The ubiquitination-deubiquitination cycle on the ribosomal protein eS7A is crucial for efficient translation.

Ubiquitination is a major post-translational modification of ribosomal proteins. The role of ubiquitination in the regulation of ribosome functions is still being elucidated. However, the importance of ribosome deubiquitination remains unclear. Here, we show that the cycle of ubiquitination and deubiquitination of the 40S ribosome subunit eS7 is important for efficient translation. eS7 ubiquitination at lysine 83 is required for efficient protein translation. We identified Otu2 and Ubp3 as the deubiquitinating enzymes for eS7. An otu2Δubp3Δ mutation caused a defect in protein synthesis. Ubp3 inhibited polyubiquitination of eS7 in polysomes to keep eS7 in a mono-ubiquitinated form, whereas Otu2 was specifically bound to the free 40S ribosome and promoted the dissociation of mRNAs from 40S ribosomes in the recycling step. Our results provide clues for understanding the molecular mechanism of the translation system via a ubiquitination-deubiquitination cycle.

Sexual dimorphism in external morphology of the American bullfrog Rana (Aquarana) catesbeiana and the possibility of sex determination based on tympanic membrane/eye size ratio.

The American bullfrog Rana (Aquarana) catesbeiana has been reported to show significant sexual dimorphism based on the size ratio between the tympanic membrane and the eye. In males the tympanic membrane is much larger than the eye, but not in females. The ratio has been used as a convenient criterion to discriminate sexes (sexing) in the American bullfrog, though its reliability is unknown. In this study, we examined 86 adult American bullfrogs to clarify whether the tympanic membrane long diameter/eye long diameter (Dtm/De) ratio is a reliable index to discriminate sexes in this species. In addition, we examined the growth of this sexually dimorphic trait. Results indicated that there is a significant difference but there is a small overlap in this ratio Dtm/De between sexes. The allometric comparisons showed the sexual dimorphism of the Dtm/De ratio was increased during growth and the dimorphism is attributable to the difference in the growth rate of the tympanic membrane (Dtm). Therefore, sex determination of American bullfrogs cannot be wholly reliably achieved by the Dtm/De ratio alone; other external morphological features are required in addition.

Suppression of peroxisome biogenesis factor 10 reduces cuticular wax accumulation by disrupting the ER network in Arabidopsis thaliana.

Peroxisome biogenesis factor 10 (PEX10) is a component of the peroxisomal matrix protein import machinery. To analyze the physiological function of PEX10, we used transgenic AtPEX10i Arabidopsis plants that had suppressed expression of the PEX10 gene due to RNA interference. AtPEX10i plants had patches of paleness on leaves, and abnormal floral organs that were typical of cuticular wax-deficient mutants. Quantitative analysis of cuticular wax revealed that the amount of wax in AtPEX10i plants was indeed lower than that in control plants. This result was confirmed by toluidine blue staining and scanning electron microscopic analysis of AtPEX10i. The CER1, CER4, WAX2 and SHN1 genes are known to be responsible for wax biosynthesis in Arabidopsis. Of these, CER1, CER4 and WAX2 were found to be localized on the endoplasmic reticulum (ER). In AtPEX10i plants, the expression of these genes was down-regulated, and CER1, CER4 and WAX2 were mislocalized to the cytosol. We also found that AtPEX10i plants had defects in ER morphology. Based on these results, we propose that PEX10 is essential for the maintenance of ER morphology and for the expression of CER1, CER4, WAX2 and SHN1 genes, which contribute to the biosynthesis of cuticular wax.

Plant catalase is imported into peroxisomes by Pex5p but is distinct from typical PTS1 import.

We have previously demonstrated that the targeting signal of pumpkin catalase, Cat1, is an internal PTS1 (peroxisomal targeting signal 1)-like sequence, QKL, located at -13 to -11 from the C-terminus, which is different from the typical PTS1 SKL motif located in the C-terminus. Here we show that Cat1 import into peroxisome is dependent on the cytosolic PTS receptor, Pex5p, in Arabidopsis, similar to typical PTS1 import, and that other components for transport of peroxisomal matrix proteins such as Pex14p, Pex13p, Pex12p and Pex10p also contribute to the import of Cat1. Interestingly, however, we found that Cat1 interacts with the N-terminal domain of Pex5p, but not the C-terminal domain for interaction with the typical PTS1, revealing that Pex5p recognizes Cat1 in a manner distinct from typical PTS1.

Gateway Vectors for Simultaneous Detection of Multiple Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation.

Bimolecular fluorescence complementation (BiFC) is widely used to detect protein-protein interactions, because it is technically simple, convenient, and can be adapted for use with conventional fluorescence microscopy. We previously constructed enhanced yellow fluorescent protein (EYFP)-based Gateway cloning technology-compatible vectors. In the current study, we generated new Gateway cloning technology-compatible vectors to detect BiFC-based multiple protein-protein interactions using N- and C-terminal fragments of enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), and monomeric red fluorescent protein (mRFP1). Using a combination of N- and C-terminal fragments from ECFP, EGFP and EYFP, we observed a shift in the emission wavelength, enabling the simultaneous detection of multiple protein-protein interactions. Moreover, we developed these vectors as binary vectors for use in Agrobacterium infiltration and for the generate transgenic plants. We verified that the binary vectors functioned well in tobacco cells. The results demonstrate that the BiFC vectors facilitate the design of various constructions and are convenient for the detection of multiple protein-protein interactions simultaneously in plant cells.

Functional classification of Arabidopsis peroxisome biogenesis factors proposed from analyses of knockdown mutants.

In higher plants, peroxisomes accomplish a variety of physiological functions such as lipid catabolism, photorespiration and hormone biosynthesis. Recently, many factors regulating peroxisomal biogenesis, so-called PEX genes, have been identified not only in plants but also in yeasts and mammals. In the Arabidopsis genome, the presence of at least 22 PEX genes has been proposed. Here, we clarify the physiological functions of 18 PEX genes for peroxisomal biogenesis by analyzing transgenic Arabidopsis plants that suppressed the PEX gene expression using RNA interference. The results indicated that the function of these PEX genes could be divided into two groups. One group involves PEX1, PEX2, PEX4, PEX6, PEX10, PEX12 and PEX13 together with previously characterized PEX5, PEX7 and PEX14. Defects in these genes caused loss of peroxisomal function due to misdistribution of peroxisomal matrix proteins in the cytosol. Of these, the pex10 mutant showed pleiotropic phenotypes that were not observed in any other pex mutants. In contrast, reduced peroxisomal function of the second group, including PEX3, PEX11, PEX16 and PEX19, was induced by morphological changes of the peroxisomes. Cells of the pex16 mutant in particular possessed reduced numbers of large peroxisome(s) that contained unknown vesicles. These results provide experimental evidence indicating that all of these PEX genes play pivotal roles in regulating peroxisomal biogenesis. We conclude that PEX genes belonging to the former group are involved in regulating peroxisomal protein import, whereas those of the latter group are important in maintaining the structure of peroxisome.

Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor.

Many peroxisomal proteins are imported into peroxisomes via recognition of the peroxisomal targeting signal (PTS1) present at the C-termini by the PTS1 receptor (Pex5p). Catalase, a peroxisomal protein, has PTS1-like motifs around or at the C-terminus. However, it remains unclear whether catalase is imported into peroxisome via the PTS1 system. In this work, we analyzed the PTS of pumpkin catalase (Cat1). A full or truncated pumpkin Cat1 cDNA fused at the 3' end of the green fluorescent protein (GFP) coding sequence was introduced and stably expressed in tobacco BY-2 (Nicotiana tabacum cv. Bright Yellow 2) cells or Arabidopsis thaliana by Agrobacterium-mediated transformation. The cellular localization of GFP was analyzed by fluorescence microscopy. The results showed that the C-terminal 10-amino acid region containing an SKL motif-like tripeptide (SHL) was not required for the import into peroxisomes. Surprisingly, the C-terminal 3-amino acid region was required for the import when the fusion proteins were transiently expressed by using particle gun bombardment, suggesting that the transient expression system is inadequate to analyze the targeting signal. We proposed that the C-terminal amino acid region from 13 to 11 (QKL), which corresponds with the PTS1 consensus sequence, may function as an internal PTS1. Analysis of the binding of Cat1 to PTS1 receptor (Pex5p) by the yeast two-hybrid system revealed that Cat1 can bind with the PTS1 receptor (Pex5p), indicating that Cat1 is imported into peroxisomes by the PTS1 system.