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.
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.
Stacks off tracks Osterrieder, Anne; Sparkes, Imogen A.; Botchway, Stan W. ...
Journal of experimental botany,
06/2017, Letnik:
68, Številka:
13
Journal Article
Recenzirano
Odprti dostop
The plant Golgi apparatus modifies and sorts incoming proteins from the endoplasmic reticulum (ER) and synthesizes cell wall matrix material. Plant cells possess numerous motile Golgi bodies, which ...are connected to the ER by yet to be identified tethering factors. Previous studies indicated a role for cis-Golgi plant golgins, which are long coiled-coil domain proteins anchored to Golgi membranes, in Golgi biogenesis. Here we show a tethering role for the golgin AtCASP at the ER-Golgi interface. Using live-cell imaging, Golgi body dynamics were compared in Arabidopsis thaliana leaf epidermal cells expressing fluorescently tagged AtCASP, a truncated AtCASP-ΔCC lacking the coiled-coil domains, and the Golgi marker STtmd. Golgi body speed and displacement were significantly reduced in AtCASP-ΔCC lines. Using a dual-colour optical trapping system and a TIRF-tweezer system, individual Golgi bodies were captured in planta. Golgi bodies in AtCASP-ΔCC lines were easier to trap and the ER-Golgi connection was more easily disrupted. Occasionally, the ER tubule followed a trapped Golgi body with a gap, indicating the presence of other tethering factors. Our work confirms that the intimate ER-Golgi association can be disrupted or weakened by expression of truncated AtCASP-ΔCC and suggests that this connection is most likely maintained by a golgin-mediated tethering complex.
Compartmentation of proteins and processes is a defining feature of eukaryotic cells. The growth and development of organisms is critically dependent on the accurate sorting of proteins within cells. ...The mechanisms by which cytosol-synthesized proteins are delivered to the membranes and membrane compartments have been extensively characterized. However, the protein complement of any given compartment is not precisely fixed and some proteins can move between compartments in response to metabolic or environmental triggers. The mechanisms and processes that mediate such relocation events are largely uncharacterized. Many proteins can in addition perform multiple functions, catalysing alternative reactions or performing structural, non-enzymatic functions. These alternative functions can be equally important functions in each cellular compartment. Such proteins are generally not dual-targeted proteins in the classic sense of having targeting sequences that direct de novo synthesized proteins to specific cellular locations. We propose that redox post-translational modifications (PTMs) can control the compartmentation of many such proteins, including antioxidant and/or redox-associated enzymes.
Gene families with multiple members are predicted to have individuals with overlapping functions. We examined all of the Arabidopsis (Arabidopsis thaliana) myosin family members for their involvement ...in Golgi and other organelle motility. Truncated fragments of all 17 annotated Arabidopsis myosins containing either the IQ tail or tail domains only were fused to fluorescent markers and coexpressed with a Golgi marker in two different plants. We tracked and calculated Golgi body displacement rate in the presence of all myosin truncations and found that tail fragments of myosins MYA1, MYA2, XI-C, XI-E, XI-I, and XI-K were the best inhibitors of Golgi body movement in the two plants. Tail fragments of myosins XI-B, XI-F, XI-H, and ATM1 had an inhibitory effect on Golgi bodies only in Nicotiana tabacum, while tail fragments of myosins XI-G and ATM2 had a slight effect on Golgi body motility only in Nicotiana benthamiana. The best myosin inhibitors of Golgi body motility were able to arrest mitochondrial movement too. No exclusive colocalization was found between these myosins and Golgi bodies in our system, although the excess of cytosolic signal observed could mask myosin molecules bound to the surface of the organelle. From the preserved actin filaments found in the presence of enhanced green fluorescent protein fusions of truncated myosins and the motility of myosin punctae, we conclude that global arrest of actomyosin-derived cytoplasmic streaming had not occurred. Taken together, our data suggest that the above myosins are involved, directly or indirectly, in the movement of Golgi and mitochondria in plant cells.
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.
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-negative myosin 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.
In many vacuolate plant cells, individual Golgi bodies appear to be attached to tubules of the pleiomorphic cortical endoplasmic reticulum (ER) network. Such observations culminated in the ...controversial mobile secretory unit hypothesis to explain transport of cargo from the ER to Golgi via Golgi attached export sites. This proposes that individual Golgi bodies and an attached‐ER exit machinery move over or with the surface of the ER whilst collecting cargo for secretion. By the application of infrared laser optical traps to individual Golgi bodies within living leaf cells, we show that individual Golgi bodies can be micromanipulated to reveal their association with the ER. Golgi bodies are physically attached to ER tubules and lateral displacement of individual Golgi bodies results in the rapid growth of the attached ER tubule. Remarkably, the ER network can be remodelled in living cells simply by movement of laser trapped Golgi dragging new ER tubules through the cytoplasm and new ER anchor sites can be established. Finally, we show that trapped Golgi ripped off the ER are ‘sticky’ and can be docked on to and attached to ER tubules, which will again show rapid growth whilst pulled by moving Golgi.
Allantoate amidohydrolases (AAHs) hydrolize the ureide allantoate to ureidoglycolate, CO₂, 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.
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).
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