Spatial and temporal variations in cuticle proteins as revealed by monoclonal antibodies. Immunoblotting analysis and ultrastructural immunolocalization in a beetle, Tenebrio molitor

A library of monoclonal antibodies (Mabs) against adult cuticle of Tenebrio was used to visualize the secretion of cuticular antigens during metamorphosis. Immunoblots of water- and urea-soluble proteins, and high resolution immunogold labelling has shown that, except in one clone, the Mabs recognize antigens in the three developmental stages. However, the MW of larval and pupal antigens are different from the adult ones, though sharing common epitopes. Blots of cuticle proteins (CPs) bound to different lectins shown few water-soluble glycosylated proteins weakly or not recognized by the Mabs, suggesting that the majority of the Mabs do not recognize glycosylated epitopes. The immunolocalization of the different antigens suggests a molecular basis for both developmental and regional variations in cuticular architecture and to the modifications due to sclerotization, which differ between pre- and postecdysial cuticles of the three developmental stages.     

Ubiquitination of transporters at the forefront of plant nutrition

In plants, the tight regulation of plasma membrane transporters is essential to maintain nutrient homeostasis. The mechanisms controlling the abundance of transporters, and other integral plasma membrane proteins, now come to light. Ubiquitination appears as a major signal initiating cargo endocytosis and sorting into multivesicular bodies prior to degradation in the vacuole. We have indeed demonstrated that the root iron transporter IRT1 is subjected to ubiquitin-dependent trafficking in root epidermal cells. This control is crucial to keep IRT1 levels at the cell surface low and to cope with the toxicity associated with other readily available metal substrates of IRT1. Our work combined with recent report on the BOR1 boron transporter establishes ubiquitination as a conserved mechanism of plasma membrane protein trafficking in plants and highlights its importance for plant nutrition.Key words: Endocytosis, Iron, IRT1, BOR1, Degradation, monoubiquitinPlasma membrane transporters play a pivotal role in all living organisms by providing the cell surface with a selective barrier for a wide range of nutrients, small molecules and ions. These transport proteins are very often under the control of sophisticated molecular mechanisms to adapt to changing environmental conditions and to ensure optimal absorption of their cognate substrates. Although the molecular events controlling the abundance of plasma membrane proteins at the cell surface are largely documented in non-plant model organisms, the situation in plants is much less understood. This is particularly true for the well-established ubiquitin-dependent endocytosis and sorting in late endosomes of plasma membrane proteins, for which evidence is still lacking in plants.Recently, we showed that the root iron transporter IRT1 from the model plant Arabidopsis thaliana, which mediates Fe²⁺ and other divalent metal absorption from the soil,¹ surprisingly localizes to the early endosomes/trans-Golgi network compartment (EE/TGN).² Pharmacological interference with vesicular trafficking highlighted the dynamic behavior of IRT1 in root epidermal cells. IRT1, although localized to EE/TGN, rapidly cycles to the plasma membrane to perform iron uptake from the soil. We demonstrated that IRT1 is ubiquitinated in vivo and carries monoubiquitin moieties on several cytosol-exposed lysine residues, a process known as multiubiquitination. Reminiscent of the ubiquitin-dependent endocytosis and degradation of animal receptor tyrosine kinases and yeast nutrient permeases,^3,4 we hypothesized that IRT1 ubiquitination may control its dynamics in the cell. Transgenic plants expressing a mutant version of IRT1 in which two lysine residues were replaced by arginine (IRT1_K154K179R) indeed show accumulation of IRT1 protein at the plasma membrane. This clearly establishes the role of ubiquitination in driving IRT1 intracellular trafficking.Whether the cell surface localization of IRT1_K154K179R reflects a role of ubiquitination in the internalization from the plasma membrane, in the sorting at the multivesicular bodies, or both is an open question. Interestingly, IRT1_K154K179R-expressing plants displayed dramatic growth reduction, likely due to uncontrolled uptake of metals. Altogether, our work demonstrates the existence of monoubiquitin-dependent endocytosis in plants and reveals its crucial role in keeping IRT1 level at the plasma membrane low to prevent metal toxicity. The uptake of Fe²⁺ by IRT1 indeed requires the prior reduction of Fe³⁺ by the FRO2 ferric chelate reductase, whose activity was shown to be limiting for iron uptake.^5–7 The amount of Fe²⁺ produced by FRO2 is therefore low and favors the uptake of other substrates of IRT1 such as Zn²⁺, Mn²⁺, Co²⁺ and Cd²⁺. Limiting the pool of IRT1 at the plasma membrane by ubiquitin-dependent endocytosis appears as a protective mechanism to limit the absorption of readily available metals other than iron. This also highlights the necessity for a strict co-regulation of FRO2 and IRT1 to optimize iron uptake. The overexpression of IRT1 in transgenic Arabidopsis plants consequently leads to a strong overload of Zn and Mn in roots and aerial tissues, while iron only very mildly accumulates. This, combined with the existence of point mutants of IRT1 with altered selectivity,⁸ offers the long term perspective of engineering transgenic plants designed to phytoremediate polluted soil for a given element.Only a handful of plasma membrane proteins were shown to be ubiquitinated in plants, including the PIN2 auxin efflux carrier,⁹ the FLS2 flagellin receptor¹⁰ and the PIP2;1 water channel.¹¹ Yet, the role of ubiquitination for such proteins remained unclear until very recently. Along with our recent report of monoubiquitin-dependent trafficking of IRT1 came a similar study on the Arabidopsis BOR1 boron transporter. Like iron, boron is essential for plants but toxic when present in excess. BOR1 has been previously shown to be regulated by boron-inducible endocytosis and degradation in the vacuole.¹² Kasai et al.¹³ now showed that high boron concentrations induced BOR1 mono- or diubiquitination and that mutation of a single lysine residue affected BOR1 trafficking to the vacuole.¹³ In yeast and mammals, ubiquitination has been shown to trigger the internalization of plasma membrane proteins from the cell surface and sorting in multivesicular bodies/late endosomes (MVBs/LEs) for targeting and degradation in the vacuole/lysosomes,^3,4 although recent reports emphasize the role of ubiquitination in later stages of endocytosis.¹⁴ Similarly, BOR1 ubiquitination is not required for endocytosis from the plasma membrane, this step being probably mediated by tyrosine-based motives found on its cytosolic face,¹⁵ but is crucial for the sorting of BOR1 in multivesicular bodies for subsequent targeting and degradation in the vacuole.¹³It will be interesting to pinpoint the exact role of IRT1 ubiquitination in either the control of its internalization from the plasma membrane, the targeting to the internal vesicles of multivesicular bodies for delivery to the vacuole, or both. Hence, ubiquitination constitutes a conserved process controlling the localization and the fate of plasma membrane proteins, and appears as a critical mechanism to maintain iron and boron homeostasis in plants (Fig. 1). The quest for the machinery driving the ubiquitination of plasma membrane proteins has now begun. Already, the PUB12 and PUB13 U-box E3 ubiquitin ligases have just been reported to polyubiquitinate the FLS2 flagellin receptor, driving its flagellin-dependent degradation and preventing excessive or prolonged activation of immune responses.¹⁶ Whether these E3 ubiquitin ligases, or members from the same family, act on other plasma membrane proteins will have to be determined in the future. This will provide breakthrough information on how the dynamics and the fate of integral plasma membrane proteins are driven, and will highlight the importance of ubiquitination in important biological processes such as nutrition, response to pathogens and hormone transport.Open in a separate windowFigure 1Ubiquitin-dependent trafficking of plasma membrane proteins in plants. Diagram illustrating the role of ubiquitination throughout the endocytic pathway of plant plasma membrane proteins with the example of IRT 1 (in orange) and BOR1 (in blue). IRT 1 multiubiquitination (red circles) controls either its internalization from the plasma membrane and/or its sorting in late endosomes required for vacuolar targeting, to maintain IRT 1 low at the plasma membrane independently of iron nutrition. BOR1 mono- or diubiquitination (yellow circle) is required when plants are exposed to high boron for BOR1 sorting in multivesicular bodies and subsequent targeting to the vacuole for degradation. The interplay of ubiquitin ligases and deubiquitinases thereby controls the dynamics of plasma membrane proteins. PM, plasma membrane; EE /TGN, Early endosome/Trans-Golgi network; MVB/LE , Multivesicular body/Late endosome compartment; B, Boron; M²⁺, Metals transported by IRT 1 in addition to Fe²⁺.     

Iron utilization and metabolism in plants

The solubilization and long-distance allocation of iron between organs and tissues, as well as its subcellular compartmentalization and remobilization, involve various chelation and oxidation/reduction steps, transport activities and association with soluble proteins that store and buffer this metal. Maintaining iron homeostasis is an important determinant in building prosthetic groups such as heme and Fe-S clusters, and in assembling them into apoproteins, which are major components of plant metabolism. Such processes require complex protein machineries located in mitochondria and plastids. An essential role for iron metabolism and utilization in plant productivity is evidenced by the strong iron requirement for proper photosynthetic reactions.     

Red-shifted aequorin-based bioluminescent reporters for in vivo imaging of Ca2 signaling

Real-time visualization of calcium (Ca(2+)) dynamics in the whole animal will enable important advances in understanding the complexities of cellular function. The genetically encoded bioluminescent Ca(2+) reporter green fluorescent protein-aequorin (GA) allows noninvasive detection of intracellular Ca(2+) signaling in freely moving mice. However, the emission spectrum of GA is not optimal for detection of activity from deep tissues in the whole animal. To overcome this limitation, two new reporter genes were constructed by fusing the yellow fluorescent protein (Venus) and the monomeric red fluorescent protein (mRFP1) to aequorin. Transfer of aequorin chemiluminescence energy to Venus (VA) is highly efficient and produces a 58 nm red shift in the peak emission spectrum of aequorin. This substantially improves photon transmission through tissue, such as the skin and thoracic cage. Although the Ca(2+)-induced bioluminescence spectrum of mRFP1-aequorin (RA) is similar to that of aequorin, there is also a small peak above 600 nm corresponding to the peak emission of mRFP1. Small amounts of energy transfer between aequorin and mRFP1 yield an emission spectrum with the highest percentage of total light above 600 nm compared with GA and VA. Accordingly, RA is also detected with higher sensitivity from brain areas. VA and RA will therefore improve optical access to Ca(2+) signaling events in deeper tissues, such as the heart and brain, and offer insight for engineering new hybrid molecules.     

The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha: molecular cloning, characterization and expression

Summary The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the Al gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 by DNA fragment extending from –982 to –634 by upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 F1 gene and of several highly expressed plant genes.     

Choice of the adequate detection time for the accurate evaluation of the efficiency of siRNA-induced gene silencing

RNA interference (RNAi) mediated by small interfering RNA (siRNA) has become a popular tool of examining the function of various genes. However, many studies have failed to identify any inhibitory effect of the siRNAs on the expression of the target gene, even though the siRNA being tested had been designed sequence-specifically. In order to determine if this failure is due to the incorrect choice of observation time rather than that of the target site of the gene of interest, this study examined the RNAi efficiency of a vector-driven siRNA targeting two different reporter proteins, EGFP and d2EGFP, whose targeted sequences were identical but the half-lives within the cells differed remarkably from each other (>24h versus 2h), during the time course after transfection. The EGFP expression levels in both cells were reduced in time-dependent manner but the reduction patterns were quite different from each other. The RNAi efficiency varied among the different observation time points and the time required for the maximum RNAi efficiency was proportional to the half-life of the target protein. Stable knocked down cell lines for EGFP expression were then established and the reduced EGFP expression levels in these cell lines were retained for a long period. These results suggest that the choice of an adequate observation time or the establishment of stable knocked down cells by antibiotic selection might be required for making an accurate evaluation of the RNAi effect on the target protein possessing a long half-life.