM type), successfully conferred the vacuolar targeting (Fig. 4B, compared with Fig. 1A and B). The localization of this chimeric protein was not completely the same as that of AtVAMP711 (Figs. 1A and 4B) because the PM and the TGN/endosome targeting was also observed. Since the plasma membrane was proposed as the default destination of singlepass membrane proteins [22], the plasma membrane localization of this chimeric protein might be caused by the default transport. Both the LD of the vacuolar type (AtvAMP711) were substituted for those of the Golgi type (AtVAMP714) and the endosome type (AtVAMP727). GFP-vacuolar-type-LD/VAMP714 and GFP-vacuolar-type-LD/VAMP727 were localized to the vacuolar membrane (Fig. 4C and D, compared with Fig. 1C
Fig. 3. Schematic drawings and subcellular localizations of the LDdeleted VAMP7 proteins and chimeric proteins. The GFP-tagged proteins were transiently expressed in the protoplasts prepared from Arabidopsis suspension cultured cells. Fluorescence and Nomarski images are shown for GFP-AtVAMP711DNT-s (A), GFP-AtVAMP712DNT-s (B) and GFP-AtVAMP711DNT-s (C). Scale bars = 10 lm.
and D). Together with all of the data, we concluded that complete LD of AtVAMP711 play a key role for the vacuolar targeting.
T. Uemura et al. / FEBS Letters 579 (2005) 2842–2846
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Fig. 4. Schematic drawings and subcellular localizations of chimeric proteins. The GFP-tagged chimeric proteins, of which LD, was replaced for with those of other VAMP7 proteins, were transiently expressed in the protoplasts prepared from Arabidopsis suspension cultured cells. Fluorescence and Nomarski images are shown for GFP-Vac-type-NT-s/AtVAMP721 (A) and GFP-Vac-type-LD/AtVAMP721 (B), GFP-Vac-type-LD/ AtVAMP714 (C) and GFP-Vac-type-LD/AtVAMP727 (D). Scale bars = 10 lm.
Fig. 5. Schematic drawings and subcellular localizations of chimeric proteins. GFP-tagged chimera proteins, of which the LD were replaced for with those of other organelle-localized VAMP7 proteins, were transiently expressed in the protoplasts from Arabidopsis suspension cultured cells. Fluorescence and Nomarski images are shown for GFP-PM-type-LD/AtVAMP711 (A), GFP-Golgi-type-LD/AtVAMP711 (B) and GFP-endosometype-LD/AtVAMP711 (C). Scale bars = 10 lm.
3.3. Role of the LD in the others organelle targeting Is the LD capable of targeting plant VAMP7 also to organelle other than the vacuole? To answer this, PM-type LD, Golgi-type LD and endosome-type-LD were attached separately to the vacuolar-type SNM/TMD, and their subcellular localization were examined. GFP-tagged PM-type-LD/AtVAMP711 was observed to the plasma membrane and TGN/ endosome like wild type AtVAMP721 (Fig. 5A, compare with Fig. 1B), implying that PM-type-LD may play a role for the plasma membrane targeting. Moreover, Golgi-type-LD/AtVAMP711 and Endosome-type-LD/AtVAMP711 was also found on the Golgi apparatus and endosomes, respectively (Fig. 5B and C, compared with Fig. 1C and D). However, interestingly, they also appeared on the vacuolar membrane. In the case of Golgi and endosome, the LD domain may not be sufficient for changing the subcellular localization completely. The SNM or TMD also might affect the subcellular localization.
4. Discussion In the experiments presented here, GFP-tagged chimeric proteins were expressed in the Arabidopsis protoplast cells under the 35S promoter. We are aware of the possibility that
GFP itself or overexpression of the fusion proteins, in general, might cause a mislocalization. However, it has been shown that GFP tagged Qa- and Qb-SNARE proteins are located to proper subcellular compartment [22–24]. In this study, to avoid the effect of overexpression, we carefully observed only weakly fluorescent cells, and found that a LD of the vacuolar localized AtVAMP711 were found to be important for the vacuolar targeting. Although we mainly focused on the vacuolar targeting, the results imply that LDs of other VAMP7 proteins also play a general role in the organelle targeting. The LD occurs only in R-SNAREs, but not in Qa-, Qb-, Qc-SNAREs or SNAPs Q-SNAREs [16]. In mammal, the LD of TI-VAMP/VAMP7 interacts with the AP-3 complex (adaptor complex-3), which targets VAMP7 to the late endosomes/lysosomes. Moreover, a LD having lost its central region lacks subcellular targeting capacity [25]. In this study, we also showed that the LD lacking its C-terminus also lost the capacity to regulate the vacuolar targeting (Fig. 4A). These results showed that a complete LD is necessary for the vacuolar sorting in plant cells like mammalian TIVAMP/VAMP7. In case of another longin Ykt6, the LD also controlled the conformation and subcellular distribution [26–28]. Our findings suggest that only a complete LD is a general determinant of subcellular sorting in not only mammalian but also plant cells.
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ER retention is considered to depend on the length of the TMD, since a shorter TMD of 17 amino acids is retained in the ER, and an artificially-made longer TMD of over 20 amino acids is accumulated in the post-Golgi organelle [22,29]. Actually, the TMD length of Arabidopsis ER-localized R-SNARE, AtSec22, is 17 amino acids, whereas the TMD lengths of postGolgi organelle localized R-SNAREs are over 20 amino acids. Therefore, we would hypothesize the vacuolar targeting mechanism of R-SNARE proteins as follows: R-SNARE proteins first enter to the ER, and remain there depending on the length of TMD. The R-SNAREs with a longer TMD will exit the ER and be transported to the Golgi apparatus. At the Golgi apparatus and the TGN, both the LD in cytoplasmic region is critically involved in the determination of the specific organelle transport. Acknowledgements: We thank Dr. Y. Niwa for kindly providing the CaMV35S-sGFP(S65T)-NOS3Õ GFP fusion plasmid. We also thank Dr. M. Umeda for Arabidopsis suspension cultured cell. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology: a Grant-in-Aid for Encouragement of Young Scientist, and Scientific Research on Priority Areas (B) (M.H.S) and a Grant-in-Aid for JSPS Research Fellowships for Young Scientists (T.U.).
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T. Uemura et al. / FEBS Letters 579 (2005) 2842–2846 [13] Tochio, H., Tsui, M.M., Banfield, D.K. and Zhang, M. (2001) An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p. Science 293, 698– 702. [14] Galli, T., Zahraoui, A., Vaidyanathan, V.V., Raposo, G., Tian, J.M., Karin, M., Niemann, H. and Louvard, D. (1998) A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells. Mol. Biol. Cell 9, 1437–1448. [15] Rossi, V., Banfield, D.K., Vacca, M., Dietrich, L.E., Ungermann, C., DÕEsposito, M., Galli, T. and Filippini, F. (2004) Longins and their longin domains: regulated SNAREs and multifunctional SNARE regulators. Trends Biochem. Sci. 29, 682–688. [16] Rossi, V., Picco, R., Vacca, M., DÕEsposito, M., DÕUrso, M., Galli, T. and Filippini, F. (2004) VAMP subfamilies identified by specific R-SNARE motifs. Biol. Cell 96, 251–256. [17] Uemura, T., Ueda, T., Ohniwa, R.L., Nakano, A., Takeyasu, K. and Sato, M.H. (2004) Systematic analysis of SNARE molecules in Arabidopsis: dissection of the post-Golgi network in plant cells. Cell Struct. Funct. 29, 49–65. [18] Higuchi, R., Krummel, B. and Saiki, R.K. (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16, 7351–7367. [19] Forreiter, C., Kirschner, M. and Nover, L. (1997) Stable transformation of an Arabidopsis cell suspension culture with firefly luciferase providing a cellular system for analysis of chaperone activity in vivo. Plant Cell 9, 2171–2181. [20] Ueda, T., Yamaguchi, M., Uchimiya, H. and Nakano, A. (2001) Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana. EMBO J. 20, 4730– 4741. [21] Ueda, T., Uemura, T., Sato, M.H. and Nakano, A. (2004) Functional differentiation of endosomes in Arabidopsis cells. Plant J. 40, 783–789. [22] Brandizzi, F., Frangne, N., Marc-Martin, S., Hawes, C., Neuhaus, J.M. and Paris, N. (2002) The destination for single-pass membrane proteins is influenced markedly by the length of the hydrophobic domain. Plant Cell 14, 1077–1092. [23] Niihama, M., Uemura, T., Saito, C., Nakano, A., Sato, M.H., Tasaka, M. and Morita, M.T. (2005) Conversion of functional specificity in Qb-SNARE VTI1 homologues of Arabidopsis. Curr. Biol. 15, 555–560. [24] Uemura, T., Yoshimura, S.H., Takeyasu, K. and Sato, M.H. (2002) Vacuolar membrane dynamics revealed by GFP-AtVam3 fusion protein. Genes Cells 7, 743–753. [25] Martinez-Arca, S., et al. (2003) A dual mechanism controlling the localization and function of exocytic v-SNAREs. Proc. Nat. Acad. Sci. USA 100, 9011–9016. [26] Fukasawa, M., Varlamov, O., Eng, W.S., Sollner, T.H. and Rothman, J.E. (2004) Localization and activity of the SNARE Ykt6 determined by its regulatory domain and palmitoylation. Proc. Nat. Acad. Sci. USA 101, 4815–4820. [27] Hasegawa, H., Zinsser, S., Rhee, Y., Vik-Mo, E.O., Davanger, S. and Hay, J.C. (2003) Mammalian ykt6 is a neuronal SNARE targeted to a specialized compartment by its profilin-like amino terminal domain. Mol. Biol. Cell 14, 698–720. [28] Hasegawa, H., Yang, Z., Oltedal, L., Davanger, S. and Hay, J.C. (2004) Intramolecular protein-protein and protein-lipid interactions control the conformation and subcellular targeting of neuronal Ykt6. J. Cell Sci. 117, 4495–4508. [29] Rayner, J.C. and Pelham, H.R. (1997) Transmembrane domaindependent sorting of proteins to the ER and plasma membrane in yeast. EMBO J. 16, 1832–1841.
FEBS 29558
The longin domain regulates subcellular targeting of VAMP7 in Arabidopsis thaliana Tomohiro Uemuraa,*, Masa H. Satob,1, Kunio Takeyasua b
a Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan Graduate School of Human and Environmental Studies, Kyoto University, Yoshidanihonmatsu, Sakyo-ku, Kyoto 606-8501, Japan
Received 1 March 2005; revised 14 April 2005; accepted 14 April 2005 Available online 26 April 2005 Edited by Ulf-Ingo Flu¨gge
Abstract SNAREs (soluble N-ethyl-maleimide sensitive factor attachment protein receptors) which locate on the specific organelle membrane assure the correct vesicular transport by mediating specific membrane fusions. SNAREs are referred to as R- or Q-SNAREs on the basis of the amino acid sequence similarities and specific conserved residues. All of the Arabidopsis RSNAREs have a N-terminal domain, called the longin domain (LD). In this study, we investigated the vacuolar targeting mechanism of Arabidopsis R-SNAREs. The vacuolar localized AtVAMP711 was used as the mother protein of GFP-tagged chimeric proteins joined to several domains such as the LD, the SNARE motif (SNM) and the transmembrane domain (TMD) of other organelle-localized R-SNAREs. The results showed that, whereas the TMD is not relevant for the vacuolar targeting, a complete LD is essential for the vacuolar and subcellular targeting. Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Membrane traffic; Vesicle fusion; R-SNARE; Longin domain; Arabidopsis
1. Introduction In eukaryotic cells, the specific delivery of soluble and membrane proteins to the distinct subcellular compartments is accomplished by means of the trafficking of the transport vesicles through the endomembrane systems. The transport vesicles budding from one compartment fuse with the membrane of another compartment and discharge the cargo molecules. The spe-
*
Corresponding author. Present address: Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Fax: +81 3 3814 1408. E-mail addresses: [email protected], [email protected] (T. Uemura).
cific membrane fusions are mediated by a family of SNAREs [1–4]. The SNARE proteins share a coiled-coil domain built upon the SNARE motif that is composed of a hydrophobic heptad repeat [5]. Based on the amino acid sequence similarity of the SNARE motifs (SNMs), the SNARE proteins have been divided into four subgroups: Qa-SNARE, Qb-SNARE, Qc-SNARE and R-SNARE [6]. A complex consisting of a cross-subfamily four-helix bundle (i.e., one R-SNAREs and three Q SNAREs (Qa, Qb and Qc)) is called the trans-SNARE complex, and governs a specific membrane fusion [7,8]. R-SNAREs are divided into two subclasses; ÔlonginsÕ that containing the N-terminal longin domain (LD) of 120–140 amino acids preceding the SNM, and ÔbrevinsÕ that lack the LD [9]. Interestingly, plants lack the brevins. The longins are classified into three groups, Sec22- [10,11], YKT6- [12,13] and VAMP7like groups [14]. The Sec22- and YKT6-like groups occur in all eukaryotes, whereas the VAMP7-like group showed a restricted distribution. Arabidopsis possess eleven genes encoding the VAMP7-like group, but yeast lacks VAMP7-like genes [9,15,16]. In order to assure the correct intra-cellular transport, each SNARE molecule must be localized to a specific subcellular compartment. We have determined the specific subcellular localization of Arabidopsis eleven VAMP7 proteins (AtVAMP711AtVAMP714, AtVAMP721-AtVAM727) [17]. In spite of the high similarities among the VAMP7 proteins, they showed different subcellular localizations. AtVAMP711, AtVAMP712 and AtVAMP713 were localized to the vacuolar membrane, AtVAMP714, AtVAMP723 and AtVAMP727 on the Golgi apparatus, the ER and the endosome, respectively. AtVAMP721, AtVAMP722, AtVAMP724, AtVAMP725 and AtVAMP726 were mainly localized to the plasma membrane and the endosome. Thus, the Arabidopsis VAMP7 proteins will be useful markers to elucidate molecular mechanisms for the specific organelle localization of the R-SNARE proteins. In this study, to identify domains determining specific targeting of VAMP7 SNAREs, a vacuolar-localized VAMP7 protein, AtVAMP711, was chosen. After chimeric recombination with other SNAREs, the domain determining the vacuolar targeting was identified by monitoring the changes of the subcellular localization.
1 Present address: Faculty of Human Environmental Sciences, Kyoto Prefectural University, Shimogamo-nakaragi-cho, Sakyo-ku, Kyoto 606-8522, Japan
2. Materials and methods
Abbreviations: SNAREs, soluble N-ethyl-maleimide sensitive factor attachment protein receptors; LD, longin domain; SNM, SNARE motif; TMD, transmembrane region; PM, plasma membrane; TGN, trans-Golgi network; GFP, green fluorescent protein
2.1. Plasmids construction of chimeric cDNAs The cDNAs for Arabidopsis VAMP7 proteins have been isolated by RT-PCR [17]. By using these cDNAs as templates, the cDNA fragment necessary for the chimeric recombination was amplified by using
0014-5793/$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.04.022
T. Uemura et al. / FEBS Letters 579 (2005) 2842–2846 sequential PCR methods [18]. Open reading frames (ORFs) of the amplified chimeric cDNA were fused to the ORFs of sGFP (synthetic GFP) of the CaMV35S-sGFP(S65T)-NOS3 0 vector (provided by Y. Niwa). For the C-terminal replacement study, cDNAs that corresponded to residues 176–219 (CT-l) and 189–219 (CT-s) of AtVAMP711 or residues 180–219 (CT-l) and 193–219 (CT-s) of AtVAMP721 were used. For the N-terminal deletion or replacement studies, cDNAs that corresponded to residues 1–111 (NT-s) and 1– 126 (NT-l) of AtVAMP711, residues 1–111 (NT-s) of AtVAMP712, residues 1–112 (NT-s) of AtVAMP713, residues 1–127 (NT-l) of AtVAMP714, residues 1- 130 (NT-l) of AtVAMP721 or residues 1–149 (NT-s) of AtVAMP727 were used. 2.2. Transient expression of the GFP-tagged protein in protoplast cells The transient expression of the GFP-fused proteins in Arabidopsis suspension cultured cells was performed as described previously [19,20]. Briefly, 2 g of cultured cells was incubated in 25 ml of enzyme solution (0.4 M mannitol, 5 mM EGTA, 1% cellulase R-10 and 0.05% pectolyase Y-23) for 1–2 h at 30 °C and filtered with a nylon mesh (125 lm pore). The resultant protoplasts were washed twice with 25 ml of solution A (0.4 M mannitol, 70 mM CaCl2, and 5 mM MES-KOH, pH 5.7) and resuspended in 1 ml of the MaMg solution (0.4 M mannitol, 15 mM MgCl2, and 5 mM MES-KOH, pH 5.7). After the addition of 2 lg of plasmid and 50 lg of carrier DNA to 100 ll of the protoplast solution, 400 ll of DNA uptake solution (0.4 M mannnitol, 40% polyethylene glycol (PEG) 6000 and 0.1 M Ca(NO3)2) was added. The protoplasts were incubated on ice for 20 min and then diluted with 10 ml of a dilution solution (0.4 M mannitol, 125 mM CaCl2, 5 mM KCl, 5 mM glucose and 1.5 mM MESKOH, pH 5.7). The collected protoplasts were resuspended in 4 ml of MS medium containing 0.4 M mannitol and incubated with gentle agitation at 23 °C for 16 h in the dark. 2.3. Confocal laser scanning microscopy Observations were done using a confocal laser scanning microscope, Zeiss LSM LSM5 PASCAL equipped with green HeNe and argon lasers.
3. Results VAMP7 proteins consist of three distinct domains, the LD, SNM and the transmembrane domain (TMD). Amino acid se-
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quence analysis showed that vacuolar localized VAMP711, AtVAMP712 and AtVAMP713 share over 90% similarities with each other, and 80% and 60% similarities with Golgi-localized VAMP714 and plasma membrane/endosome localized VAMP7 proteins, respectively. In spite of the high homology of the VAMP7s proteins, they showed different subcellular localizations [17]. To determine the roles played by the three different domains (LD, SNM and TMD) of VAMP7 proteins in vacuolar sorting, AtVAMP711 was used as a model of vacuolar localized protein (Fig. 1A). AtVAMP721, AtVAMP714 and AtVAMP727 were used as the PM-, Golgi-, and endosome-localized proteins, respectively (Fig. 1B, C and D). We have already shown that the dot structures of GFP-AtVAMP714 and GFP-VAMP727 were merged with the fluorescence of SYP31 (Golgi marker) and endocytic dye FM4-64 (endosome marker), respectively [17,21]. Chimeric proteins were generated by replacing the corresponding domains of these proteins with that of AtVAMP711. 3.1. TMD is not the determinant for vacuolar localization To examine whether the TMD is the determinant for the vacuolar targeting or not, the GFP tagged chimeric AtVAMP711 proteins replacing the C-terminal (CT) region with those of PM-localized AtVAMP721, were transiently expressed in the protoplast of Arabidopsis suspension cells. Two different CT fragment were used in the experiments: CT-s (short), including TMD and intravesicular tail, and CT-l (long), including further 13 residues from the coiled coil domain. GFP fluorescence of GFP-AtVAMP711/PM-typeCT-s and GFP-AtVAMP711/PM-type-CT-l was observed on the vacuolar membrane like wild type AtVAMP711 (Fig. 2A and B, compared with Fig. 1A). GFP-AtVAMP721/ Vac-type-CT-s and GFP-AtVAMP721/Vac-type-CT-l which contain the vacuolar-type of CT-s and CT-l, respectively, were appeared on the plasma membrane, but not on the vacuolar membrane (Fig. 2C and D). These results suggest that the CT containing TMD is not a vacuolar targeting determinant.
Fig. 1. Schematic drawings and subcellular localizations of the VAMP7 proteins used in this study. The GFP-tagged VAMP7 proteins were transiently expressed in the protoplasts prepared from Arabidopsis suspension cultured cells. Fluorescence and Nomarski images are shown for GFPAtVAMP711 (A), GFP-AtVAMP721 (B), GFP-AtVAMP714 (C) and GFP-AtVAMP727 (D). Arrowheads in (C) show the dot-like structures of Golgi apparatus. Arrowheads in (D) show the dot-like structures of endosomes as confirmed in [15]. Scale bars = 10 lm.
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T. Uemura et al. / FEBS Letters 579 (2005) 2842–2846
Fig. 2. Schematic drawings and subcellular localizations of chimeric proteins. The GFP-tagged chimeric proteins, of which the TMDs were replaced for with those of other VAMP7 proteins, were transiently expressed in the protoplasts prepared from Arabidopsis suspension cultured cells. Fluorescence and Nomarski images are shown for GFP-AtVAMP711/PM-type-CT-s (A) and GFP-AtVAMP711/PM-type-CT-l (B), GFPAtVAMP721/Vac-type-CT-s (C) and GFP-AtVAMP721/Vac-type-CT-l (D). Scale bars = 10 lm.
3.2. The LD is critical for the vacuolar localization GFP-tagged truncated AtVAMP711(GFP-AtVAMP7 11DNTs), lacking the N-terminal short fragment (NT-s) which is corresponded to the truncated LD, lost the vacuolar-targeting ability and appeared on the plasma membrane and the small dot structures which are considered to be trans-Golgi network (TGN), or endosome (Fig. 3A, compared with Fig. 1A). GFP-AtVAMP712DNT-s and GFP-AtVAMP713DNT-s, which were the NT-s-deleted proteins of other vacuolar-localized VAMP7 proteins, also lost their vacuolar-targeting properties and were localized on the plasma membrane and the TGN/endosome (Fig. 3B and C). These results imply that the LD may be important for the vacuolar targeting of the vacuolar-localized VAMPs (AtVAMP711, AtVAMP712 and AtVAMP713). However, a substitution of the NT-s of AtVAMP721 (PM) with that of AtVAMP711 (vacuolar NT-s) (GFP-tagged vacuolar-type-NTs/VAMP721) did not affect the PM and the TGN/endosome localization, resulting in no vacuolar localization (Fig. 4A, compared with Fig. 1A and B). Further addition of 15 amino acids to the NT-s (which is corresponded to the complete LD [10,13] in an additional chimera), GFP-vacuolar-type-LD/VAMP721 (containing of the LD of the vacuolar type and the SNM and the TMD of the PM type), successfully conferred the vacuolar targeting (Fig. 4B, compared with Fig. 1A and B). The localization of this chimeric protein was not completely the same as that of AtVAMP711 (Figs. 1A and 4B) because the PM and the TGN/endosome targeting was also observed. Since the plasma membrane was proposed as the default destination of singlepass membrane proteins [22], the plasma membrane localization of this chimeric protein might be caused by the default transport. Both the LD of the vacuolar type (AtvAMP711) were substituted for those of the Golgi type (AtVAMP714) and the endosome type (AtVAMP727). GFP-vacuolar-type-LD/VAMP714 and GFP-vacuolar-type-LD/VAMP727 were localized to the vacuolar membrane (Fig. 4C and D, compared with Fig. 1C
Fig. 3. Schematic drawings and subcellular localizations of the LDdeleted VAMP7 proteins and chimeric proteins. The GFP-tagged proteins were transiently expressed in the protoplasts prepared from Arabidopsis suspension cultured cells. Fluorescence and Nomarski images are shown for GFP-AtVAMP711DNT-s (A), GFP-AtVAMP712DNT-s (B) and GFP-AtVAMP711DNT-s (C). Scale bars = 10 lm.
and D). Together with all of the data, we concluded that complete LD of AtVAMP711 play a key role for the vacuolar targeting.
T. Uemura et al. / FEBS Letters 579 (2005) 2842–2846
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Fig. 4. Schematic drawings and subcellular localizations of chimeric proteins. The GFP-tagged chimeric proteins, of which LD, was replaced for with those of other VAMP7 proteins, were transiently expressed in the protoplasts prepared from Arabidopsis suspension cultured cells. Fluorescence and Nomarski images are shown for GFP-Vac-type-NT-s/AtVAMP721 (A) and GFP-Vac-type-LD/AtVAMP721 (B), GFP-Vac-type-LD/ AtVAMP714 (C) and GFP-Vac-type-LD/AtVAMP727 (D). Scale bars = 10 lm.
Fig. 5. Schematic drawings and subcellular localizations of chimeric proteins. GFP-tagged chimera proteins, of which the LD were replaced for with those of other organelle-localized VAMP7 proteins, were transiently expressed in the protoplasts from Arabidopsis suspension cultured cells. Fluorescence and Nomarski images are shown for GFP-PM-type-LD/AtVAMP711 (A), GFP-Golgi-type-LD/AtVAMP711 (B) and GFP-endosometype-LD/AtVAMP711 (C). Scale bars = 10 lm.
3.3. Role of the LD in the others organelle targeting Is the LD capable of targeting plant VAMP7 also to organelle other than the vacuole? To answer this, PM-type LD, Golgi-type LD and endosome-type-LD were attached separately to the vacuolar-type SNM/TMD, and their subcellular localization were examined. GFP-tagged PM-type-LD/AtVAMP711 was observed to the plasma membrane and TGN/ endosome like wild type AtVAMP721 (Fig. 5A, compare with Fig. 1B), implying that PM-type-LD may play a role for the plasma membrane targeting. Moreover, Golgi-type-LD/AtVAMP711 and Endosome-type-LD/AtVAMP711 was also found on the Golgi apparatus and endosomes, respectively (Fig. 5B and C, compared with Fig. 1C and D). However, interestingly, they also appeared on the vacuolar membrane. In the case of Golgi and endosome, the LD domain may not be sufficient for changing the subcellular localization completely. The SNM or TMD also might affect the subcellular localization.
4. Discussion In the experiments presented here, GFP-tagged chimeric proteins were expressed in the Arabidopsis protoplast cells under the 35S promoter. We are aware of the possibility that
GFP itself or overexpression of the fusion proteins, in general, might cause a mislocalization. However, it has been shown that GFP tagged Qa- and Qb-SNARE proteins are located to proper subcellular compartment [22–24]. In this study, to avoid the effect of overexpression, we carefully observed only weakly fluorescent cells, and found that a LD of the vacuolar localized AtVAMP711 were found to be important for the vacuolar targeting. Although we mainly focused on the vacuolar targeting, the results imply that LDs of other VAMP7 proteins also play a general role in the organelle targeting. The LD occurs only in R-SNAREs, but not in Qa-, Qb-, Qc-SNAREs or SNAPs Q-SNAREs [16]. In mammal, the LD of TI-VAMP/VAMP7 interacts with the AP-3 complex (adaptor complex-3), which targets VAMP7 to the late endosomes/lysosomes. Moreover, a LD having lost its central region lacks subcellular targeting capacity [25]. In this study, we also showed that the LD lacking its C-terminus also lost the capacity to regulate the vacuolar targeting (Fig. 4A). These results showed that a complete LD is necessary for the vacuolar sorting in plant cells like mammalian TIVAMP/VAMP7. In case of another longin Ykt6, the LD also controlled the conformation and subcellular distribution [26–28]. Our findings suggest that only a complete LD is a general determinant of subcellular sorting in not only mammalian but also plant cells.
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ER retention is considered to depend on the length of the TMD, since a shorter TMD of 17 amino acids is retained in the ER, and an artificially-made longer TMD of over 20 amino acids is accumulated in the post-Golgi organelle [22,29]. Actually, the TMD length of Arabidopsis ER-localized R-SNARE, AtSec22, is 17 amino acids, whereas the TMD lengths of postGolgi organelle localized R-SNAREs are over 20 amino acids. Therefore, we would hypothesize the vacuolar targeting mechanism of R-SNARE proteins as follows: R-SNARE proteins first enter to the ER, and remain there depending on the length of TMD. The R-SNAREs with a longer TMD will exit the ER and be transported to the Golgi apparatus. At the Golgi apparatus and the TGN, both the LD in cytoplasmic region is critically involved in the determination of the specific organelle transport. Acknowledgements: We thank Dr. Y. Niwa for kindly providing the CaMV35S-sGFP(S65T)-NOS3Õ GFP fusion plasmid. We also thank Dr. M. Umeda for Arabidopsis suspension cultured cell. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology: a Grant-in-Aid for Encouragement of Young Scientist, and Scientific Research on Priority Areas (B) (M.H.S) and a Grant-in-Aid for JSPS Research Fellowships for Young Scientists (T.U.).
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