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Transcript
A Long and Winding Road SEB Symposium on Membrane Trafficking in Plants Federica Brandizzi1 and Chris Hawes2 1 Biology Dept., University of Saskatchewan, Saskatoon SK S7N 5E2 Canada, Tel. 13069664404, e-mail: [email protected] 2 BMS, Oxford Brookes University, Oxford, OX3 0BP, UK, Tel. 441865 483266, e-mail: [email protected] 1 Introduction In August 2003, the plant endomembrane community gathered under the auspices of the SEB to consider the latest developments in the rapidly growing field of the plant secretory pathway. The meeting was preceded by the annual two day gathering of the European Plant Secretory Pathway Group where postgrads. and postdocs. presented their latest data. Here we consider highlights of the meeting and the controversies surrounding the mechanisms by which the plant cell exports material from sites of synthesis to sites of action and back in again from the external environment. The diversity of subjects covered means that we cannot report on all the speakers’ talks in the detail they deserve. Rather, we have chosen themes of particular interest for emerging areas with significant and exciting advances. So our apologies go to all those excellent speakers whose contributions due space constraints prevent us from discussing. Molecular Interactions in the ER The field of plant endomembrane biology is moving ahead very rapidly in various directions. No longer are the discovery of molecular regulators and the characterisation of the component structures the central themes. Instead, the investigation of molecular interactions and their influence on transport pathways is the focus of many recent studies of the secretory pathway. Starting at the endoplasmic reticulum (ER), A. Vitale (Milan, Italy) addressed the issue of the sequences recognised in vivo by BiP, a major ER chaperone that promotes folding of proteins. BiP binds to exposed hydrophobic amino acids in folding and assembly intermediates, or in defective proteins, a 2 conclusion derived from work on synthetic peptides. As a biologically relevant model for investigating BiP partners, A.V. used different phaseolin constructs and GFP fusion proteins. Monomers of the homotrimeric storage protein phaseolin associate with BiP and trimerisation of phaseolin abolishes the interaction. BiP-association with phaseolin or with secretory GFP fusions results from the interaction of one of the two alpha-helical regions of polypeptide in contact in phaseolin trimers. One of the regions of monomer contact is a BiP binding determinant and during the synthesis of phaseolin, the association with BiP and trimer formation may be competing events. A.V. also showed that the other, internal region of contact between monomers is necessary for phaseolin assembly in vivo and contains one potential BiP binding site. J. Denecke (Leeds, UK) introduced the new concept that BiP is transported to the vacuole when HDEL-mediated retention fails. Through systematic deletion analysis and transplantation experiments, the vacuolar sorting signal of BiP was identified and shown to contain the predicted features of a BP80ligand. Currently, it is unknown how the ER quality control machinery distinguishes between folding intermediates from permanently unfolded proteins that are targeted for degradation. The duration of the interaction of BiP with potential ligands may be the key to answering this question. Persistent binding to the chaperone could simply result in vacuolar transport via association with BiP, as HDEL-mediated retrieval is saturable and likely to be streched to the limit during ER stress and the unfolded protein response. The drug wortmannin was shown to divert the BiP route from the vacuole to 3 the apoplast. Wortmannin also induced hypersecretion of soluble vacuolar cargo carrying sequence-specific vacuolar sorting signals, but wild type BP80 over- expression reduced this effect. Interestingly, co-expression of a GFP fusion to the transmembrane domain and cytosolic tail of BP80 results in a wortmannin-like effect and increases the secretion of soluble vacuolar proteins. By this approach, J.D. has shown that recycling of BP-80 between the PVC and the GA is a saturable pathway. Competition of the truncated receptor with endogenous BP80 compromises efficient recycling of endogenous BP80 from the endosome to the GA. Wortmannin causes almost complete missorting of the hybrid receptor to the vacuole, suggesting that wortmannin interferes with the retrograde transport of the receptor. Exit from the ER Exactly how and where proteins leave the ER for the GA is still an open question that two groups have addressed. D. Robinson (Heidelberg, Germany) suggested that plants most likely utilise both COPI and COPII coat proteins in ER and GA protein transport. Using BY2 cells and Sec13-GFP expression under the control of an inducible promoter, he showed that Sec13 punctate structures, interpreted as ER exit sites, co-localised 50% with immuno-labelled Sar1p containing structures. Interestingly Sar1p labelling rarely colocalised with the GA and the conclusion was that GA must move over the ER to find exit sites. In contrast to this, F. Brandizzi (Oxford, UK) proposed a new model of a motile export competent complex, comprising the 4 Golgi stack and export site. She used a photobleaching technique based on the bleaching of one fluorochrome (YFP) on a Golgi stack labelled with both CFP and YFP, to follow the recovery of fluorescence on the organelle still visible through the CFP channel (Brandizzi et al., 2002). She showed that Golgi bodies moving over the cortical ER network collect membrane cargo without stopping (Fig 1). Further evidence on the co-location of the Sar1p GTPase and mutant forms with the Golgi supported her model. Whether the differences reported here simply reflect two different experimental systems has yet to be proven. Trafficking to the PM and Endocytosis Despite the fact that most of the molecular machinery needed for endocytosis in plants was described a number of years ago (Hawes et al., 1991), until recently the necessary probes and physiological markers have not been available to functionally dissect the endocytic pathway. Recently there has been a resurgence of interest in the pathway with reports of PM protein recyling such as the auxin efflux carrier PIN 1 (Geldner et al. 2001) and the use of the FM family of styryl dyes as reporters of endocytic compartments. U. Homann (Darmstadt, Germany) investigated the osmotically driven swelling and shrinkage of guard cells by patch-clamp measurements to determine that capacitance changes result from increase and decrease of the PM surface area. She reported that in guard cells changes in the PM surface area are accomplished by fusion and fission of small exo- and endocytic vesicles and not by PM stretching. By expressing GFP constructs of the 5 inward K+ channel Kat1 in protoplasts, it was shown that pressure driven increases in the PM surface area induces exocytosis of Kat1 containing vesicles and unstimulated protoplasts show Kat1 containing vesicles colocalising with the endocytic marker FM4-64, suggesting a cycling of K+ channels at the PM. Two presentations at the meeting offered conflicting data on both the location and function of Rab5-GTPase homologues, which are involved in trafficking in the early mammalian endosomal pathway. T. Ueda (Riken, Japan) used fluorescent protein tagged versions of two arabidopsis Rab5 homologues, Ara6 and Ara7. Ara6 is a member of a novel class of plant Rabs lacking the Cterminal region for membrane attachment and is uniquely modified at the Nterminus for N-myristoylation and palmitoylation (Ueda et al., 2001). Ara7 is a conventional Rab5 homologue. In arabidopsis protoplasts, Ara7-mRFP localises on putative endosomal compartments and with a putative endosomal SNARE AtVAMP27 (Fig. 2). Ara6-GFP co-localised with a compartment marked by AtPEP12, a prevacuolar t-SNARE. In the mutant cells of GNOM, an ARF-GEF which is involved in recycling plasma membrane proteins to an endosomal-like compartment (Geldner et al., 2003), Ara7 locates to a deformed membrane along with AtVAMP727. A proposal was made that the two Rab proteins mark a continuum in the development of a pathway from endocytic vesicle, to an endosomal compartment through to the prevacuole and that Rabs regulate membrane fusion along this pathway. In contrast, S. Bolte (Gif-sur-Yvette, France) showed that a Rab closely related to Ara6, isolated from Mesembryanthemum crystallinum (m-Rabmc), which also has the unique N-terminal myristoylation site, is involved in 6 vacuolar trafficking (Bolte et al., 2003). In BY2 cells and arabidopsis protoplasts this Rab co-locates with the prevacuole marker BP80 (Fig. 3) and partially with the GA. Its role in vacuolar transport was shown by expression of a non-functional mutant resulting in blockage of the lytic vacuole targeted protein aleurain-GFP. In addition, at the secretory pathway meeting 3 days earlier A. Kotzer (Oxford, UK) presented new data suggesting that AtRabF2b (Ara7) may also be involved in vacuolar trafficking in tobacco. It is obvious that more work is needed to clarify the roles of these closely related Rabs. Microdomains and Macrodomains The maintenance of organelle function in multi-compartmentalised cells requires specific protein sorting and delivery mechanisms to ensure efficient movement of proteins to their respective destinations. An increasing number of studies demonstrate that proteins may target specific domains of certain organelles. G. Hinz (Gottingen, Germany) reported on the sorting of vacuolar storage proteins in the GA. An initial observation suggested that spatial separation of vacuolar sorting events takes place in the GA of developing pea cotyledons (Hillmer et al., 2001). These contain protein storage vacuoles (PSV), and lytic vacuoles. Lumenal and membrane proteins of the PSV exit the GA in dense vesicles (DVs) and not in clathrin-coated vesicles. The GA works as an efficient protein sorter by precisely separating proteins destined for the ER, the PM or different types of vacuoles. G.H. proposed that major storage proteins segregate as early as the cis-Golgi. A model based on intracisternal lipid rafts involved in molecular sorting could explain how the GA efficiently minimises mis-sorting. To test this model, G.H. used prolegumins, 7 which are transported to DVs en route to the SV by a 53kDa protein. Lipid rafts may be vectors transporting the 53kDa protein and lipodomains may form the DVs. Such lipid domains may exclude other secretory proteins from DVs thus ensuring an early and precise segregation of proteins destined to the PSV. P. Dupree (Cambridge, UK) introduced PM lipid rafts and suggested that these may be involved in defining different areas of the surface in polarised cells. The PM is enriched in glycosylphosphatidylinositol (GPI) anchored proteins (GAP) which represent an alternative means of attaching a protein to a membrane and may be used to target a specific subset of proteins to the cell surface for extracellular matrix remodelling and signalling. The C-terminus of a GAP is covalently attached via phosphoethanolamine and a conserved glycan to phosphatidylinositol or a ceramide. Recent reports confirm the existence of lipid rafts in plants and that GAPs may associate with them, indicating that GPI anchored proteins are important in cell surface function, remodelling and also in cell polarisation. P.D. demonstrated that fusing a reporter protein PAT to the hydrophobic C-terminal domain of an arabidopsis GAP was sufficient to target it to the PM. Interestingly the protein associated with detergent resistant membranes indicating the presence of PM lipid rafts. An arabidopsis genome search of secretory proteins with a hydrophobic Cterminus and no TMD, but with a putative cleavage site, revealed248 potential GPI anchored proteins. These correspond to a variety of proteins, including putative receptors, proteases, oxidases, extensins, and lipid-transfer-proteins. A proteomic analysis subsequently identified 39 of these proteins. Lipid- based sorting is obviously an area ripe for further research. For instance do 8 other components of the endomembrane system use such mechanisms? A few years ago it was reported that phosphatidylserine-rich vesicles can be produced in vitro by ER membranes (Sturbois-Balcerzak et al., 1999). Could these play a role in sorting cargo destined for the Golgi? The Influence of Regulatory Proteins on Whole Plants The mechanisms by which membrane traffic integrates with other cellular function such as mitosis, and whole plant development were addressed. Intracellular traffic has long been studied as a separate functional issue, but it is obviously closely co-ordinated with other cellular functions in living cells. G. Jürgens (Tübingen, Germany) reviewed the advances made by his group in understanding the regulation of plant cell division. He focussed on cytokinesis as a system to analyse syntaxin specificity in membrane fusion. G.J. described the regulatory molecules of this complex process using arabidopsis embryos as a model. The arabidopsis KNOLLE (KN) gene encodes a cytokinesis-specific syntaxin that localises to the division plane and mediates cell-plate formation. He identified mutants defective in regulation of the phragmoplast vesicle fusion machinery and cell division mutants that do not progress in division at very early stages of embryogenesis. For example, KEULE and KN cooperate to promote vesicle fusion in the cell division plane. In mutants, unfused vesicles accumulate in the plane of cell division and strong cytokinesis defects are visible and result in seedling lethality. G.J. highlighted the specificity of the interaction of these molecules to organise cytokinesis with experiments based on rescue of mutant phenotypes with selective regulators. For example, some syntaxins such as AtSNAP33 are 9 capable of interaction with KN but are not necessarily exclusively cytokinesis proteins as deduced from ubiquitous expression during the cell cycle and subcellular distribution at the PM as well as at the cell plate. To test the specificity of KN in membrane recognition and fusion in cytokinesis, other syntaxins were tested for mediation of KN functionality when expressed under the control of KN cis-regulatory sequences (Fig. 4, Muller at al., 2003). By proving that only one non-essential and non-cytokinesis-related syntaxin, SYP112 (37% identity and 62% similarity to KN), was targeted to the cell division plane and was able to rescue phenotypically kn mutants, the specificity of KN in regulating cytokinesis was confirmed. Other syntaxins such as the prevacuolar PEP12, and SYR1 (which shares with KN a co-localisation plus 44% identity and 64% similarity) were unable to rescue kn homozygous mutants. G.J. demonstrated that syntaxins have three requirements for the fine tuning of cytokinesis: expression of at M phase, absence of targeting signals to membrane compartments different from the plane of cell division, and a sequence to mediate functional specificity in the process of membrane fusion. Integrating Trafficking with Growth and Development A hallmark of all eukaryotic cells is the compartmentalisation of the secretory system into organelles to host specific biochemical reactions, whilst still enabling communication by means of vectors, such as vesicles, tubules or direct connections. This is achieved by cargo loading into vectors, and by tightly regulated interactions between donor and acceptor membranes. The machinery involved in this regulation comprises different groups of proteins 10 such as the SNAREs, ubiquitous integral membrane proteins that are required for correct vesicle sorting and trafficking and fusion. T. Sanderfoot (Minneapolis, USA) reviewed the functionality of plant specific SNAREs in the GA/endosomal system. T.S. highlighted the vital importance of these SNAREs at an organismal level. Twenty-four syntaxins are grouped into 10 gene families in arabidopsis, each group containing one to five paralogous members. Their role in a cell is essential, however their abundance does not reflect a degree of redundancy, despite the high degree of sequence similarity between the gene family members. For example, disruption of individual syntaxins form SYP2 and SYP4 gene families is lethal, indicating essential and unique functions of the syntaxins of these families. Similarly, F. Assaad (Stanford, USA) illustrated the importance of SNAREs in the plant response to abiotic and biotic stresses. For example, SYP121 and SYP122 are essential genes that, despite their sequence similarity, are differentially regulated upon biotic stress in the host-pathogen interaction. M. Kalde (Norwich, UK) also suggested that a regulatory link might exist between elicitor-induced calcium fluxes and a rapid phosphorylation of SYP122. This may have a direct effect in the host defence response to a pathogen attack mediated by exocytosis of defence-related compounds. Advances in Microscopy The ability to witness the dynamics of the secretory pathway in vivo is redefining our concept of cellular organisation and as expected the application of fluorescent protein technology pervaded every aspect of the meeting. For example, I. Moore (Oxford, UK) reported on signal sequence-GFP expression 11 as a non-invasive live cell assay of secretion in arabidopsis. The intracellular retention of a secreted GFP in a stable line can be used as a tool for forward and reverse genetic analysis of membrane traffic and endomembrane organisation. Such a line was used both for screening for secretory mutants and for identifying a role for RHD3 (Root Hair Defective) in the secretory pathway (Zheng et al., 2003). With the latest advances in technology, the resolution by which we can follow cellular events is rapidly improving. M. Oheim (Paris, France) introduced 2PEF evanescent-field microscopy, by which organelle tracking and imaging single-molecule dynamics with millisecond time resolution becomes feasible in mono and dual fluorochrome imaging mode. A. Nakano (Tokyo, Japan) demonstrated the advantage of using the spinning Nipkow disk confocal in combination with the ultrahigh-sensitivity, high-speed and high-resolution camera system. This now allows observation of the dynamic movement of small vesicles in living yeast in real time (Fig. 5). Without doubt, our knowledge of the dynamics of the secretory pathway and its regulation mechanism will increase exponentially once the identity of more proteins and signals are uncovered. However, the undoubted advantages of the resolution offered by EM still have yet to be fully harnessed by the endomembrane community. To unravel the complexities of protein distribution within many of the compartments reported on at the meeting such as the putative endosomes and prevacuoles, will require faithful and reproducible immunogold labelling. We are still waiting for much of this data. 12 Conclusions M. Blatt and G. Thiel assembled an exciting and stimulating group of speakers that described significant advances in our understanding of the mechanisms regulating the biology of the plant secretory membranes. Overall, the meeting brought together researchers working on apparently disparate subjects but linked by a common theme of the secretory pathway. The array of techniques applied clearly illustrated strength in diversity and the importance of using complementary methodologies in order to penetrate and understand such a complex system. Acknowledgements: The meeting was generously supported by the SEB, Gatsby Charitable Foundation, Carl Zeiss, Conviron and Universal Imaging. We thank various contributors for supplying us with micrographs for this report, and A. Kotzer for reading the manuscript. Finally, we thank Jennifer Lippincott-Schwartz (NIH, Maryland, USA) for an inspirational opening address on live cell imaging and the animal secretory pathway. References Bolte, S. Brown, S. and Satiat-Jeunemaitre, B. (2003) The N-myristoylated Rab-GTPase m-Rabmc is involved in post-Golgi trafficking events to the lytic vacuole in plants. J. Cell Sci. In press. Brandizzi, F., Snapp, E.L., Roberts, A.G., Lippincott-Schwartz, J. and Hawes, C. (2002) Membrane protein transport between the endoplasmic 13 reticulum and the Golgi in tobacco leaves is energy dependent but cytoskeleton independent: evidence from selective photobleaching. Plant Cell, 14, 1293-1309. Geldner, N., Anders, N., Wolters, H., Keicher, J., Kornberger, W., Muller, P., Delbarre, A., Ueda, T., Nakano, A. & Jurgens, G. (2003). The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell, 112, 141-142. Geldner, N., Friml, J., Stierhof, Y.D., Jurgens, G. and Palme, K. (2001) Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature, 413, 425-428. Hawes, C.R., Coleman, J.O.D. & Evans, D.E. (1991) Endocytosis, Exocytosis and vesicle traffic in plants. Cambridge University Press, Cambridge, UK. Hillmer, S., Movafeghi, A., Robinson, D.G. and Hinz, G. (2001) Vacuolar storage proteins are sorted in the cis-cisternae of the pea cotyledon Golgi apparatus. J Cell Biol, 152, 41-50. Muller, I., Wagner, W., Volker, A., Schellmann, S., Nacry, P., Kuttner, F., Schwarz-Sommer, Z., Mayer, U. and Jurgens, G. (2003) Syntaxin specificity of cytokinesis in Arabidopsis. Nat Cell Biol, 5, 531-534. Sturbois-Balcerzak. B., Vincent. P, Maneta-Peyret. L., Duvert. M., SatiatJeunemaitre. B., Cassagne. C. and Moreau. P. (1999) ATP-dependent formation of phosphatidylserine-rich vesicles from the endoplasmic reticulum of leek cells. Plant Phys. 120, 245-256. 14 Ueda, T., Yamaguchi, M., Uchimiya, H. & Nakano, A. (2001). Ara6, a plantunique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana. EMBO Journal, 20, 4730-4741. Zheng, H., Kunst, L., Hawes, C. and Moore, I. (2004). A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus. Plant J. in press. Figure legends: Fig. 1. Recovery after photobleaching of an ERD2-YFP Golgi (red) against ST-CFP (green) targeted to the same Golgi. Fluorescence recovery indicates protein transport into moving Golgi (Micrograph of F. Brandizzi). Fig. 2. Colocation of Ara7 and AtVamp727 in arabidopsis protoplasts (Courtesy of T. Ueda). Fig. 3. Immunofluorescence of m-Rabmc and BP80 showing co-location on the prevacuolar compartment (Courtesy of S. Bolte). Bar = 10 ųm Fig. 4. Subcellular localisation of Myc-tagged syntaxins (Muller et al., 2003). Immunofluorescence images of seedling root cytokinetic cells immunostained with anti-KN serum (green; a, d, g, j, m) and with an anti-Myc monoclonal antibody (red; b, e, h, k, n). Overlays with DAPI-stained chromatin are shown (blue; c, f, i, l, o). Transgenic genotypes were: Myc–KN (a–c); Myc–AmKN (d–f); Myc–PEP12 (g–i); Myc–SYR1 (j–l); Myc–SYP112 (m–o). Asterisk in c marks the cell plate. Note lack of colocalisation between KN and Myc–PEP12 15 in i. Scale bars = 10 ųm. (Courtesy of G. Jurgens, reproduced with permission). Fig. 5. Real-time observation of COPI in living yeast cells expressing GFPRer1p. The observation was conducted using a spinning-disk confocal microscope with a Harp camera (Courtesy of A. Nakano, reproduced with permission). Bar = 5 ųm. 16