Bioscience Horizons Advance Access originally published online on April 28, 2009
Bioscience Horizons 2009 2(2):205-211; doi:10.1093/biohorizons/hzp024
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Differential localization of two plant homologues of Rab5 GTPases in the secretory pathway
Undergraduate School of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK
* Corresponding author: 1A High Street, Upton, Nr Pontefract, West Yorkshire WF9 1HR, UK. Tel: +44 7729413670. Email: hani19866{at}hotmail.com
Supervisor: Prof. Jurgen Denecke, Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Clarendon Way, Leeds LS2 9JT, UK.
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Abstract |
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Rab5 GTPases are key regulators of vesicular transport. Known plant Rab5 homologues ARA6 and RHA1 have been assigned to the endocytic and/or biosynthetic vesicular trafficking pathway, but conflicting reports in the literature justify further work on these two GTPases. In this project, binary vectors for Agrobacterium-mediated plant transformation were constructed to drive expression in tobacco leaf epidermis of GTP-trapped mutants of ARA6-green fluorescent protein (GFP) and Venus-RHA1. Confocal laser scanning microscopy revealed key differences in the subcellular localization of the two fluorescently tagged GTPase mutants. When present in the GTP-locked configuration ARA6-GFP is primarily found associated with the tonoplast, whereas Venus-RHA1 is significantly cytosolic. Co-expression of the two fluorescently tagged mutant GTPases with the Golgi markers ST-YFP and ST-CFP, respectively, suggest that neither ARA6 nor RHA1 mutants are mis-targeted to the Golgi apparatus and moreover, they do not show any significant colocalization with each other. The results are consistent with the notion that they differ in their roles within the endocytic and biosynthetic vesicular pathway. A future focus to continue this project would be to use GFP-tagged ARA7, the third plant Rab5 homologue, to verify if the high sequence homology between RHA1 and ARA7 warrants overlapping redundant functions or not.
Key words: ARA6, RHA1, endocytic pathway, biosynthetic pathway
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Introduction |
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Rab GTPases are molecular switches that alternate between an active GTP and an inactive GDP bound form. They are central regulators of all aspects of vesicle trafficking: vesicle formation/budding, motility via the cells cytoskeleton through interaction with motor proteins1 docking and membrane remodelling and fusion. As a result it is thought that each organelle within the endomembrane system carries its own set of Rab proteins, which ensures the specificity of the intracellular membrane transport.2 The functions of Rab are controlled by downstream regulators that mediate not only nucleotide recycling but also their cycling between membranes.3
In Arabidopsis thaliana, AtRabF2a (RHA1) and AtRabF2b (ARA7) are very closely related to each other and correspond best to the Rab5 molecules in yeasts and animals. Interestingly, the AtRabF1 GTPase (ARA6) appears to be an abnormal member of the RabF class which has been proposed to be specific to plants,4 although its closest mammalian homologue is still believed to be Rab5 due to high sequence homology.
The effector domain is what subdivides the Rab proteins and determines their function, thus this domain in ARA7 is related to that of Rab5c.5 However, the effector domain of ARA6 differs from ARA7 and other Rab GTPases in that it has an extra stretch of amino acids at the N terminus which contains putative N-myristoylation and palmitoylation sites. ARA6 also lacks the conserved cysteine residues at the C terminus, usually employed by all other Rabs for isoprenylation and membrane anchoring. Consequently, ARA6 has been termed the bona fide novel plant subtype.4
As a result of their structural differences and the unique nature of ARA6, it has suggested they have specific roles within the endosomal compartments, as illustrated by different temporal and spatial patterns of expression.4 However, their definite roles are still unclear. Double-staining experiments have demonstrated that Arabidopsis thaliana cells have two distinct populations of early endosomes and that ARA6 is localized to only one4 and ARA7 and RHA1 are thought to be located on a partially overlapping but different population of earlier endosomes involved in the recycling of plasma membrane proteins.6 The plant Rab5 proteins were fluorescently tagged with GFP and visualization under a confocal microscope illustrated that ARA6-GFP and GFP-ARA7 are associated with very similar compartments within an early step of the endocytic pathway, probably the early endosome.4
Further refined studies using the fluorescent internalization marker FM4-646 indicated that ARA6 and ARA7 are indeed linked to the same endosome, although ARA6-GFP was not localized on all of the early endosomal compartments highlighted by the FM4-64 marker.4 Hence, FM4-64 fluorescence visualized on the ring-like structures did not show considerable evidence of any ARA6-GFP staining leading to the conclusion that Arabidopsis thaliana has two distinct endosomal structures and that ARA6 is only associated with one of these,4 whereas ARA7 accumulates on the other. However, the use of FM4-64 as an endosomal marker has been disputed, because it does not only associate with endosomes and pre-vacuolar compartments (PVCs), but also labels Golgi bodies and tonoplast.7–9
The early work on plant Rab5 members was strongly influenced by the known fact that Rab5 is localized to early endosomes in mammalian cells. More recent investigations confirmed that this initial hypothesis about plant Rab5 members may be too simplistic. Good evidence was presented showing that GFP-ARA7 labels the PVC, which has been suggested to be spatially related to the late endosome.9 Thus, GFP-ARA7 has been hypothesized to cycle between the GA and the PVC, a pattern which is also observed for BP80.9 In contrast to these experiments, another investigation using Venus-tagged SNARE proteins AtSyp41 and AtSyp61, known markers of the trans-golgi network in plants10 did not reveal any colocalization with the GFP-tagged ARA7. Moreover, the SNARE proteins AtSyp21 (AtPep12) and AtSyp22 (AtVam3), which evidently function and associate with the PVC, also showed no sign of colocalization with GFP-ARA7 or GFP-RHA1. However, they did show strong colocalization with Ara6-GFP. This suggested firstly that the two RabF subfamilies are found on different endosomal domains but ARA7 does not cycle between the GA and the PVC.6 In the contrasting experiments, this cycling behaviour was accepted by experiments on the GDP bound ARA7[S42 N], which is restricted to the GA and the GTP-bound form ARA7[Q69L] which localizes to the PVCs.9 This localization suggests that ARA7 is initially targeted to the GA then on to the PVC. In summary, these studies have illustrated that ARA7 acts on a pathway between the GA and the PVC.
Additional research on the RabF class in plants has focused attention on an ARA6 orthologue isolated from the salinity resistant plant Mesembryanthmum crystallinum.11 Like ARA6, it represents another example of the plant unique new subclass of Rab proteins, and was termed m-Rabmc. Through confocal laser scanning microscopy (CLSM), m-Rabmc is evidently predominantly located on the PVC of the lytic vacuole and partially on the GA of Arabidopsis protoplasts.12 But more recently, all three of the GFP-tagged Arabidopsis thaliana Rab5 homologues (ARA7, RhaI, ARA6) were shown to colocalize with GFP-SKD1 on the multivesicular bodies,13 suggesting the PVC as a major localization for the Rab5 group members in plants. But none of the more recent studies using statistically meaningful fluorescence microscopy in epidermis cells have involved a systematic comparison of the F1 and F2 group of plant Rab5 GTPases. For these reasons, the aim of this project was to re-evaluate the putative function and localization of the two types of Rab5 homologues found in plants, ARA6 (F1 type) and RHA1/ARA7 (F2 type) in the secretory pathway. Owing to the interesting property of the GTP-trapped (GTP hydrolysis) mutant to accumulate in a distal compartment in the pathway, specific fluorescently tagged GTP-locked Rab5 fusions, ARA6(Q93L)-GFP and Venus-RHA1(Q69L), were created and analyzed by microscopy, to allow a better comparison of the presumed differential function of the two types of GTPase.
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Materials and Methods |
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Recombinant Plasmid Construction
All DNA manipulations were done according to established procedures, except for the use of low EDTA-strength TE (10 mM Tris–HCl and 0.1 mM EDTA pH 8) and loading dye (24% sucrose, 0.1% Bromophenol blue and 40 mM EDTA). Buffers for restriction digests, dephosphorylation or ligation reactions were used at 1x concentration. Escherichia coli MC1061 (Casadaban and Cohen, 1980) was used for all transformations of ligation mixtures due to its superior competence and faster growth compared with strains such as DH5
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Preparative restriction digests for vector generation and fragment isolation were routinely done in a total volume of 50 µl, containing 10–20 µg of plasmid DNA, digested with 10–20 U of the appropriate restriction enzyme, supplemented with the appropriate restriction buffers according to manufacturers instructions, brought to 50 µl with TE and incubated at the appropriate temperature (usually 37°C). To monitor the progress of the restriction, 1 µl samples were taken at 0, 20 and 40 min incubation, supplemented with 5 µl of loading dye and analyzed by gel electrophoresis together with a molecular weight marker (phage 
digested with PSTI). Preparative gel-electrophoresis and gel extraction using QIA quick spin column kit (Quiagen) was performed according to manufacturer's instructions. Dephosphorylation using calf intestinal alkaline phosphatase was performed by supplementing the 47 µl of the master mix left from the preparative digest after testing, 43 µl of TE, 5 µl of 10x Cip buffer and 5 µl of the Cip enzyme (1 U/µl) to yield a total of 100 µl. This was then left at 37°C for 30 min.
Ligations included vector alone without ligase, vector with ligase and vector + fragment(s) + ligase and incubated for a minimum of 1 h at 22°C. After routine transformation of competent E. coli MC1061, selective plates were incubated overnight and testing of recombinants was carried out by growing 2 ml overnight shaking cultures inoculated with single isolated colonies, followed by DNA extraction and restriction analysis. High quality DNA for sequencing and Agrobacterium tumefaciens transformation was carried out using the ‘Wizard prep’ procedure according to manufacturer's instructions (Promega). Table 1 shows a list of recombinant plasmid names followed by a short description.
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Agrobacterium Transformation
The competent Agrobacterium was thawed on ice and split up into four tubes with 90 µl in each. Then 3 µl of plasmid was added to 90 µl of cells. These tubes were then frozen at –80°C for 30 min and then thawed for 4 min at 37°C in a heat clock. The 1 ml of LB medium with no antibiotics was added and transferred to a Falcon tube. This was then incubated with shaking at 26°C for 4 h. Meanwhile the plates containing streptomycin, spectinomycin and rifampicin were dried out and after the 4 h incubation the culture was added directly and left to dry again. When dry they were incubated at 28°C and incubated for 2 days. Single colonies were streaked on new plates containing streptomycin, spectinomycin and rifampicin and incubated at 25°C for a further 2 days. A liquid culture for infiltration was made by inoculating 2.5 ml of MGL (2.5 g/l yeast extract, 5.0 g/l of tryptone, NaCl and mannitol, 1.16 g/l of Monosodium Glutamate, 0.25 g/l KH2PO4, 0.1 g/l MgSO4·7H2O and 1.0 g/l Biotin, pH 7.0) with a single colony and incubated on a shaking incubator at 25°C overnight.
Infiltration
For both constructs, the infiltration protocol was carried out the same. One millilitre of the liquid Agrobacterium (see above) culture was transferred into an Eppendorf tube and spun for 5 min at 5000 r.p.m. The supernatant was then removed and the pellet re-suspended in 1 ml of infiltration buffer (50 mM MES (pH 5.6), 2 mM Na3PO4, 0.5% glucose, 100 µM Acetosyringone) and vortexed. The optical density (OD600) was then measured. The suspensions were diluted to obtain an OD600 of 0.1 with infiltration buffer. The wild-type GTPases and the mutant GTPases ARA6(Q93L)-GFP and Venus-RHA1(Q69L) were then mixed separately with their Golgi markers ST-YFP and ST-CFP, respectively, and made up to 1 ml. The Agrobacterium suspensions were infiltrated via a syringe into small insertions made in the abaxial epidermis of healthy tobacco leaves. The outline of the infiltrated area was marked.
Confocal Laser Scanning Microscopy
After 48 h of normal growing conditions, a scalpel was used to cut out a small square from the leaf, from the marked area, mounted in tap water on a slide with the lower epidermis facing the cover glass (22 3 50 mm; no. 0). Images were taken with a Zeiss LSM 510 META laser scanning microscope with a Plan-Neofluar 340/1.3 oil DIC objective. For GFP and CFP constructs, excitation lines of an argon ion laser of 458 nm were used, whereas Venus was excited with the 514 nm laser line with alternate line switching on the multitrack facility of the microscope. Fluorescence was detected using a 545 nm dichroic beam splitter and a 475–525 nm band-pass filter for GFP/CFP and a 560–615 nm band-pass filter for YFP. Post-acquisition image processing was performed with the LSM 5 image browser (Zeiss).
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Results |
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A main objective of this project was to construct GTP-trapped mutants ARA6(Q93L)-GFP and Venus-RHA1(Q96L) within the plant binary transformation vector pTJLH20. These were then used to visually compare wild-type Rab5 localizations with mutant Rab5 localizations in Tobacco leaf cells. Furthermore, these mutant GTPases were co-expressed with their corresponding Golgi markers to reveal any targeting to the Golgi apparatus.
Rather than using protoplasts as test objects as in previous studies, the localization experiments were carried out with Agrobacterium infiltrated leaves. This technique allows stable integration of a few copies of the DNA, leading to a more homogenous expression profile from cell to cell and ruling out over expression artefacts. In addition, the lower leaf epidermis cells form ideal objects for live imaging, because they contain no chloroplasts and display a thin cytosol layer between tonoplast and plasma membrane.
Subcloning of an ARA6(Q93L)-GFP Expression Construct into a Binary Plant Transformation Vector
In order to carry out transient expression experiments in tobacco leaf epidermis cells, it was necessary to subclone a previously constructed fusion between the mutant GTP-trapped ARA6 and the GFP. The existing chimaeric gene, termed ARA6(Q93L)-GFP had to be isolated from a small pUC19 based vector (pFB15) containing ARA6(Q93L)-GFP. This is under the control of the cauliflower mosaic virus 35S promoter flanked by a 3' untranslated end of nopaline polyadenylation signal. This chimaeric gene was then inserted into the binary plant expression vector pTJLH20 (based on pDE7001 containing BP80). pTJLH20 is easy to clone when a visible fragment is removed from the vector. Once the ARA6(Q93L)-GFP was inserted into pTJLH20 Agrobacterium-mediated transformation was carried out. The subcloning strategy is based on the fact that unique restriction sites that are also present in the plant expression vector pTJLH20 flank the chimaeric gene. The chimaeric gene present in pTJLH20 is significantly larger than the ARA6(Q93L)-GFP expression construct and recombinants can be identified without difficulty. After testing and subsequent transformation of Agrobacterium tumefaciens, a test infiltration showed that GTP-trapped ARA6 was partially expressed on the tonoplast (Fig. 1A and B).
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Creating a Fusion Between Venus and RHA1(Q69L)
In the case of the second Rab5 GTPase (RHA1), a fluorescent fusion protein with the mutant GTP-trapped mutant did not exist. Therefore, a fragment carrying the fluorescent protein with the mutant GTP-trapped molecule was to be ligated with a fragment carrying the mutant GTPase RHA1(Q69L). The fusion was directly subcloned into the plant expression vector pTJLH20, used in the first construct, via a three fragment approach. Since RHA1 is linked to membranes via a C-terminal isoprenylation in contrast to ARA6, which is membrane anchored via its N terminus through mryistoylation and palmitoylation (Ueda et al.6) the fluorescent marker Venus was fused at the N terminus. Test-infiltrations indicated that GTP-trapped RHA1 was mainly cytosolic (Fig. 2A and B), revealing a discrepancy between the patterns obtained with the two mutant GTPases.
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The Wild-Types ARA6-GFP and Venus-RHA1 Expression with their Respective GA Markers
The preliminary data suggested the relevance of re-evaluating the wild-type Rabs ARA6-GFP (Fig. 3) and Venus-RHA1 (Fig. 4) with the Golgi (GA) markers ST-YFP and ST-CFP, respectively. As observed in Image 3 ARA6wt-GFP, 72 h after Agrobacterium inoculation, the expression is primarily cytosolic although there is some tonoplast colocalization observed on the bulb (but this is very weak). The closed arrows indicated on the images demonstrates firstly that there is little if any bleeding through of the red into the green and moreover, that the mutant ARA6-GFP does not show any colocalization with the GA marker ST-YFP. This is further confirmed with the merged image showing simultaneous expression of ARA6-GFP and ST-YFP. The co-expression of the construct with its corresponding GA marker ST-CFP was also observed with wild-type Venus-RHA1 (Fig. 4). Venus-RHA1wt was significantly more cytosolic as ARA6-GFP but shows the same lack of colocalization with the GA marker. The results indicate that the two GTPases partition differently between the cytosol and their target organelle.
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ARA6(Q93L)-GFP and Venus-RHA1(Q69L) Expression with their Respective GA Markers
Similar co-expression experiments between the Golgi marker and the fluorescent GTP-trapped GTPase fusions illustrated a much more dramatic difference in behaviour between ARA6 and RHA1. ARA6 is strongly re-localized to the tonoplast, whereas RHA1 appears to be mostly cytosolic when trapped in its GTP-locked form. Relocalization of ARA6(Q93L)-GFP to the tonoplast is evident from Fig. 5 in that there are extendable membrane protrusions facing inwards from the cell boundary that are only visible with the expression on the tonoplast. This is in sharp contrast to plasma membrane labelling that would show a defined line exclusively at the boundary, without any inward intrusions of any kind. Furthermore, the green fluorescence is wrapped around the bulb that is pointed out by the closed arrow in Fig. 5, this again would not be present if this were plasma membrane expression. What is also made apparent from Fig. 5 is that ARA6(Q93L)-GFP is not localized on the GA. With respect to the second construct, Venus-RHA1(Q69L) expression is evidently cytosolic. This can be fully appreciated when focusing on the obvious negative staining (dark punctate) caused by organelles embedded in the cytosol (i.e. mitochondria, peroxisomes) that exclude the cytosolic fluorescence. In addition, no punctate GFP fluorescence is noticeable for the mutant (Fig. 6 closed arrow), in sharp contrast to the wild-type. As observed for ARA6(Q93L)-GFP, Venus-RHA1(Q69L) is also not localized on the GA. In conclusion, the two Rab5 GTPases ARA6 and RHA1 show little similarities in their expression within the secretory pathway, suggesting a differential function in the vacuolar route.
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Discussion |
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Rab GTPases are key regulators of vesicular transport in eukaryotic organisms. To date, there have been 60 mammalian Rabs identified, thus a picture of all the Rabs with their functions and interactions has now started to emerge, allowing the way for homologues to be revealed in other kingdoms and potential difference to be uncovered. ARA6, ARA7 and RHA1, the Rab5 homologues identified in plants, have been assigned to the endocytic and the vacuolar trafficking pathway.9 Their specific location within these pathways has been narrowed to the endosomal compartments. In order to extend these observations, GTP-hydrolysis mutants were created and compared with the wild-type.
Disrupted GTP Hydrolysis Leads to Redistribution of ARA6 and RHA1 to Different Subcellular Localizations
Both chimaeric genes were successfully created during the first half of the project and tested. The initial microscopy experiments (Figs 1A and B and 2A and B) illustrate that these constructs did get expressed within the tobacco leaves. Figures 1A and B and 2A and B illustrate, at different magnifications, the expression of ARA6(Q93L)-GFP. These images show that this mutant is primarily expressed in the tonoplast. Whereas, the initial CLSM experiments illustrated that Venus-RHA1(Q69L) was primarily expressed in the cytosol. However, there was also evidence of tonoplast expression, which has been seen in other recent experiments, suggesting that this may be due to vesicles distributed from the PVC containing bound RHA1 may then travel to the tonoplast.5 These initial images make it apparent that ARA6(Q93L)-GFP and Venus-RHA1(Q69L) are not identical in where they express. Owing to rapid progress with the sub-cloning steps, sufficient time was left for thorough colocalization experiments using a reference marker for the Golgi apparatus. This involved the co-expression of ARA6(Q93L)-GFP and VenusRHA1(Q69L) with the Golgi marker ST-YFP and ST-CFP, respectively. In order to make comparisons, the GTP-tagged wild-type fusions ARA6-GFP and Venus-RHA1 were also co-expressed with the corresponding Golgi markers. The confocal images show no evidence of the wild-types ARA6-GTP or Venus-RHA1 converging to the Golgi markers, thus the punctate dots do not overlap (Figs 3 and 4, merge). Furthermore, ARA6(Q93L)-GFP and VenusRHA1(Q69L) also failed to colocalize with the GA. Instead, the colocalization experiments confirmed the preliminary analysis (Fig. 1A and B), showing that ARA6(Q93L)-GFP is mainly tonoplast localized, whereas VenusRHA1(Q69L) is mainly cytosolic (Fig. 2A and B). In both cases, the Golgi marker appears to be unaffected. The results obtained for ARA6 are similar to the experiments carried out with the mutant mRabmc(N147I) in Arabidopsis thaliana protoplasts.11 This mutant was expressed with the same Golgi marker ST-YFP and moreover, a comparison was made to the wild-type m-Rabmc. In direct comparison to ARA6 in this project, m-Rabmc(N147I) and the wild-type m-Rabmc did not affect the distribution of ST-YFP.11 As a result, it was suggested that the mutant m-Rabmc(N147I) does not affect the movement of transport proteins in the ER-Golgi pathway.
RHA1 and ARA7 May not Perform Exactly the Same Function
The third Rab5 homologue ARA7 is believed to be identical in its localization within the secretory pathway with RHA1.6 With this proposed similarity, it would be reasonable to suggest that the outcome of experiments using the ARA7 would correspond to those presented here for RHA1. An experiment using GTP-tagged ARA7 illustrated two sets of punctuate staining one that associated with an unknown organelle and one that colocalized with the GA marker ST-YFP.9 This result was also observed with the GTP-trapped mutant AtRabF2b[S24 N] when transiently expressed in tobacco epidermal cells along side ST-YFP.9 However, the observations made with RHA1 in this project do not compare with this experiment, there is a possibility that the difference in the constructs used may be the cause of these dissimilarities, although it is more likely that ARA7 and RHA1 are in actual fact not as similar as previously predicted.6
To rule out non-redundancy completely, it would be important to carry out knock-out analysis, followed by detailed phenotypic analysis. In addition, the project could be further explored by a direct comparison between the already prepared VenusRHA1(Q69L) and a fluorescent fusion with the GTP-trapped mutant ARA7 together with GA markers ST-CFP and ST-YFP, respectively, would be necessary to reveal whether these GTPases do in actual act show any difference in their colocalization. Since the RHA1(Q69L) mutant was specifically created with the spectral variant Venus, it should also be possible to directly compare it with a GFP fusions of the GTP-trapped ARA7 in double labelling experiments.
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Acknowledgements |
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I would like to make a special thank you to my supervisor Professor Jurgen Denecke for guiding me through this project. It has been a pleasure to work with Professor Denecke and his research team. I would also like to thank Francesca Bottanelli for providing the plasmids needed to make my constructs.
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References |
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Hannah Lee studied Biology as an undergraduate at the University of Leeds. She is currently studying for a PhD at the University of Warwick and hopes to become a lecturer in Biological Sciences.
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