Organelle-targeting Signal Research Articles

Modular Engineering of DNAzyme-Based Sensors for Spatioselective Imaging of Metal Ions in Mitochondria

DNAzyme-based sensors remain at the forefront of metal-ion imaging efforts, but most lack the subcellular precision necessary to their applications in specific organelles. Here, we seek to overcome this limitation by presenting a DNAzyme-based biosensor technology for spatiotemporally controlled imaging of metal ions in mitochondria. A DNA nanodevice was constructed by integrating an optically activatable DNAzyme sensor and an upconversion nanoparticle with an organelle-targeting signal. We exemplify that this approach allows for mitochondria-specific imaging of Zn2+ in living cells in a near-infrared light-controlled manner. Based on this, the system is used for the monitoring of mitochondrial Zn2+ during drug treatment in a cellular model of ischemia insult. Furthermore, the DNA nanodevice is employed to assess dynamic Zn2+ change and pharmacological interventions in an injury cell model of Zn2+ toxicity. This method paves the way for engineering of DNAzyme sensors to investigate the pathophysiological roles of metal ions at the subcellular level.

Stable expression of bacterial transporter ArsB attached to SNARE molecule enhances arsenic accumulation in Arabidopsis

ABSTRACT Acute and chronic arsenic (As) toxicity is a global health issue affecting millions of people, which leads to inactivation of over 200 enzymes, particularly those involved in cellular energy pathways and DNA synthesis and repair. The fern Pteris vittata acts as a hyperaccumulator of As and may be useful for phytoremediation to reduce disposal risks by utilizing metal-enriched plant biomass in energy and metal recovery. However, these ferns grow in limited environments and its transplantation and transport can be challenging. Therefore, we generated a transgenic Arabidopsis plant as a seed plant model, capable of accumulating As in their vacuole lumen. This was achieved by transforming the As-resistant bacterial As transporter, ArsB, via fusion with a organelle-targeting signal to the vacuolar membrane, N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs) protein, VAMP711. In this study, we developed the iVenus assay as a method for detecting whether the N- or C-terminus of a membrane protein is located on the cytoplasmic or exoplasmic side, and from the result of the iVenus assay, we generated the transgenic plant introduced N-terminal end of ArsB with VAMP711, localized to the central vacuolar membrane to accumulate As in the shoot and differentiation zone of root.

Aromatization of natural products by a specialized detoxification enzyme.

In plants, lineage-specific metabolites can be created by activities derived from the catalytic promiscuity of ancestral proteins, although examples of recruiting detoxification systems to biosynthetic pathways are scarce. The ubiquitous glyoxalase (GLX) system scavenges the cytotoxic methylglyoxal, in which GLXI isomerizes the α-hydroxy carbonyl in the methylglyoxal-glutathione adduct for subsequent hydrolysis. We show that GLXIs across kingdoms are more promiscuous than recognized previously and can act as aromatases without cofactors. In cotton, a specialized GLXI variant, SPG, has lost its GSH-binding sites and organelle-targeting signal, and evolved to aromatize cyclic sesquiterpenes bearing α-hydroxyketones to synthesize defense compounds in the cytosol. Notably, SPG is able to transform acetylated deoxynivalenol, the prevalent mycotoxin contaminating cereals and foods. We propose that detoxification enzymes are a valuable source of new catalytic functions and SPG, a standalone enzyme catalyzing complex reactions, has potential for toxin degradation, crop engineering and design of novel aromatics.

Gateway binary vectors with organelle-targeted fluorescent proteins for highly sensitive reporter assay in gene expression analysis of plants

Fluorescent proteins are valuable tools in the bioscience field especially in subcellular localization analysis of proteins and expression analysis of genes. Fusion with organelle-targeting signal accumulates fluorescent proteins in specific organelles, increases local brightness, and highlights the signal of fluorescent proteins even in tissues emitting a high background of autofluorescence. For these advantages, organelle-targeted fluorescent proteins are preferably used for promoter:reporter assay to define organ-, tissue-, or cell-specific expression pattern of genes in detail. In this study, we have developed a new series of Gateway cloning technology-compatible binary vectors, pGWBs (attR1-attR2 acceptor sites) and R4L1pGWB (attR4-attL1 acceptor sites), carrying organelle-targeted synthetic green fluorescent protein with S65T mutation (sGFP) (ER-, nucleus-, peroxisome-, and mitochondria-targeted sGFP) and organelle-targeted tag red fluorescent protein (TagRFP) (nucleus-, peroxisome-, and mitochondria-targeted TagRFP). These are available for preparation of promoter:reporter constructs by an LR reaction with a promoter entry clone attL1-promoter-attL2 (for pGWBs) or attL4-promoter-attR1 (for R4L1pGWBs), respectively. A transient expression experiment with particle bombardment using cauliflower mosaic virus 35S promoter-driven constructs has confirmed the correct localization of newly developed organelle-targeted TagRFPs by a co-localization analysis with the previously established organelle-targeted sGFPs. More intense and apparent fluorescence signals were detected by the nucleus- and peroxisome-targeted sGFPs than by the normal sGFPs in the promoter assay using transgenic Arabidopsis thaliana. The new pGWBs and R4L1pGWBs developed here are highly efficient and may serve as useful platforms for more accurate observation of GFP and RFP signals in gene expression analyses of plants.

Development of an R4 dual-site (R4DS) gateway cloning system enabling the efficient simultaneous cloning of two desired sets of promoters and open reading frames in a binary vector for plant research.

Vast numbers of proteins work cooperatively to exert their functions in various cells. In order to understand the functions and molecular mechanisms of these proteins in plants, analyses of transgenic plants that concomitantly express two protein-coding genes are often required. We developed a novel Gateway cloning technology-compatible binary vector system, the R4 dual-site (R4DS) Gateway cloning system, which enables the easy and efficient cloning of two desired sets of promoters and open reading frames (ORFs) into a binary vector using promoter and ORF entry clones. In this system, C-terminal fusions with 17 kinds of tags including visible reporters and epitope tags are available for each ORF, and selection by four kinds of resistance markers is possible. We verified that the R4DS Gateway cloning system functioned well in Arabidopsis thaliana by observing the expression and localization patterns of fluorescent proteins fused with organelle-targeting signals and driven by stomatal-lineage specific promoters. We also confirmed that the two cloning sites in the R4DS Gateway cloning system were equivalent and independently regulated. The results obtained indicate that the R4DS Gateway cloning system facilitates detailed comparisons of the expression patterns of two promoters as well as co-localization and interaction analyses of two proteins in specific cells in plants.

Capsid protease domain as a tool for assessing protein-domain folding during organelle import of nascent polypeptides in living cells.

Various proteins synthesized by ribosomes are imported into specific organelles. To elucidate the behavior of protein domains during import, we developed a folding probe, in which the capsid protease (CP) domain of the Semliki Forest virus was connected to enhanced green fluorescent protein (EGFP). The probe was fused to appropriate N-terminal organelle-targeting signal sequences and expressed in cultured cells. When the entire CP-domain was present in the cytosol, it became folded and cleaved off the following EGFP-domain. Once cleaved, EGFP stability was not affected by upstream sequences. Based on EGFP localization, we estimated the extent of CP-domain folding in the cytosolic space. When fused to mitochondrial hydrophobic multispanning membrane protein ABCB10, more than half of the EGFP remained in the cytoplasm, whereas most of the CP-portion was in the mitochondrial fraction. When fused to the endoplasmic reticulum (ER) signal, the cleaved EGFP was observed only in the ER fraction, confirming that the CP-domain cannot fold on the cytoplasmic side during cotranslational ER translocation. Thus, import of the ABCB10 molecule was not as tightly coupled with chain elongation as ER translocation. Use of this probe to quantitatively examine stop-translocation at the ER translocon in living cells revealed that positively charged residues on the translocating nascent chain stall at the ER translocon.

Differential age-dependent import regulation by signal peptides.

Author SummaryIt is well known that some genes are preferentially transcribed in young tissues and others are specifically expressed in aging tissue, but protein import into organelles is generally thought to be constitutive and independent of age. In this study, we find that, contrary to expectation, in higher plants the import of proteins into chloroplasts is indeed dependent on the age of the organelle. We find that chloroplast precursor proteins can be divided into three age-selective groups, with each having a preference for chloroplasts of a different age. The age-selective signal is located within the signal peptide of each protein that controls organelle import, and we further identify a motif that is necessary to make a signal peptide target older chloroplasts preferentially. We show that different members of a gene family often belong to different age-selective groups, and that changing the sequence motifs within a protein's signal peptide can change its age selectivity. These results indicate that organelle-targeting signal peptides are one set of tools available to multicellular organisms for differential age-specific regulation.

Multiple organelle-targeting signals in the N-terminal portion of peroxisomal membrane protein PMP70

Most membrane proteins are recognized by a signal recognition particle and are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, whereas almost all peroxisomal membrane proteins are posttranslationally targeted to the destination. Here we examined organelle-targeting properties of the N-terminal portions of the peroxisomal isoform of the ABC transporter PMP70 (ABCD3) using enhanced green fluorescent protein (EGFP) fusion. When the N-terminal 80 amino acid residue (N80)-segment preceding transmembrane segment (TM) 1 was deleted and the TM1-TM2 region was fused to EGFP, the TM1 segment induced ER-targeting and integration in COS cells. When the N80-segment was fused to EGFP, the fusion protein was targeted to the outer mitochondrial membrane. When both the N80-segment and the following TM1-TM2 region were present, the fusion located exclusively to the peroxisome. The full-length PMP70 molecule was clearly located in the ER in the absence of the N80-segment, even when multiple peroxisome-targeting signals were retained. We concluded that the TM1 segment possesses a sufficient ER-targeting function and that the N80-segment is critical for suppressing the ER-targeting function to allow the TM1-TM2 region to localize to the peroxisome. Cooperation of the organelle-targeting signals enables PMP70 to correctly target to peroxisomal membranes.

An Arabidopsis mutant resistant to thaxtomin A, a cellulose synthesis inhibitor from Streptomyces species.

Thaxtomin A is a phytotoxin produced by Streptomyces scabies and other Streptomyces species, the causative agents of common scab disease in potato and other taproot crops. At nanomolar concentrations, thaxtomin causes dramatic cell swelling, reduced seedling growth, and inhibition of cellulose synthesis in Arabidopsis. We identified a mutant of Arabidopsis, designated txr1, that exhibits increased resistance to thaxtomin as a result of a decrease in the rate of toxin uptake. The TXR1 gene was identified by map-based cloning and found to encode a novel, small protein with no apparent motifs or organelle-targeting signals. The protein, which has homologs in all fully sequenced eukaryotic genomes, is expressed in all tissues and during all developmental stages analyzed. Microarray transcript profiling of some 14,300 genes revealed two stomatin-like genes that were expressed differentially in the txr1 mutant and the wild type. We propose that TXR1 is a regulator of a transport mechanism.

Novel organelle-targeting signals in viral proteins.

Peroxisomes are vital cellular organelles controllingimportant metabolic functions like hydrogen peroxidedegradation, fatty acid metabolism, gluconeogenesis,toxin degradation, etc. (Flatmark et al., 1988; van denBosch et al., 1992; Masters, 1997; Lopez-Huertas etal., 2000). Certain human metabolic disorders such asZellweger syndrome, neonatal adrenoleukodystrophy,infantile Refsum disease and rhizomelic chondrodysplasiapunctata are associated with the peroxisomal defect inprotein import and/or biogenesis (Gould and Valle, 2000).Intracellular transport of proteins into peroxisomes de-pends on either type 1 peroxisomal targeting signal (PTS1)or, type 2 (PTS 2). Type 1 signal sequence is a tripeptidemotif present in the carboxy terminal of the protein(consensus sequence [STAGQCN]-[KRH]-[LIVMAFY];Gould et al., 1987, 1989; Swinkels et al., 1991; Olivieret al., 2000). Whereas, PTS2 constitutes a combina-tion of nine amino acid bipartite sequences (consensus[RKS]-[ILVH]-x(5)-[QH]-[LAE]) in the N-terminal halfof the protein (Gould et al., 1989; Swinkels et al., 1991;Terlecky et al., 1996). Receptors for PTS1 and PTS2 havebeen identified as PEX5 and PEX7 respectively (Terleckyet al., 1996; Lametschwandtner et al., 1998; Subramani,1998). The first report of PTS was in the firefly luciferasegene more than a decade ago (Gould et al., 1987, 1989).Following its discovery, except for viruses, virtually alleukaryotes, parasites and fungi have been identified tohave PTS-containing proteins and demonstrated to targetsuch proteins to the organelle (Fung and Clayton, 1991;Aitchison et al., 1992; Subramani, 1998). Recently weidentified the presence of a functional and conserved PTS1in one of the rotaviral proteins (VP4) which prompted usto look into other viral protein sequences available in thedatabase for potential PTS1 and PTS2 motifs.

Membrane Targeting Sequences in Tombusvirus Infections

Infection ofNicotiana benthamianacells with cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) viruses results in the formation of conspicuous membranous bodies [multivesicular bodies (MVBs)], which develop from modified peroxisomes or mitochondria, respectively. The organelle targeting signal is located in the proteins of 33 kDa (CymRSV) or 36 kDa (CIRV) encoded by ORF 1, which contain an N-terminal hydrophilic portion followed by two predicted hydrophobic transmembrane segments. Biochemical analysis showed that the 33- and 36-kDa proteins are integral membrane proteins. By exchanging small portions of the ORF 1 sequence between the infectious full-length clones of the two viruses, hybrid constructs were obtained of which thein vitrosynthesized RNA was inoculated toN. benthamianaplants and protoplasts. The structure of infectious clones suggested that both the N-terminal hydrophilic region and the transmembrane segments of the ORF 1-encoded proteins specify which organelle is involved in the synthesis of MVBs. Mutational analysis of the CIRV 36-kDa protein also suggested the presence of an internal mitochondrial targeting sequence similar to that found in several normal host proteins that are synthesized in the cytoplasm and transported to mitochondria. The CymRSV 33-kDa protein did not contain the obvious consensus signals thought to be characteristic of proteins targeted to peroxisomes, and an mitochondrial targeting sequence motif was not evident.