MAIGO5 Functions in Protein Export from Golgi-Associated Endoplasmic Reticulum Exit Sites in Arabidopsis

Plant cells face unique challenges to efficiently export cargo from the endoplasmic reticulum (ER) to mobile Golgi stacks. Coat protein complex II (COPII) components, which include two heterodimers of Secretory23/24 (Sec23/24) and Sec13/31, facilitate selective cargo export from the ER; however, little is known about the mechanisms that regulate their recruitment to the ER membrane, especially in plants. Here, we report a protein transport mutant of Arabidopsis thaliana, named maigo5 (mag5), which abnormally accumulates precursor forms of storage proteins in seeds. mag5-1 has a deletion in the putative ortholog of the Saccharomyces cerevisiae and Homo sapiens Sec16, which encodes a critical component of ER exit sites (ERESs). mag mutants developed abnormal structures (MAG bodies) within the ER and exhibited compromised ER export. A functional MAG5/SEC16A–green fluorescent protein fusion localized at Golgi-associated cup-shaped ERESs and cycled on and off these sites at a slower rate than the COPII coat. MAG5/SEC16A interacted with SEC13 and SEC31; however, in the absence of MAG5/SEC16A, recruitment of the COPII coat to ERESs was accelerated. Our results identify a key component of ER export in plants by demonstrating that MAG5/SEC16A is required for protein export at ERESs that are associated with mobile Golgi stacks, where it regulates COPII coat turnover.     

Quality Evaluation of Steel,Aluminum, and Road Material Recycled from End‐of‐Life Urban Buildings in Japan in Terms of Total Material Requirement

In this study we introduce the concept of total material requirement (TMR) to quantify the quality of materials from end‐of‐life buildings. The TMRs for the recycling of materials (urban ore TMR [UO‐TMR]) from four types of Japanese buildings ( Japanese traditional wooden structure [ JTWS], wooden frame with walls structure [ WFS ], reinforced‐concrete structure [RCS], and steel‐based structure [SS]) have been estimated and the trade‐off between the increase in function of recycled materials such as steel made from scrap and the additional inputs of energy and materials required to create the increase in function were evaluated. Steel made from scrap, aluminum made from scrap, and road material are assumed to be recycled from steel products, aluminum products, and aggregate and cement concrete in the buildings, respectively. Case study analyses were carried out to determine the effect of recycling only aboveground materials compared to recycling both aboveground and subsurface materials. Also, the effect of varying the recycling rate of wooden demolition debris is determined. The UO‐TMRs of steel made from scrap range from 4.7 kilograms per kilogram (kg/kg) to 18.2 kg/kg. Urban tailings (unrecycled components) account for the greatest proportion of the UO‐TMR of steel made from scrap, and the next largest contributor is the recycling process. In the case of aluminum made from scrap, the UO‐TMRs range from 22 to 196 kg/kg, with the contribution of urban tailings generally dominant, and the second largest contributor being on‐site demolition and shredding. The UO‐TMRs of recycled road material range from 1.04 to 1.16 kg/kg and are similar for different recycling cases and types of buildings.     

Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1^−/−) cell clones, derived from a heterozygous LAT1^+/− clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1^−/− DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1^−/− cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1^−/− DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1^−/− DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1^+/− DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.     

Downregulation of Notch mediates the seamless transition of individual Drosophila neuroepithelial progenitors into optic medullar neuroblasts during prolonged G1

The first step in the development of the Drosophila optic medullar primordia is the expansion of symmetrically dividing neuroepithelial cells (NEs); this step is then followed by the appearance of asymmetrically dividing neuroblasts (NBs). However, the mechanisms responsible for the change from NEs to NBs remain unclear. Here, we performed detailed analyses demonstrating that individual NEs are converted into NBs. We also showed that this transition occurs during an elongated G1 phase. During this G1 phase, the morphological features and gene expressions of each columnar NE changed dynamically. Once the NE-to-NB transition was completed, the former NE changed its cell-cycling behavior, commencing asymmetric division. We also found that Notch signaling pathway was activated just before the transition and was rapidly downregulated. Furthermore, the clonal loss of the Notch wild copy in the NE region near the medial edge caused the ectopic accumulation of Delta, leading to the precocious onset of transition. Taken together, these findings indicate that the activation of Notch signaling during a finite window coordinates the proper timing of the NE-to-NB transition.     

Distal transport of exogenously applied jasmonoyl-isoleucine with wounding stress

Determining the mobile signal used by plants to defend against biotic and abiotic stresses has proved elusive, but jasmonic acid (JA) and its derivatives appear to be involved. Using deuterium-labeled analogs, we investigated the distal transport of JA and jasmonoyl-isoleucine (JA-Ile) in response to leaf wounding in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum) plants. We recovered [(2)H(2)-2]JA ([(2)H(2)]JA) and [(2)H(3)-12]JA-Ile ([(2)H(3)]JA-Ile) in distal leaves of N. tabacum and S. lycopersicum after treating wounded leaves with [(2)H(2)]JA or [(2)H(3)]JA-Ile. We found that JA-Ile had a greater mobility than JA, despite its lower polarity, and that application of exogenous JA-Ile to wounded leaves of N. tabacum led to a higher accumulation of JA and JA-Ile in distal leaves compared with wounded control plants. We also found that exudates from the stem of S. lycopersicum plants with damaged leaflets contained JA and JA-Ile at higher levels than in an undamaged plant, and a significant difference in the levels of JA-Ile was observed 30 min after wounding. Based on these results, it was found that JA-Ile is a transportable compound, which suggests that JA-Ile is a signaling cue involved in the resistance to biotic and abiotic stresses in plants.     

Involvement of CD56brightCD11c+ cells in IL-18-mediated expansion of human γδ T cells

γδ T cells are considered to be innate lymphocytes that play an important role in host defense against tumors and infections. We recently reported that IL-18 markedly amplified γδ T cell responses to zoledronate (ZOL)/IL-2. In an extension of this finding, we analyzed the mechanism underlying the IL-18-mediated expansion of γδ T cells. After incubation of PBMCs with ZOL/IL-2/IL-18, the majority of the cells expressed γδ TCR, and the rest mostly exhibited CD56(bright)CD11c(+) under the conditions used in this study. CD56(bright)CD11c(+) cells were derived from a culture of CD56(int)CD11c(+) cells and CD14(+) cells in the presence of IL-2 and IL-18 without the addition of ZOL. They expressed IL-18Rs, HLA-DR, CD25, CD80, CD83, CD86, and CD11a/CD18. In addition, they produced IFN-γ, TNF-α, but not IL-12, when treated with IL-2/IL-18, and they exerted cytotoxicity against K562 cells, thus exhibiting characteristics of both NK cells and dendritic cells. Incubation of purified γδ T cells with CD56(bright)CD11c(+) cells in the presence of ZOL/IL-2/IL-18 resulted in the formation of massive cell clusters and led to the marked expansion of γδ T cells. However, both conventional CD56(-/int)CD11c(high) dendritic cells induced by GM-CSF/IL-4 and CD56(+)CD11c(-) NK cells failed to support the expansion of γδ T cells. These results strongly suggest that CD56(bright)CD11c(+) cells play a key role in the IL-18-mediated proliferation of γδ T cells.     

Su(dx) E3 ubiquitin ligase-dependent and -independent functions of polychaetoid, the Drosophila ZO-1 homologue

Zona occludens (ZO) proteins are molecular scaffolds localized to cell junctions, which regulate epithelial integrity in mammals. Using newly generated null alleles, we demonstrate that polychaetoid (pyd), the unique Drosophila melanogaster ZO homologue, regulates accumulation of adherens junction-localized receptors, such as Notch, although it is dispensable for epithelial polarization. Pyd positively regulates Notch signaling during sensory organ development but acts negatively on Notch to restrict the ovary germline stem cell niche. In both contexts, we identify a core antagonistic interaction between Pyd and the WW domain E3 ubiquitin ligase Su(dx). Pyd binds Su(dx) directly, in part through a noncanonical WW-binding motif. Pyd also restricts epithelial wing cell numbers to control adult wing shape, a function associated with the FERM protein Expanded and independent of Su(dx). As both Su(dx) and Expanded regulate trafficking, we propose that a conserved role of ZO proteins is to coordinate receptor trafficking and signaling with junctional organization.