Download to get the file - Oxford Brookes University

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