Publications by year
2018
Lin C, Lemarchand L, Euler R, Sparkes I (2018). Modeling the Geometry and Dynamics of the Endoplasmic Reticulum Network.
IEEE/ACM Transactions on Computational Biology and Bioinformatics,
15(2), 377-386.
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Sparkes I, White RR, Coles B, Botchway SW, Ward A (2018). Using Optical Tweezers Combined with Total Internal Reflection Microscopy to Study Interactions Between the ER and Golgi in Plant Cells.
Methods Mol Biol,
1691, 167-178.
Abstract:
Using Optical Tweezers Combined with Total Internal Reflection Microscopy to Study Interactions Between the ER and Golgi in Plant Cells.
Optical tweezers have been used to trap and micromanipulate several biological specimens ranging from DNA, macromolecules, organelles to single celled organisms. Using a combination of the refraction and scattering of laser light from a focused laser beam, refractile objects are physically captured and can be moved within the surrounding media. The technique is routinely used to determine biophysical properties such as the forces exerted by motor proteins. Here, we describe how optical tweezers combined with total internal reflection fluorescence (TIRF) microscopy can be used to assess physical interactions between organelles, more specifically the ER and Golgi bodies in plant cells.
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2017
Bayer EM, Sparkes I, Vanneste S, Rosado A (2017). From shaping organelles to signalling platforms: the emerging functions of plant ER–PM contact sites.
Current Opinion in Plant Biology,
40, 89-96.
Abstract:
From shaping organelles to signalling platforms: the emerging functions of plant ER–PM contact sites
The plant endoplasmic reticulum (ER) defines the biosynthetic site of lipids and proteins destined for secretion, but also contains important signal transduction and homeostasis components that regulate multiple hormonal and developmental responses. To achieve its various functions, the ER has a unique architecture, both reticulated and highly plastic, that facilitates the spatial-temporal segregation of biochemical reactions and the establishment of inter-organelle communication networks. At the cell cortex, the cortical ER (cER) anchors to and functionally couples with the PM through largely static structures known as ER–PM contact sites (EPCS). These spatially confined microdomains are emerging as critical regulators of the geometry of the cER network, and as highly specialized signalling hubs. In this review, we share recent insights into how EPCS regulate cER remodelling, and discuss the proposed roles for plant EPCS components in the integration of environmental and developmental signals at the cER–PM interface.
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Lin C, White RR, Sparkes I, Ashwin PB (2017). Modelling endoplasmic reticulum network maintenance in a plant cell.
Biophysical Journal,
113, 214-222.
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Griffing LR, Lin C, Perico C, White RR, Sparkes I (2017). Plant ER geometry and dynamics: biophysical and cytoskeletal control during growth and biotic response.
Protoplasma,
254(1), 43-56.
Abstract:
Plant ER geometry and dynamics: biophysical and cytoskeletal control during growth and biotic response.
The endoplasmic reticulum (ER) is an intricate and dynamic network of membrane tubules and cisternae. In plant cells, the ER 'web' pervades the cortex and endoplasm and is continuous with adjacent cells as it passes through plasmodesmata. It is therefore the largest membranous organelle in plant cells. It performs essential functions including protein and lipid synthesis, and its morphology and movement are linked to cellular function. An emerging trend is that organelles can no longer be seen as discrete membrane-bound compartments, since they can physically interact and 'communicate' with one another. The ER may form a connecting central role in this process. This review tackles our current understanding and quantification of ER dynamics and how these change under a variety of biotic and developmental cues.
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Osterrieder A, Sparkes IA, Botchway SW, Ward A, Ketelaar T, de Ruijter N, Hawes C (2017). Stacks off tracks: a role for the golgin AtCASP in plant endoplasmic reticulum-Golgi apparatus tethering.
Journal of Experimental Botany,
68(13), 3339-3350.
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2016
Gao H, Metz J, Teanby NA, Ward AD, Botchway SW, Coles B, Pollard MR, Sparkes I (2016). In Vivo Quantification of Peroxisome Tethering to Chloroplasts in Tobacco Epidermal Cells Using Optical Tweezers.
Plant Physiol,
170(1), 263-272.
Abstract:
In Vivo Quantification of Peroxisome Tethering to Chloroplasts in Tobacco Epidermal Cells Using Optical Tweezers.
Peroxisomes are highly motile organelles that display a range of motions within a short time frame. In static snapshots, they can be juxtaposed to chloroplasts, which has led to the hypothesis that they are physically interacting. Here, using optical tweezers, we tested the dynamic physical interaction in vivo. Using near-infrared optical tweezers combined with TIRF microscopy, we were able to trap peroxisomes and approximate the forces involved in chloroplast association in vivo in tobacco (Nicotiana tabacum) and observed weaker tethering to additional unknown structures within the cell. We show that chloroplasts and peroxisomes are physically tethered through peroxules, a poorly described structure in plant cells. We suggest that peroxules have a novel role in maintaining peroxisome-organelle interactions in the dynamic environment. This could be important for fatty acid mobilization and photorespiration through the interaction with oil bodies and chloroplasts, highlighting a fundamentally important role for organelle interactions for essential biochemistry and physiological processes.
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Wang P, Richardson C, Hawkins TJ, Sparkes I, Hawes C, Hussey PJ (2016). Plant VAP27 proteins: domain characterization, intracellular localization and role in plant development.
New Phytologist,
210(4), 1311-1326.
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Sparkes I (2016). Using Optical Tweezers to Characterize Physical Tethers at Membrane Contact Sites: Grab it, Pull it, Set it Free?.
Front Cell Dev Biol,
4 Author URL.
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2015
Knox K, Wang P, Kriechbaumer V, Tilsner J, Frigerio L, Sparkes I, Hawes C, Oparka K (2015). Putting the Squeeze on Plasmodesmata: a Role for Reticulons in Primary Plasmodesmata Formation.
Plant Physiol,
168(4), 1563-1572.
Abstract:
Putting the Squeeze on Plasmodesmata: a Role for Reticulons in Primary Plasmodesmata Formation.
Primary plasmodesmata (PD) arise at cytokinesis when the new cell plate forms. During this process, fine strands of endoplasmic reticulum (ER) are laid down between enlarging Golgi-derived vesicles to form nascent PD, each pore containing a desmotubule, a membranous rod derived from the cortical ER. Little is known about the forces that model the ER during cell plate formation. Here, we show that members of the reticulon (RTNLB) family of ER-tubulating proteins in Arabidopsis (Arabidopsis thaliana) may play a role in the formation of the desmotubule. RTNLB3 and RTNLB6, two RTNLBs present in the PD proteome, are recruited to the cell plate at late telophase, when primary PD are formed, and remain associated with primary PD in the mature cell wall. Both RTNLBs showed significant colocalization at PD with the viral movement protein of Tobacco mosaic virus, while superresolution imaging (three-dimensional structured illumination microscopy) of primary PD revealed the central desmotubule to be labeled by RTNLB6. Fluorescence recovery after photobleaching studies showed that these RTNLBs are mobile at the edge of the developing cell plate, where new wall materials are being delivered, but significantly less mobile at its center, where PD are forming. A truncated RTNLB3, unable to constrict the ER, was not recruited to the cell plate at cytokinesis. We discuss the potential roles of RTNLBs in desmotubule formation.
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2014
Rodríguez-Serrano M, Pazmiño DM, Sparkes I, Rochetti A, Hawes C, Romero-Puertas MC, Sandalio LM (2014). 2,4-Dichlorophenoxyacetic acid promotes S-nitrosylation and oxidation of actin affecting cytoskeleton and peroxisomal dynamics.
J Exp Bot,
65(17), 4783-4793.
Abstract:
2,4-Dichlorophenoxyacetic acid promotes S-nitrosylation and oxidation of actin affecting cytoskeleton and peroxisomal dynamics.
2,4-Dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin used as a herbicide to control weeds in agriculture. A high concentration of 2,4-D promotes leaf epinasty and cell death. In this work, the molecular mechanisms involved in the toxicity of this herbicide are studied by analysing in Arabidopsis plants the accumulation of reactive oxygen species (ROS) and nitric oxide (NO), and their effect on cytoskeleton structure and peroxisome dynamics. 2,4-D (23 mM) promotes leaf epinasty, whereas this process was prevented by EDTA, which can reduce ·OH accumulation. The analysis of ROS accumulation by confocal microscopy showed a 2,4-D-dependent increase in both H2O2 and O2·(-), whereas total NO was not affected by the treatment. The herbicide promotes disturbances on the actin cytoskeleton structure as a result of post-translational modification of actin by oxidation and S-nitrosylation, which could disturb actin polymerization, as suggested by the reduction of the F-actin/G-actin ratio. These effects were reduced by EDTA, and the reduction of ROS production in Arabidopsis mutants deficient in xanthine dehydrogenase (Atxdh) gave rise to a reduction in actin oxidation. Also, 2,4-D alters the dynamics of the peroxisome, slowing the speed and shortening the distances by which these organelles are displaced. It is concluded that 2,4-D promotes oxidative and nitrosative stress, causing disturbances in the actin cytoskeleton, thereby affecting the dynamics of peroxisomes and some other organelles such as the mitochondria, with xanthine dehydrogenase being involved in ROS production under these conditions. These structural changes in turn appear to be responsible for the leaf epinasty.
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Griffing LR, Gao HT, Sparkes I (2014). ER network dynamics are differentially controlled by myosins XI-K, XI-C, XI-E, XI-I, XI-1, and XI-2.
Frontiers in Plant Science,
5(MAY).
Abstract:
ER network dynamics are differentially controlled by myosins XI-K, XI-C, XI-E, XI-I, XI-1, and XI-2
The endoplasmic reticulum (ER) of higher plants is a complex network of tubules and cisternae. Some of the tubules and cisternae are relatively persistent, while others are dynamically moving and remodeling through growth and shrinkage, cycles of tubule elongation and retraction, and cisternal expansion and diminution. Previous work showed that transient expression in tobacco leaves of the motor-less, truncated tail of myosin XI-K increases the relative area of both persistent cisternae and tubules in the ER. Likewise, transient expression of XI-K tail diminishes the movement of organelles such as Golgi and peroxisomes. To examine whether other class XI myosins are involved in the remodeling and movement of the ER, other myosin XIs implicated in organelle movement, XI-1 (MYA1),XI-2 (MYA2), XI-C, XI-E, XI-I, and one not, XI-A, were expressed as motor-less tail constructs and their effect on ER persistent structures determined. Here, we indicate a differential effect on ER dynamics whereby certain class XI myosins may have more influence over controlling cisternalization rather than tubulation. © 2014 Griffing, Gao and Sparkes.
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Lemarchand L, Euler R, Lin C, Sparkes I (2014). Modeling the geometry of the endoplasmic reticulum network.
Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics),
8542 LNBI, 131-145.
Abstract:
Modeling the geometry of the endoplasmic reticulum network
We have studied the network geometry of the endoplasmic reticulum by means of graph theoretical and integer programming models. The purpose is to represent this structure as close as possible by a class of finite, undirected and connected graphs the nodes of which have to be either of degree three or at most of degree three. We determine plane graphs of minimal total edge length satisfying degree and angle constraints, and we show that the optimal graphs are close to the ER network geometry. Basically, two procedures are formulated to solve the optimization problem: a binary linear program, that iteratively constructs an optimal solution, and a linear program, that iteratively exploits additional cutting planes from different families to accelerate the solution process. All formulations have been implemented and tested on a series of real-life and randomly generated cases. The cutting plane approach turns out to be particularly efficient for the real-life testcases, since it outperforms the pure integer programming approach by a factor of at least 10. © 2014 Springer International Publishing.
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Sparkes I, Gao H (2014). Plant peroxisome dynamics: Movement, positioning and connections. In (Ed)
Molecular Machines Involved in Peroxisome Biogenesis and Maintenance, 461-477.
Abstract:
Plant peroxisome dynamics: Movement, positioning and connections
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Lin C, Zhang Y, Sparkes I, Ashwin P (2014). Structure and dynamics of ER: minimal networks and biophysical constraints.
Biophys J,
107(3), 763-772.
Abstract:
Structure and dynamics of ER: minimal networks and biophysical constraints.
The endoplasmic reticulum (ER) in live cells is a highly mobile network whose structure dynamically changes on a number of timescales. The role of such drastic changes in any system is unclear, although there are correlations with ER function. A better understanding of the fundamental biophysical constraints on the system will allow biologists to determine the effects of molecular factors on ER dynamics. Previous studies have identified potential static elements that the ER may remodel around. Here, we use these structural elements to assess biophysical principles behind the network dynamics. By analyzing imaging data of tobacco leaf epidermal cells under two different conditions, i.e. native state (control) and latrunculin B (treated), we show that the geometric structure and dynamics of ER networks can be understood in terms of minimal networks. Our results show that the ER network is well modeled as a locally minimal-length network between the static elements that potentially anchor the ER to the cell cortex over longer timescales; this network is perturbed by a mixture of random and deterministic forces. The network need not have globally minimum length; we observe cases where the local topology may change dynamically between different Euclidean Steiner network topologies. The networks in the treated cells are easier to quantify, because they are less dynamic (the treatment suppresses actin dynamics), but the same general features are found in control cells. Using a Langevin approach, we model the dynamics of the nonpersistent nodes and use this to show that the images can be used to estimate both local viscoelastic behavior of the cytoplasm and filament tension in the ER network. This means we can explain several aspects of the ER geometry in terms of biophysical principles.
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Wang P, Hawkins TJ, Richardson C, Cummins I, Deeks MJ, Sparkes I, Hawes C, Hussey PJ (2014). The plant cytoskeleton, NET3C, and VAP27 mediate the link between the plasma membrane and endoplasmic reticulum.
Curr Biol,
24(12), 1397-1405.
Abstract:
The plant cytoskeleton, NET3C, and VAP27 mediate the link between the plasma membrane and endoplasmic reticulum.
The cortical endoplasmic reticulum (ER) network in plants is a highly dynamic structure, and it contacts the plasma membrane (PM) at ER-PM anchor/contact sites. These sites are known to be essential for communication between the ER and PM for lipid transport, calcium influx, and ER morphology in mammalian and fungal cells. The nature of these contact sites is unknown in plants, and here, we have identified a complex that forms this bridge. This complex includes (1) NET3C, which belongs to a plant-specific superfamily (NET) of actin-binding proteins, (2) VAP27, a plant homolog of the yeast Scs2 ER-PM contact site protein, and (3) the actin and microtubule networks. We demonstrate that NET3C and VAP27 localize to puncta at the PM and that NET3C and VAP27 form homodimers/oligomers and together form complexes with actin and microtubules. We show that F-actin modulates the turnover of NET3C at these puncta and microtubules regulate the exchange of VAP27 at the same sites. Based on these data, we propose a model for the structure of the plant ER-PM contact sites.
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2013
Lee H, Sparkes I, Gattolin S, Dzimitrowicz N, Roberts LM, Hawes C, Frigerio L (2013). An Arabidopsis reticulon and the atlastin homologue RHD3-like2 act together in shaping the tubular endoplasmic reticulum.
New Phytologist,
197(2), 481-489.
Abstract:
An Arabidopsis reticulon and the atlastin homologue RHD3-like2 act together in shaping the tubular endoplasmic reticulum
The endoplasmic reticulum (ER) is a network of membrane sheets and tubules connected via three-way junctions. A family of proteins, the reticulons, are responsible for shaping the tubular ER. Reticulons interact with other tubule-forming proteins (Dp1 and Yop1p) and the GTPase atlastin. The Arabidopsis homologue of Dp1/Yop1p is HVA22. We show here that a seed-specific isoform of HVA22 labels the ER in tobacco (Nicotiana tabacum) cells but its overexpression does not alter ER morphology. The closest plant homologue of atlastin is RHD3. We show that RHD3-like 2 (RL2), the seed-specific isoform of RHD3, locates to the ER without affecting its shape or Golgi mobility. Expression of RL2-bearing mutations within its GTPase domain induces the formation of large ER strands, suggesting that a functional GTPase domain is important for the formation of three-way junctions. Coexpression of the reticulon RTNLB13 with RL2 resulted in a dramatic alteration of the ER network. This alteration did not depend on an active GTPase domain but required a functional reticulon, as no effect on ER morphology was seen when RL2 was coexpressed with a nonfunctional RTNLB13. RL2 and its GTPase mutants coimmunoprecipitate with RTNLB13. These results indicate that RL2 and RTNLB13 act together in modulating ER morphology. © 2012 the Authors. New Phytologist © 2012 New Phytologist Trust.
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Werner AK, Medina-Escobar N, Zulawski M, Sparkes IA, Cao FQ, Witte CP (2013). The ureide-degrading reactions of purine ring catabolism employ three amidohydrolases and one aminohydrolase in arabidopsis, soybean, and rice.
Plant Physiology,
163(2), 672-681.
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The ureide-degrading reactions of purine ring catabolism employ three amidohydrolases and one aminohydrolase in arabidopsis, soybean, and rice
Several ureides are intermediates of purine base catabolism, releasing nitrogen from the purine nucleotides for reassimilation into amino acids. In some legumes like soybean (Glycine max), ureides are used for nodule-to-shoot translocation of fixed nitrogen. Four enzymes of Arabidopsis (Arabidopsis thaliana), (1) allantoinase, (2) allantoate amidohydrolase (AAH), (3) ureidoglycine aminohydrolase, and (4) ureidoglycolate amidohydrolase (UAH), catalyze the complete hydrolysis of the ureide allantoin in vitro. However, the metabolic route in vivo remains controversial. Here, in growth and metabolite analyses of Arabidopsis mutants, we demonstrate that these enzymes are required for allantoin degradation in vivo. Orthologous enzymes are present in soybean, encoded by one to four gene copies. All isoenzymes are active in vitro, while some may be inefficiently translated in vivo. Surprisingly, transcript and protein amounts are not significantly regulated by nitrogen fixation or leaf ureide content. A requirement for soybean AAH and UAH for ureide catabolism in leaves has been demonstrated by the use of virus-induced gene silencing. Functional AAH, ureidoglycine aminohydrolase, and UAH are also present in rice (Oryza sativa), and orthologous genes occur in all other plant genomes sequenced to date, indicating that the amidohydrolase route of ureide degradation is universal in plants, including mosses (e.g. Physcomitrella patens) and algae (e.g. Chlamydomomas reinhardtii). © 2013 American Society of Plant Biologists. All Rights Reserved.
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2012
Sparkes I, Brandizzi F (2012). Fluorescent protein-based technologies: shedding new light on the plant endomembrane system.
Plant J,
70(1), 96-107.
Abstract:
Fluorescent protein-based technologies: shedding new light on the plant endomembrane system.
Without doubt, GFP and spectral derivatives have revolutionized the way biologists approach their journey toward the discovery of how plant cells function. It is fascinating that in its early days GFP was used merely for localization studies, but as time progressed researchers successfully explored new avenues to push the power of GFP technology to reach new and exciting research frontiers. This has had a profound impact on the way we can now study complex and dynamic systems such as plant endomembranes. Here we briefly describe some of the approaches where GFP has revolutionized in vivo studies of protein distribution and dynamics and focus on two emerging approaches for the application of GFP technology in plant endomembranes, namely optical tweezers and forward genetics approaches, which are based either on the light or on genetic manipulation of secretory organelles to gain insights on the factors that control their activities and integrity.
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2011
Sparkes IA, Graumann K, Martinire A, Schoberer J, Wang P, Osterrieder A (2011). Bleach it, switch it, bounce it, pull it: Using lasers to reveal plant cell dynamics. Journal of Experimental Botany, 62(1), 1-7.
Francin-Allami M, Saumonneau A, Lavenant L, Bouder A, Sparkes I, Hawes C, Popineau Y (2011). Dynamic trafficking of wheat γ-gliadin and of its structural domains in tobacco cells, studied with fluorescent protein fusions.
Journal of Experimental Botany,
62(13), 4507-4520.
Abstract:
Dynamic trafficking of wheat γ-gliadin and of its structural domains in tobacco cells, studied with fluorescent protein fusions
Prolamins, the main storage proteins of wheat seeds, are synthesized and retained in the endoplasmic reticulum (ER) of the endosperm cells, where they accumulate in protein bodies (PBs) and are then exported to the storage vacuole. The mechanisms leading to these events are unresolved. To investigate this unconventional trafficking pathway, wheat γ-gliadin and its isolated repeated N-terminal and cysteine-rich C-terminal domains were fused to fluorescent proteins and expressed in tobacco leaf epidermal cells. The results indicated that γ-gliadin and both isolated domains were able to be retained and accumulated as protein body-like structures (PBLS) in the ER, suggesting that tandem repeats are not the only sequence involved in γ-gliadin ER retention and PBLS formation. The high actin-dependent mobility of γ-gliadin PBLS is also reported, and it is demonstrated that most of them do not co-localize with Golgi body or pre-vacuolar compartment markers. Both γ-gliadin domains are found in the same PBLS when co-expressed, which is most probably due to their ability to interact with each other, as indicated by the yeast two-hybrid and FRET-FLIM experiments. Moreover, when stably expressed in BY-2 cells, green fluorescent protein (GFP) fusions to γ-gliadin and its isolated domains were retained in the ER for several days before being exported to the vacuole in a Golgi-dependent manner, and degraded, leading to the release of the GFP 'core'. Taken together, the results show that tobacco cells are a convenient model to study the atypical wheat prolamin trafficking with fluorescent protein fusions. © 2011 the Author(s).
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Sparkes I, Hawes C, Frigerio L (2011). FrontiERs: movers and shapers of the higher plant cortical endoplasmic reticulum.
Curr Opin Plant Biol,
14(6), 658-665.
Abstract:
FrontiERs: movers and shapers of the higher plant cortical endoplasmic reticulum.
The endoplasmic reticulum (ER) in higher plants performs many important functions, yet our understanding of how its intricate network shape and dynamics relate to function is very limited. Recent work has begun to unpick key molecular players in the generation of the pleomorphic, highly dynamic ER network structure that pervades the entire cytoplasm. ER movement is acto-myosin dependent. ER shape is dependent on RHD3 (Root Hair Defective 3) and a family of proteins called reticulons. The major challenge that lies ahead is understanding how factors that control ER shape and movement are regulated and how this relates to the numerous functions of the ER.
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Wang P, Hummel E, Osterrieder A, Meyer AJ, Frigerio L, Sparkes I, Hawes C (2011). KMS1 and KMS2, two plant endoplasmic reticulum proteins involved in the early secretory pathway.
Plant J,
66(4), 613-628.
Abstract:
KMS1 and KMS2, two plant endoplasmic reticulum proteins involved in the early secretory pathway.
We have identified two endoplasmic reticulum (ER)-associated Arabidopsis proteins, KMS1 and KMS2, which are conserved among most species. Fluorescent protein fusions of KMS1 localised to the ER in plant cells, and over-expression induced the formation of a membrane structure, identified as ER whorls by electron microscopy. Hydrophobicity analysis suggested that KMS1 and KMS2 are integral membrane proteins bearing six transmembrane domains. Membrane protein topology was assessed by a redox-based topology assay (ReTA) with redox-sensitive GFP and confirmed by a protease protection assay. A major loop domain between transmembrane domains 2 and 3, plus the N- and C-termini were found on the cytosolic side of the ER. A C-terminal di(tri)-lysine motif is involved in retrieval of KMS1 and deletion led to a reduction of the GFP-KMS1 signal in the ER. Over-expression of KMS1/KMS2 truncations perturbed ER and Golgi morphology and similar effects were also seen when KMS1/KMS2 were knocked-down by RNA interference. Microscopy and biochemical experiments suggested that expression of KMS1/KMS2 truncations inhibited ER to Golgi protein transport.
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Sparkes I (2011). Recent advances in understanding plant myosin function: life in the fast lane.
Mol Plant,
4(5), 805-812.
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Recent advances in understanding plant myosin function: life in the fast lane.
Plant myosins are required for organelle movement, and a role in actin organization has recently come to light. Myosin mutants display several gross morphological phenotypes, the most severe being dwarfism and reduced fecundity, and there is a correlation between reduced organelle movement and morphological defects. This review aims to discuss recent findings in plants relating to the role of myosins in actin dynamics, development, and organelle movement, more specifically the endoplasmic reticulum (ER). One overarching theme is that there still appear to be more questions than answers relating to plant myosin function and regulation.
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2010
Sparkes I, Tolley N, Aller I, Svozil J, Osterrieder A, Botchway S, Mueller C, Frigerio L, Hawes C (2010). Five Arabidopsis reticulon isoforms share endoplasmic reticulum location, topology, and membrane-shaping properties.
Plant Cell,
22(4), 1333-1343.
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Five Arabidopsis reticulon isoforms share endoplasmic reticulum location, topology, and membrane-shaping properties.
The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved in shaping ER tubules. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. We show by Förster resonance energy transfer and fluorescence lifetime imaging microscopy that several RTNs have the capacity to interact with themselves and each other, and we suggest that oligomerization is responsible for their residence in the ER membrane. We also show that a complete reticulon homology domain is required for both RTN residence in high-curvature ER membranes and ER tubule constriction, yet it is not necessary for homotypic interactions.
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Sparkes IA (2010). Motoring around the plant cell: Insights from plant myosins.
Biochemical Society Transactions,
38(3), 833-838.
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Motoring around the plant cell: Insights from plant myosins
Organelle movement in plants cells is extremely dynamic. Movement is driven by the acto-myosin system. Higher plant myosins fall into two classes: classes XI and VIII. Localization studies have highlighted that myosins are present throughout the cytosol, label motile puncta and decorate the nuclear envelope and plasma membrane. Functional studies through expression of dominant-negativemyosin variants, RNAi (RNA interference) and T-DNA insertional analysis have shown that class XI myosins are required for organelle movement. Intriguingly, organelle movement is also linked to Arabidopsis growth and development. The present review tackles current findings relating to plant organelle movement and the role of myosins. ©The Authors.
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Hawes C, Osterrieder A, Sparkes IA, Ketelaar T (2010). Optical tweezers for the micromanipulation of plant cytoplasm and organelles.
Current Opinion in Plant Biology,
13(6), 731-735.
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Optical tweezers for the micromanipulation of plant cytoplasm and organelles
Laser trapping of micron-sized particles can be achieved utilizing the radiation pressure generated by a focused infrared laser beam. Thus, it is theoretically possible to trap and manipulate organelles within the cytoplasm and remodel the architecture of the cytoplasm and membrane systems. Here we describe recent progress, using this under utilized technology, in the manipulation of cytoplasmic strands and organelles in plant cells. © 2010 Elsevier Ltd.
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Baker A, Sparkes IA, Brown LA, O'Leary-Steele C, Warriner SL (2010). Peroxisome biogenesis and positioning.
Biochemical Society Transactions,
38(3), 807-816.
Abstract:
Peroxisome biogenesis and positioning
Plant peroxisomes are extremely dynamic, moving and undergoing changes of shape in response to metabolic and environmental signals. Matrix proteins are imported via one of two import pathways, depending on the targeting signal within the protein. Each pathway has a specific receptor but utilizes common membrane-bound translocation machinery. Current models invoke receptor recycling, which may involve cycles of ubiquitination. Some components of the import machinery may also play a role in proteolytic turnover of matrix proteins, prompting parallels with the endoplasmic-reticulum-associated degradation pathway. Peroxisome membrane proteins, some of which are imported post-translationally, others of which may traffic to peroxisomes via the endoplasmic reticulum, use distinct proteinaceous machinery. The isolation of mutants defective in peroxisome biogenesis has served to emphasize the important role of peroxisomes at all stages of the plant life cycle. ©The Authors.
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Mitsuya S, El-Shami M, Sparkes IA, Charlton WL, Lousa CDM, Johnson B, Baker A (2010). Salt stress causes peroxisome proliferation, but inducing peroxisome proliferation does not improve NaCI tolerance in Arabidopsis thaliana.
PLoS ONE,
5(2).
Abstract:
Salt stress causes peroxisome proliferation, but inducing peroxisome proliferation does not improve NaCI tolerance in Arabidopsis thaliana
The PEX11 family of peroxisome membrane proteins have been shown to be involved in regulation of peroxisome size and number in plant, animals, and yeast cells. We and others have previously suggested that peroxisome proliferation as a result of abiotic stress may be important in plant stress responses, and recently it was reported that several rice PEX11 genes were up regulated in response to abiotic stress. We sought to test the hypothesis that promoting peroxisome proliferation in Arabidopsis thaliana by over expression of one PEX11 family member, PEX11e, would give increased resistance to salt stress. We could demonstrate up regulation of PEX11e by salt stress and increased peroxisome number by both PEX11e over expression and salt stress, however our experiments failed to find a correlation between PEX11e over expression and increased peroxisome metabolic activity or resistance to salt stress. This suggests that although peroxisome proliferation may be a consequence of salt stress, it does not affect the ability of Arabidopsis plants to tolerate saline conditions. © 2010 Mitsuya et al.
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Tolley N, Sparkes I, Craddock CP, Eastmond PJ, Runions J, Hawes C, Frigerio L (2010). Transmembrane domain length is responsible for the ability of a plant reticulon to shape endoplasmic reticulum tubules in vivo.
Plant Journal,
64(3), 411-418.
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Transmembrane domain length is responsible for the ability of a plant reticulon to shape endoplasmic reticulum tubules in vivo
Reticulons are integral endoplasmic reticulum (ER) membrane proteins that have the ability to shape the ER into tubules. It has been hypothesized that their unusually long conserved hydrophobic regions cause reticulons to assume a wedge-like topology that induces membrane curvature. Here we provide proof of this hypothesis. When over-expressed, an Arabidopsis thaliana reticulon (RTNLB13) localized to, and induced constrictions in, cortical ER tubules. Ectopic expression of RTNLB13 was sufficient to induce ER tubulation in an Arabidopsis mutant (pah1 pah2) whose ER membrane is mostly present in a sheet-like form. By sequential shortening of the four transmembrane domains (TMDs) of RTNLB13, we show that the length of the transmembrane regions is directly correlated with the ability of RTNLB13 to induce membrane tubulation and to form low-mobility complexes within the ER membrane. We also show that full-length TMDs are necessary for the ability of RTNLB13 to reside in the ER membrane. © 2010 Blackwell Publishing Ltd.
Abstract.
2009
Avisar D, Abu-Abied M, Belausov E, Sadot E, Hawes C, Sparkes IA (2009). A comparative study of the involvement of 17 arabidopsis myosin family members on the motility of Golgi and other organelles. Plant Physiology, 150(2), 700-709.
Sparkes IA, Ketelaar T, De Ruijter NCA, Hawes C (2009). Grab a Golgi: Laser trapping of Golgi bodies reveals in vivo interactions with the endoplasmic reticulum. Traffic, 10(5), 567-571.
Sparkes I, Runions J, Hawes C, Griffing L (2009). Movement and remodeling of the endoplasmic reticulum in nondividing cells of tobacco leaves.
Plant Cell,
21(12), 3937-3949.
Abstract:
Movement and remodeling of the endoplasmic reticulum in nondividing cells of tobacco leaves.
Using a novel analytical tool, this study investigates the relative roles of actin, microtubules, myosin, and Golgi bodies on form and movement of the endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) leaf epidermal cells. Expression of a subset of truncated class XI myosins, which interfere with the activity of native class XI myosins, and drug-induced actin depolymerization produce a more persistent network of ER tubules and larger persistent cisternae. The treatments differentially affect two persistent size classes of cortical ER cisternae, those >0.3 microm(2) and those smaller, called punctae. The punctae are not Golgi, and ER remodeling occurs in the absence of Golgi bodies. The treatments diminish the mobile fraction of ER membrane proteins but not the diffusive flow of mobile membrane proteins. The results support a model whereby ER network remodeling is coupled to the directionality but not the magnitude of membrane surface flow, and the punctae are network nodes that act as foci of actin polymerization, regulating network remodeling through exploratory tubule growth and myosin-mediated shrinkage.
Abstract.
Author URL.
Rodríguez-Serrano M, Romero-Puertas MC, Sparkes I, Hawes C, del Río LA, Sandalio LM (2009). Peroxisome dynamics in Arabidopsis plants under oxidative stress induced by cadmium.
Free Radical Biology and Medicine,
47(11), 1632-1639.
Abstract:
Peroxisome dynamics in Arabidopsis plants under oxidative stress induced by cadmium
Peroxisomes are organelles with an essentially oxidative metabolism that are involved in various metabolic pathways such as fatty acid β-oxidation, photorespiration, and metabolism of reactive oxygen species (ROS) and reactive nitrogen species. These organelles are highly dynamic but there is little information about the regulation of, and the effects of environment on, peroxisome movement. In this work a stable Arabidopsis line expressing the GFP-SKL peptide targeted to peroxisomes was characterized. Peroxisome-associated fluorescence was observed in all tissues, including leaves (mesophyll and epidermal cells, trichomes, and stomata) and roots. The dynamics of peroxisomes in epidermal cells was examined by confocal laser microscope, and various types of movement were observed. The speed of movement differed depending on the plant age. Treatment of plants with CdCl. (100 μM) produced a significant increase in speed, which was dependent on endogenous ROS and Ca , but was not related to actin cytoskeleton modifications. In light of the results obtained, it is proposed that the increase in peroxisomal motility observed in Arabidopsis plants could be a cellular mechanism of protection against the Cd-imposed oxidative stress. Other possible roles for the enhanced peroxisome movement in plant cell physiology are discussed. © 2009 Elsevier Inc. All rights reserved. 2 2+
Abstract.
Sparkes IA, Frigerio L, Tolley N, Hawes C (2009). The plant endoplasmic reticulum: a cell-wide web.
Biochemical Journal,
423(2), 145-155.
Abstract:
The plant endoplasmic reticulum: a cell-wide web
The ER (endoplasmic reticulum) in higher plants forms a pleomorphic web of membrane tubules and small cisternae that pervade the cytoplasm, but in particular form a polygonal network at the cortex of the cell which may be anchored to the plasma membrane. The network is associated with the actin cytoskeleton and demonstrates extensive mobility, which is most likely to be dependent on myosin motors. The ER is characterized by a number of domains which may be associated with specific functions such as protein storage, or with direct interaction with other organelles such as the Golgi apparatus, peroxisomes and plastids. In the present review we discuss the nature of the network, the role of shape-forming molecules such as the recently described reticulon family of proteins and the function of some of the major domains within the ER network. © the Authors Journal compilation. © 2009 Biochemical Society.
Abstract.
2008
Hawes C, Osterrieder A, Sparkes I (2008). Features of the Plant Golgi Apparatus. In Mironov A (Ed) The Golgi Apparatus, Springer Wein, New York.
Werner AK, Sparkes IA, Romeis T, Witte CP (2008). Identification, biochemical characterization, and subcellular localization of allantoate amidohydrolases from arabidopsis and soybean.
Plant Physiology,
146(2), 418-430.
Abstract:
Identification, biochemical characterization, and subcellular localization of allantoate amidohydrolases from arabidopsis and soybean
Allantoate amidohydrolases (AAHs) hydrolize the ureide allantoate to ureidoglycolate, CO , and two molecules of ammonium. Allantoate degradation is required to recycle purine-ring nitrogen in all plants. Tropical legumes additionally transport fixed nitrogen via allantoin and allantoate into the shoot, where it serves as a general nitrogen source. AAHs from Arabidopsis (Arabidopsis thaliana; AtAAH) and from soybean (Glycine max; GmAAH) were cloned, expressed in planta as StrepII-tagged variants, and highly purified from leaf extracts. Both proteins form homodimers and release 2 mol ammonium/mol allantoate. Therefore, they can truly be classified as AAHs. The kinetic constants determined and the half-maximal activation by 2 to 3 μM manganese are consistent with allantoate being the in vivo substrate of manganese-loaded AAHs. The enzymes were strongly inhibited by micromolar concentrations of fluoride as well as by borate, and by millimolar concentrations of L-asparagine and L-aspartate but not D-asparagine. L-Asparagine likely functions as competitive inhibitor. An Ataah T-DNA mutant, unable to grow on allantoin as sole nitrogen source, is rescued by the expression of StrepII-tagged variants of AtAAH and GmAAH, demonstrating that both proteins are functional in vivo. Similarly, an allantoinase (aln) mutant is rescued by a tagged AtAln variant. Fluorescent fusion proteins of allantoinase and both AAHs localize to the endoplasmic reticulum after transient expression and in transgenic plants. These findings demonstrate that after the generation of allantoin in the peroxisome, plant purine degradation continues in the endoplasmic reticulum. © 2007 American Society of Plant Biologists. 2
Abstract.
Tolley N, Sparkes IA, Hunter PR, Craddock CP, Nuttall J, Roberts LM, Hawes C, Pedrazzini E, Frigerio L (2008). Overexpression of a plant reticulon remodels the lumen of the cortical endoplasmic reticulum but does not perturb protein transport.
Traffic,
9(1), 94-102.
Abstract:
Overexpression of a plant reticulon remodels the lumen of the cortical endoplasmic reticulum but does not perturb protein transport
We have cloned a member of the reticulon (RTN) family of Arabidopsis thaliana (RTNLB13). When fused to yellow fluorescent protein (YFP) and expressed in tobacco leaf epidermal cells, RTNLB13 is localized in the endoplasmic reticulum (ER). Coexpression of a soluble ER luminal marker reveals that YFP-tagged, myc-tagged or untagged RTNLB13 induces severe morphological changes to the lumen of the ER. We show, using fluorescence recovery after photobleaching (FRAP) analysis, that RTNLB13 overexpression greatly reduces diffusion of soluble proteins within the ER lumen, possibly by introducing constrictions into the membrane. In spite of this severe phenotype, Golgi shape, number and dynamics appear unperturbed and secretion of a reporter protein remains unaffected. © 2007 the Authors Journal compilation.
Abstract.
Hawes C, Osterrieder A, Hummel E, Sparkes I (2008). The plant ER-Golgi interface.
Traffic,
9(10), 1571-1580.
Abstract:
The plant ER-Golgi interface.
The interface between the endoplasmic reticulum (ER) and the Golgi apparatus is a critical junction in the secretory pathway mediating the transport of both soluble and membrane cargo between the two organelles. Such transport can be bidirectional and is mediated by coated membranes. In this review, we consider the organization and dynamics of this interface in plant cells, the putative structure of which has caused some controversy in the literature, and we speculate on the stages of Golgi biogenesis from the ER and the role of the Golgi and ER on each other's motility.
Abstract.
Author URL.
Sparkes IA, Teanby NA, Hawes C (2008). Truncated myosin XI tail fusions inhibit peroxisome, Golgi, and mitochondrial movement in tobacco leaf epidermal cells: a genetic tool for the next generation.
Journal of Experimental Botany,
59(9), 2499-2512.
Abstract:
Truncated myosin XI tail fusions inhibit peroxisome, Golgi, and mitochondrial movement in tobacco leaf epidermal cells: a genetic tool for the next generation
Although organelle movement in higher plants is predominantly actin-based, potential roles for the 17 predicted Arabidopsis myosins in motility are only just emerging. It is shown here that two Arabidopsis myosins from class XI, XIE, and XIK, are involved in Golgi, peroxisome, and mitochondrial movement. Expression of dominant negative forms of the myosin lacking the actin binding domain at the amino terminus perturb organelle motility, but do not completely inhibit movement. Latrunculin B, an actin destabilizing drug, inhibits organelle movement to a greater extent compared to the effects of AtXIE-T/XIK-T expression. Amino terminal YFP fusions to XIE-T and XIK-T are dispersed throughout the cytosol and do not completely decorate the organelles whose motility they affect. XIE-T and XIK-T do not affect the global actin architecture, but their movement and location is actin-dependent. The potential role of these truncated myosins as genetically encoded inhibitors of organelle movement is discussed. © 2008 the Author(s).
Abstract.
2007
Scott I, Sparkes IA, Logan DC (2007). The missing link: inter-organellar connections in mitochondria and peroxisomes?. Trends in Plant Science, 12(9), 380-381.
2006
Hadden DA, Phillipson BA, Johnston KA, Brown LA, Manfield IW, El-Shami M, Sparkes IA, Baker A (2006). Arabidopsis PEX19 is a dimeric protein that binds the peroxin PEX10.
Molecular Membrane Biology,
23(4), 325-336.
Abstract:
Arabidopsis PEX19 is a dimeric protein that binds the peroxin PEX10
Peroxisomes are organelles found in all eukaryotic cells. Peroxisomes import integral membrane proteins post-translationally, and PEX19 is a predominantly cytosolic, farnesylated protein of mammalian and yeast cells that binds multiple peroxisome membrane proteins and is required for their correct targeting/insertion to the peroxisome membrane. We report the characterisation of the Arabidopsis thaliana homologue of PEX19 which is a predominantly cytosolic protein. AtPEX19 is encoded by two genes (designated AtPEX19-1 and AtPEX19-2) that are expressed in all tissues and at all developmental stages of the plant. Quantitative real time PCR shows that AtPEX19-1 and AtPEX19-2 have distinct expression profiles. Using in vitro translation and co-immunoprecipitation AtPEX19-1 was shown to bind to the Arabidopsis peroxisomal membrane protein PEX10. Additionally, bacterially expressed recombinant AtPEX19-1 was able to bind a fusion protein consisting of the C-terminus of PEX10 and glutathione S-transferase in pull-down assays, thereby demonstrating that non-farnesylated AtPEX19 can interact with the C-terminus of AtPEX10. Purified recombinant AtPEX19-1 was analysed by gel filtration chromatography and was found to have a molecular weight consistent with it forming a dimer and a dimer was detected in Arabidopsis cell extracts that was slightly destabilised in the presence of DTT. Moreover, cross-linking studies of native AtPEX19 suggest that in vivo it is the dimeric species of the protein that preferentially forms complexes with other proteins. © 2006 Informa UK Ltd.
Abstract.
Sparkes IA, Runions J, Kearns A, Hawes C (2006). Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.
Nature Protocols,
1(4), 2019-2025.
Abstract:
Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants
Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months.
Abstract.
2005
Sparkes IA, Hawes C, Baker A (2005). AtPEX2 and AtPEX10 are targeted to peroxisomes independently of known endoplasmic reticulum trafficking routes.
Plant Physiology,
139(2), 690-700.
Abstract:
AtPEX2 and AtPEX10 are targeted to peroxisomes independently of known endoplasmic reticulum trafficking routes
Controversy exists in the literature over the involvement of the endoplasmic reticulum (ER) in the delivery of membrane proteins to peroxisomes. In this study the involvement of the ER in the trafficking of two Arabidopsis (Arabidopsis thaliana) peroxisomal membrane proteins was investigated using confocal laser scanning microscopy of living cells expressing fusions between enhanced yellow fluorescent protein (eYFP) and AtPEX2 and AtPEX10. The fusion proteins were always detected in peroxisomes and cytosol irrespective of the location of the eYFP tag or the level of expression. The cytosolic fluorescence was not due to cleavage of the eYFP reporter from the C-terminal fusion proteins. Blocking known ER transport routes using the fungal metabolite Brefeldin a or expressing dominant negative mutants of Sar1 or RabD2a had no effect on the trafficking of AtPEX2 and AtPEX10 to peroxisomes. We conclude that AtPEX2 and AtPEX10 are inserted into peroxisome membranes directly from the cytosol. © 2005 American Society of Plant Biologists.
Abstract.
Baker A, Sparkes IA (2005). Peroxisome protein import: Some answers, more questions.
Current Opinion in Plant Biology,
8(6), 640-647.
Abstract:
Peroxisome protein import: Some answers, more questions
Recent advances in the study of plant peroxisomes are shedding new light on the importance of these organelles for plant development, and are revealing similarities and differences in peroxisome protein import pathways between plants, animals and fungi. For example, the import of matrix proteins that carry the PTS1 and PTS2 targeting signals is coupled in plants as it is in mammals, whereas these import pathways are separate in fungi. The expression of a human peroxisomal ATPase partially rescues the equivalent Arabidopsis mutant. Ubiquitination might play a role in receptor recycling in Saccharomyces cerevisiae and exciting progress is being made through studies of the targeting of membrane proteins. © 2005 Elsevier Ltd. All rights reserved.
Abstract.
2003
Sparkes IA, Brandizzi F, Slocombe SP, El-Shami M, Hawes C, Baker A (2003). An Arabidopsis pex10 Null Mutant is Embryo Lethal, Implicating Peroxisomes in an Essential Role during Plant Embryogenesis.
Plant Physiology,
133(4), 1809-1819.
Abstract:
An Arabidopsis pex10 Null Mutant is Embryo Lethal, Implicating Peroxisomes in an Essential Role during Plant Embryogenesis
Peroxisomes participate in many important functions in plants, including seed reserve mobilization, photorespiration, defense against oxidative stress, and auxin and jasmonate signaling. In mammals, defects in peroxisome biogenesis result in multiple system abnormalities, severe developmental delay, and death, whereas in unicellular yeasts, peroxisomes are dispensable unless required for growth of specific substrates. PEX10 encodes an integral membrane protein required for peroxisome biogenesis in mammals and yeast. To investigate the importance of PEX10 in plants, we characterized a Ds insertion mutant in the PEX10 gene of Arabidopsis (AfPEX10). Heterozygous AtPEX10::dissociation element mutants show normal vegetative phenotypes under optimal growth conditions, but produce about 20% abnormal seeds. The embryos in the abnormal seeds are predominantly homozygous for the disruption allele. They show retarded development and some morphological abnormalities. No viable homozygous mutant plants were obtained. AtPEX10 fused to yellow fluorescent protein colocalized with green fluorescent protein-serine-lysine-leucine, a well-documented peroxisomal marker, suggesting that AtPEX10 encodes a peroxisomal protein that is essential for normal embryo development and viability.
Abstract.
2002
Sparkes IA, Baker A (2002). Peroxisome biogenesis and protein import in plants, animals and yeasts: Enigma and variations? (review).
Molecular Membrane Biology,
19(3), 171-185.
Abstract:
Peroxisome biogenesis and protein import in plants, animals and yeasts: Enigma and variations? (review)
Peroxisomes are found in virtually all eukaryotic cells, where they play varied but essential metabolic roles, exemplified by the catastrophic effects of mutations that compromise peroxisome biogenesis and function. This review will aim to provide an accessible introduction to peroxisome biogenesis and protein import for the non-specialist, and draws together recent advances in peroxisome protein targeting and import in plants, animals and yeasts, seeking to define common themes and highlight variations. Despite much progress, many aspects of peroxisome biology remain an enigma, and current questions and controversies in the field are highlighted and discussed.
Abstract.
2000
Baker A, Charlton W, Johnson B, Lopez-Huertas E, Oh J, Sparkes I, Thomas J (2000). Biochemical and molecular approaches to understanding protein import into peroxisomes.
Biochem Soc Trans,
28(4), 499-504.
Abstract:
Biochemical and molecular approaches to understanding protein import into peroxisomes.
Peroxisomes are eukaryotic organelles that perform diverse and variable functions. Although genetic studies in yeasts and mammals have identified approximately 20 genes (PEX genes) required for the biogenesis of this important organelle, biochemical studies of protein targeting and import have lagged behind and in many cases we have no idea of the function of the PEX gene products (peroxins). Using an import assay in vitro derived from sunflower cotyledon cells and recombinant proteins, we have obtained translocation intermediates on the peroxisome import pathway and are using cross-linking to identify interacting partners. We have also used antibodies raised against human PEX14 to inhibit the import of matrix proteins in this system. To obtain homologous antibodies for inhibition experiments, to immunoprecipitate cross-linked products and to enable us to study the import pathways of peroxins we have cloned and characterized plant orthologues of three PEX genes, PEX6, PEX10 and PEX14.
Abstract.
Author URL.