Arabidopsis ENDOMEMBRANE PROTEIN 12 contributes to the endoplasmic reticulum stress response by regulating K/HDEL receptor trafficking.

ENDOMEMBRANE PROTEIN 70 (EMP70) proteins constitute a 12-member superfamily in Arabidopsis thaliana, and are the most abundant protein species in plant Golgi proteomes. However, the physiological functions of EMPs in plants remain largely unknown. Here we have demonstrated that two AtEMP12 T-DNA insertion mutants are sensitive to ER (endoplasmic reticulum) stress as induced by tunicamycin and dithiothreitol treatments. Interestingly, the unfolded protein response (UPR) is constitutively activated in the knockout mutant emp12-1 under normal growth conditions, suggesting that the activation is a result of insufficient chaperones in the ER to aid protein folding. Indeed, we have further shown that BiP is secreted into the apoplast in emp12-1, while the K/HDEL receptor ERD2a, which regulates BiP trafficking, is exclusively localized in the ER in emp12-1, instead of its known ER-Golgi dual-localization. Given an enhanced retrograde transport of ERD2a, along with less dimerized receptor formed in the absence of EMP12, ERD2a may be prematurely returned to the ER without its bound ligands. Therefore, we propose that EMP12 may act as a novel regulator of the K/HDEL receptor to ensure an effective retrograde transport of K/HDEL ligands.

RST1 Is a FREE1 Suppressor That Negatively Regulates Vacuolar Trafficking in Arabidopsis.

FYVE domain protein required for endosomal sorting1 (FREE1), a plant-specific endosomal sorting complex required for transport-I component, is essential for the biogenesis of multivesicular bodies (MVBs), vacuolar degradation of membrane protein, cargo vacuolar sorting, autophagic degradation, and vacuole biogenesis in Arabidopsis (Arabidopsis thaliana). Here, we report the characterization of RESURRECTION1 (RST1) as a suppressor of free1 that, when mutated as a null mutant, restores the normal MVB and vacuole formation of a FREE1-RNAi knockdown line and consequently allows survival. RST1 encodes an evolutionarily conserved multicellular organism-specific protein, which contains two Domain of Unknown Function 3730 domains, showing no similarity to known proteins, and predominantly localizes in the cytosol. The depletion of FREE1 causes substantial accumulation of RST1, and transgenic Arabidopsis plants overexpressing RST1 display retarded seedling growth with dilated MVBs, and inhibition of endocytosed FM4-64 dye to the tonoplast, suggesting that RST1 has a negative role in vacuolar transport. Consistently, enhanced endocytic degradation of membrane vacuolar cargoes occurs in the rst1 mutant. Further transcriptomic comparison of rst1 with free1 revealed a negative association between gene expression profiles, demonstrating that FREE1 and RST1 have antagonistic functions. Thus, RST1 is a negative regulator controlling membrane protein homeostasis and FREE1-mediated functions in plants.

Subcellular Localization of Rice Acyl-CoA-Binding Proteins ACBP4 and ACBP5 Supports Their Non-redundant Roles in Lipid Metabolism.

Acyl-CoA-binding proteins (ACBPs), conserved at the acyl-CoA-binding domain, can bind acyl-CoA esters as well as transport them intracellularly. Six ACBPs co-exist in each model plant, dicot Arabidopsis thaliana (thale cress) and monocot Oryza sativa (rice). Although Arabidopsis ACBPs have been studied extensively, less is known about the rice ACBPs. OsACBP4 is highly induced by salt treatment, but down-regulated following pathogen infection, while OsACBP5 is up-regulated by both wounding and pathogen treatment. Their differential expression patterns under various stress treatments suggest that they may possess non-redundant functions. When expressed from the CaMV35S promoter, OsACBP4 and OsACBP5 were subcellularly localized to different endoplasmic reticulum (ER) domains in transgenic Arabidopsis. As these plants were not stress-treated, it remains to be determined if OsACBP subcellular localization would change following treatment. Given that the subcellular localization of proteins may not be reliable if not expressed in the native plant, this study addresses OsACBP4:GFP and OsACBP5:DsRED expression from their native promoters to verify their subcellular localization in transgenic rice. The results indicated that OsACBP4:GFP was targeted to the plasma membrane besides the ER, while OsACBP5:DsRED was localized at the apoplast, in contrast to their only localization at the ER in transgenic Arabidopsis. Differences in tagged-protein localization in transgenic Arabidopsis and rice imply that protein subcellular localization studies are best investigated in the native plant. Likely, initial targeting to the ER in a non-native plant could not be followed up properly to the final destination(s) unless it occurred in the native plant. Also, monocot (rice) protein targeting may not be optimally processed in a transgenic dicot (Arabidopsis), perhaps arising from the different processing systems for routing between them. Furthermore, changes in the subcellular localization of OsACBP4:GFP and OsACBP5:DsRED were not detectable following salt and pathogen treatment, respectively. These results suggest that OsACBP4 is likely involved in the intracellular shuttling of acyl-CoA esters and/or other lipids between the plasma membrane and the ER, while OsACBP5 appears to participate in the extracellular transport of acyl-CoA esters and/or other lipids, suggesting that they are non-redundant proteins in lipid trafficking.

Isolation of the Plant Exocyst Complex.

The exocyst, conserved from yeast to plants to mammals, is a hetero-octameric complex that mediates tethering of secretory vesicles to designated sites on the plasma membrane during polarized exocytosis. Because structural studies of the intact exocyst complex have been greatly limited by the low yields of purified proteins, many aspects of the exocyst functions remain poorly understood. Here, we present the protocols for the isolation and purification of the recombinant and the native plant exocyst complex. Given the known diversification of the exocyst subunits in plants, our protocols will likely open the possibility of unraveling the functional significance of these subunits in the context of the fully assembled exocyst complex.

Understanding copper sensitivity in zebrafish (Danio rerio) through the intracellular localization of copper transporters in a hepatocyte cell-line ZFL and the tissue expression profiles of copper transporters.

Zebrafish (Danio rerio) is a freshwater fish species of Cyprinidae known for its copper (Cu) intolerance, yet the underlying mechanisms of the sensitivity remain unclear. In this study, we examined the highly conserved molecular machineries in the copper handling system, namely ATOX1, ATP7A, ATP7B, and CTR1, by profiling their gene expression patterns among tissues before and after acute waterborne Cu exposure, and investigating their intracellular localization patterns using a zebrafish hepatocyte cell line, ZFL. We found that ATP7B was weak in its response toward Cu exposure to elicit its copper efflux function. Tissue distribution of these Cu transporters, however, revealed a distinct expression profile compared with mammals and other fish, particularly ATP7A, which unlike ATP7B was highly expressed in the liver, while ATP7B, not ATP7A, was specifically expressed in the intestine. ATOX1 transcript expression was also found to be significantly up-regulated with acute waterborne Cu, in contrast to the decreased expression found in other fish. A possible explanation for the Cu sensitivity in zebrafish is discussed.