Inhwan Hwang's scientific contributions

Publications (128)

Publications citing this author (6378)

    • Interestingly each individual GTPase domain is dispensable for the plant (Agne et al., 2009; Aronsson et al., 2010), however, a viable plant lacking both domains from both receptors could not yet be isolated. The minimal structure required for sufficient assembly of the TOC complex and to support protein import is the M-domain of Toc159, which can partially complement the loss of Toc159 in ppi2 mutant plants (Lee et al., 2003). Toc34 is believed to exist as a homodimer in its GDPbound state, which exhibits a preprotein-binding site in its GTPase domain (Sun et al., 2002).
    [Show abstract] [Hide abstract] ABSTRACT: Chloroplasts originated from an endosymbiotic event in which a free-living cyanobacterium was engulfed by an ancestral eukaryotic host. During evolution the majority of the chloroplast genetic information was transferred to the host cell nucleus. As a consequence, proteins formerly encoded by the chloroplast genome are now translated in the cytosol and must be subsequently imported into the chloroplast. This process involves three steps: (i) cytosolic sorting procedures, (ii) binding to the designated receptor-equipped target organelle and (iii) the consecutive translocation process. During import, proteins have to overcome the two barriers of the chloroplast envelope, namely the outer envelope membrane (OEM) and the inner envelope membrane (IEM). In the majority of cases, this is facilitated by two distinct multiprotein complexes, located in the OEM and IEM, respectively, designated TOC and TIC. Plants are constantly exposed to fluctuating environmental conditions such as temperature and light and must therefore regulate protein composition within the chloroplast to ensure optimal functioning of elementary processes such as photosynthesis. In this review we will discuss the recent models of each individual import stage with regard to short-term strategies that plants might use to potentially acclimate to changes in their environmental conditions and preserve the chloroplast protein homeostasis.
    Full-text · Article · Feb 2017
    • Transformation of dicotyledonous plants via Agrobacterium tumefaciens is still far from routine and there is not a universal protocol suitable to transform genetically different cultivars within species (Romero et al., 2001; Lee et al., 2004). Application of cell and molecular biology techniques for the genetic improvement of this crop has been limited because of difficulties in plant regeneration and ultimately in linking regeneration with transformation (Lim et al., 1999). Regeneration in many different varieties of chilli was reported via shoot organogenesis in cotyledon and hypocotyl explants.
    [Show abstract] [Hide abstract] ABSTRACT: A reproducible and efficient protocol for two varieties of chilli (Capsicum frutescens L.) varieties Nepali and NARC-IV using different bacterial dilutions was optimized to assess its effect on transformation efficiency. The hypocotyl segments were inoculated with Agrobacterium tumefaciens strain EHA 101 harboring binary vector pTCL5. Plant response in culture was highly dependent on infection of hypocotyls with different bacterial densities. Proliferation and differentiation of calli forming plantlet as well as percentage transformation efficiency was strongly affected by the bacterial dilutions. Drastic increase in plant production from calli along with transformation efficiency was achieved at low bacterial density as compared to high density which in consequences brought about the necrosis and death of calli.
    Article · Dec 2008
    • Nicotiana benthamiana CHARGED MULTIVESICULAR BODY PROTEIN/CHROMATIN MODIFYING PROTEIN 1 (CHMP1; Yang et al. 2004), and in Arabidopsis AtCHMP1A and 1B (Spitzer et al. 2009) have been identified and characterized. AtCHMP1A and 1B show molecular weights, domain organization and charge distribution, similar to their yeast and mammalian homologous, as well as homologous binding partners as they interact with AtSKD1 and AtLIP5.
    [Show abstract] [Hide abstract] ABSTRACT: Being sessile organisms, plants evolved an unparalleled plasticity in their post-embryonic development, allowing them to adapt and fine-tune their vital parameters to an ever-changing environment. Crosstalk between plants and their environment requires tight regulation of information exchange at the plasma membrane (PM). PM proteins mediate such communication, by sensing variations in nutrient availability, external cues as well as by controlled solute transport across the membrane border. Localization and steady-state levels are essential for PM protein function and ongoing research identified cis- and trans-acting determinants, involved in control of plant PM protein localization and turnover. In this overview, we summarize recent progress in our understanding of plant PM protein sorting and degradation via ubiquitylation, a post-translational and reversible modification of proteins. We highlight characterized components of the machinery involved in sorting of ubiquitylated PM proteins and discuss consequences of protein ubiquitylation on fate of selected PM proteins. Specifically, we focus on the role of ubiquitylation and PM protein degradation in the regulation of polar auxin transport (PAT). We combine this regulatory circuit with further aspects of PM protein sorting control, to address the interplay of events that might control PAT and polarized growth in higher plants.
    Full-text · Article · May 2013
    • It indicates that ARS1 as a C2H2 zinc finger protein may be located either in the nucleus or cytoplasm and may translocate under certain conditions. To reveal the intracellular localization of ARS1, we used protoplast transient expression co-transforming an ARS1::sGFP construct and a chimeric construct containing the NLS domain fused to RFP (NLS::RFP) as a nuclear marker (Lee et al., 2001). As shown inFigure 2C, the intracellular distribution of green and red fluorescent signals overlapped, indicating that ARS1 localizes to the nucleus.
    [Show abstract] [Hide abstract] ABSTRACT: The phytohormone abscisic acid (ABA) induces accumulation of reactive oxygen species (ROS), which can disrupt seed dormancy and plant development. Here, we report the isolation and characterization of an Arabidopsis thaliana mutant called ars1 (aba and ros sensitive 1) that showed hypersensitivity to ABA during seed germination and to methyl viologen (MV) at the seedling stage. ARS1 encodes a nuclear protein with one zinc finger domain, two nuclear localization signal (NLS) domains, and one nuclear export signal (NES). The ars1 mutants showed reduced expression of a gene for superoxide dismutase (CSD3) and enhanced accumulation of ROS after ABA treatment. Transient expression of ARS1 in Arabidopsis protoplasts strongly suppressed ABA-mediated ROS production. Interestingly, nuclear-localized ARS1 translocated to the cytoplasm in response to treatment with ABA, H2O2, or MV. Taken together, these results suggest that ARS1 modulates seed germination and ROS homeostasis in response to ABA and oxidative stress in plants.
    Full-text · Article · Nov 2015
    • This redundancy was first discovered in single and double mutant analyses of the yeast Arf GAPs Gsc1 and Glo3 which act at the cis Golgi (Poon et al., 1999), and was then found among three Arf GAPs in mammalian cell culture; ARFGAP1 (homolog of Gsc1), ARFGAP2 and ARFGAP3 (homologs of Glo3) (Frigerio et al., 2007). Redundancy may also explain the viability of Drosophila mutants for Gap69C (homolog of Gsc1/ARFGAP1) (Frolov and Alatortsev, 2001) and is evident for homologs of Glo3 in Arabidopsis (Min et al., 2013). These Arf1 GAPs may thus play redundant, constitutive roles for Golgi function.
    [Show abstract] [Hide abstract] ABSTRACT: Biosynthetic traffic from the Golgi drives plasma membrane growth. For Drosophila embryo cleavage, this growth is rapid, but regulated, for cycles of furrow ingression and regression. The highly conserved small G protein Arf1 organizes Golgi trafficking. Arf1 is activated by guanine nucleotide exchange factors, but essential roles for Arf1 GTPase activating proteins (GAPs) are less clear. We report that the conserved Arf GAP Asap is required for cleavage furrow ingression in the early embryo. Since Asap can affect multiple sub-cellular processes, we used genetic approaches to dissect the primary effect of Asap. Our data argue against cytoskeletal or endocytic involvement, and reveal a common role for Asap and Arf1 in Golgi organization. Although Asap lacked Golgi enrichment, it was necessary and sufficient for Arf1 accumulation at the Golgi, and a conserved Arf1-Asap binding site was required for Golgi organization and output. Notably, Asap re-localized to the nuclear region at metaphase, a shift that coincided with subtle Golgi re-organization preceding cleavage furrow regression. We conclude that Asap is essential for Arf1 to function at the Golgi for cleavage furrow biosynthesis. Asap may recycle Arf1 to the Golgi from post-Golgi membranes, providing optimal Golgi output for specific stages of the cell cycle.
    Article · Aug 2016
    • On the contrary, when the two receptors were overexpressed in the same cells, AtRMR2 was produced in sufficient amounts for AtRMR1 re-localization to the TGN. In A. thaliana AtRMR1 and -2 have similar tissue expression profiles with a 30-old excess of AtRMR2 mRNA (as compiled by Genevestigator, NEBION AG, Zurich, Switzerland), and therefore, under normal conditions AtRMR1 probably co-localizes with AtRMR2 in the membrane of TGN, supporting the result of Park et al. [24] obtained in A. thaliana protoplasts. By a BiFC assay we confirmed the AtRMR1–AtRMR2 interaction and also demonstrated the formation of AtRMR2 homodimers (Figure 8b,c,e,f respectively).
    [Show abstract] [Hide abstract] ABSTRACT: In Arabidopsis thaliana, different types of vacuolar receptors were discovered. The AtVSR (Vacuolar Sorting Receptor) receptors are well known to be involved in the traffic to lytic vacuole (LV), while few evidences demonstrate the involvement of the receptors from AtRMR family (Receptor Membrane RING-H2) in the traffic to the protein storage vacuole (PSV). In this study we focused on the localization of two members of AtRMR family, AtRMR1 and -2, and on the possible interaction between these two receptors in the plant secretory pathway. Our experiments with agroinfiltrated Nicotiana benthamiana leaves demonstrated that AtRMR1 was localized in the endoplasmic reticulum (ER), while AtRMR2 was targeted to the trans-Golgi network (TGN) due to the presence of a cytosolic 23-amino acid sequence linker. The fusion of this linker to an equivalent position in AtRMR1 targeted this receptor to the TGN, instead of the ER. By using a Bimolecular Fluorescent Complementation (BiFC) technique and experiments of co-localization, we demonstrated that AtRMR2 can make homodimers, and can also interact with AtRMR1 forming heterodimers that locate to the TGN. Such interaction studies strongly suggest that the transmembrane domain and the few amino acids surrounding it, including the sequence linker, are essential for dimerization. These results suggest a new model of AtRMR trafficking and dimerization in the plant secretory pathway.
    Full-text · Article · Sep 2016
    • This provides evidence that overexpression of THI1 enhances the plant's sensitivity to ABA and improve its tolerance to drought stress. THI1 is targeted to both the mitochondrion and the chloroplast (Chabregas et al., 2001Chabregas et al., , 2003 Jin et al., 2003). Consistent with the previous results, only punctate distribution was found when pTHI1::THI-GFP was stably expressed in Arabidopsis.
    [Show abstract] [Hide abstract] ABSTRACT: Thiamine is required for both plant growth and development. Here, the involvement of a thiamine thiazole synthase, THI1, has been demonstrated in both guard cell abscisic acid (ABA) signaling and the drought response in Arabidopsis thaliana. THI1 over-expressors proved to be more sensitive to ABA than the wild type with respect to both the activation of guard cell slow type anion channels and stomatal closure; this effectively reduced the rate of water loss from the plant and thereby enhanced its level of drought tolerance. A yeast two-hybrid strategy was used to screen a cDNA library from epidermal strips of leaves for THI1 regulatory factors, and identified CPK33, a Ca2+-dependent protein kinase, as interactor with THI1 in a plasma membrane-delimited manner. Loss-of-function cpk33 mutants were hypersensitive to ABA activation of slow type anion channels and ABA induced stomatal closure, while the CPK33 overexpression lines showed opposite phenotypes. CPK33 kinase activity was essential for ABA-induced stomatal closure. Consistent with their contrasting regulatory role over stomatal closure, THI1 suppressed CPK33 kinase activity in vitro. Together, our data reveal a novel regulatory role of thiamine thiazole synthase to kinase activity in guard cell signaling.
    Full-text · Article · Dec 2015
    • It is proposed that SPHK/phyto-S1P acts upstream of PLD/PA, and PA is involved in promoting SPHK activity to mediate ABA-induced stomatal closure [41,64]. PA binds to the AGD7 [115]. The PA binding activates AGD7 hydrolysis of GTP in ARF, thus turning off ARF function.
    [Show abstract] [Hide abstract] ABSTRACT: Phospholipases D (PLD) and C (PLC) hydrolyze the phosphodiesteric linkages of the head group of membrane phospholipids. PLDs and PLCs in plants occur in different forms: the calcium-dependent phospholipid binding domain-containing PLDs (C2-PLDs), the plekstrin homology and phox homology domain-containing PLDs (PX/PH-PLDs), phosphoinositide-specific PLC (PI-PLC), and non-specific PLC (NPC). They differ in structures, substrate selectivities, cofactor requirements, and/or reaction conditions. These enzymes and their reaction products, such as phosphatidic acid (PA), diacylglycerol (DAG), and inositol polyphosphates, play important, multifaceted roles in plant response to abiotic and biotic stresses. Here, we review biochemical properties, cellular effects, and physiological functions of PLDs and PLCs, particularly in the context of their roles in stress response along with advances made on the role of PA and DAG in cell signaling in plants. The mechanism of actions, including those common and distinguishable among different PLDs and PLCs, will also be discussed.
    Full-text · Article · Jan 2016
    • The TP FGLK physicochemical domains have an established role in import efficiency (Chotewutmontri et al., 2012; Lee et al., 2006; Pilon et al., 1992a); these domains often contain Pro residues. Pro residues can exist in two isomeric forms, trans and more uncommonly cis (Song et al., 2006).
    [Show abstract] [Hide abstract] ABSTRACT: Eukaryotic organisms are defined by their endomembrane system and various organelles. The membranes that define these organelles require complex protein sorting and molecular machines that selectively mediate the import of proteins from the cytosol to their functional location inside the organelle. The plastid possibly represents the most complex system of protein sorting, requiring many different translocons located in the three membranes found in this organelle. Despite having a small genome of its own, the vast majority of plastid-localized proteins is nuclear encoded and must be posttranslationally imported from the cytosol. These proteins are encoded as a larger molecular weight precursor that contains a special “zip code,” a targeting sequence specific to the intended final destination of a given protein. The “zip code” is located at the precursor N-terminus, appropriately called a transit peptide (TP). We aim to provide an overview of plastid trafficking with a focus on the mechanism and regulation of the general import pathway, which serves as a central import hub for thousands of proteins that function in the plastid. We extend comparative analysis of plant proteomes to develop a better understanding of the evolution of TPs and differential TP recognition. We also review alternate import pathways, including vesicle-mediated trafficking, dual targeting, and import of signal-anchored and tail-anchored proteins.
    Full-text · Chapter · Jan 2017
    • Here, we describe an optimized protocol for immunoprecipitation (IP) followed by (label-free) tandem mass spectrometry ( MS/MS ) that allows the identifi cation of proteins interacting with a fl uorescently labeled bait in a non-biased and (semi-) quantitative fashion. This methodology has been successfully used to identify complexes of multiple types of proteins, including transcription factors and other nuclear proteins [4, 5] , membraneassociated proteins [6], trans-membrane proteins [6], and cytosolic proteins [7]. When considering IP-MS/ MS for the identifi cation of interacting proteins to a protein of interest, it is important to keep in mind that this is not a saturated identifi cation method.
    [Show abstract] [Hide abstract] ABSTRACT: Individual proteins often function as part of a protein complex. The identification of interacting proteins is therefore vital to understand the biological role and function of the studied protein. Here we describe a method for the in vivo identification of nuclear, cytoplasmic, and membrane-associated protein complexes from plant tissues using a strategy of immunoprecipitation followed by tandem mass spectrometry. By performing quantitative mass spectrometry measurements on biological triplicates, relative abundance of proteins in GFP-tagged complexes compared to background controls can be statistically evaluated to identify high-confidence interactors. We detail the entire workflow of this approach.
    Full-text · Chapter · Nov 2017 · BMC Plant Biology
    • Saito et al. reported that expression of the CYP707A genes, encoding ABA 8'-OH, was ubiquitous in various organs with different transcript accumulation levels; for example, in roots, CYP707A1 and CYP707A3 were moderately expressed, CYP707A2 was expressed weakly, and no expression of CYP707A4 was detected [31]. The conjugation of ABA with glucose results in ABA inactivation [17], but β-glucosidase can hydrolyze glucose-conjugated, biologically inactive ABA to produce active ABA [18] . In our study, TaGLU1 and TaGLU4, which function in ABA reactivation, were upregulated in the NaHS+PEG in the treatment in leaves while no obvious increase in expression was observed in roots.
    [Show abstract] [Hide abstract] ABSTRACT: Little information is available describing the effects of exogenous H2S on the ABA pathway in the acquisition of drought tolerance in wheat. In this study, we investigated the physiological parameters, the transcription levels of several genes involved in the abscisic acid (ABA) metabolism pathway, and the ABA and H2S contents in wheat leaves and roots under drought stress in response to exogenous NaHS treatment. The results showed that pretreatment with NaHS significantly increased plant height and the leaf relative water content of seedlings under drought stress. Compared with drought stress treatment alone, H2S application increased antioxidant enzyme activities and reduced MDA and H2O2 contents in both leaves and roots. NaHS pretreatment increased the expression levels of ABA biosynthesis and ABA reactivation genes in leaves; whereas the expression levels of ABA biosynthesis and ABA catabolism genes were up-regulated in roots. These results indicated that ABA participates in drought tolerance induced by exogenous H2S, and that the responses in leaves and roots are different. The transcription levels of genes encoding ABA receptors were up-regulated in response to NaHS pretreatment under drought conditions in both leaves and roots. Correspondingly, the H2S contents in leaves and roots were increased by NaHS pretreatment, while the ABA contents of leaves and roots decreased. This implied that there is complex crosstalk between these two signal molecules, and that the alleviation of drought stress by H2S, at least in part, involves the ABA signaling pathway.
    Full-text · Article · Sep 2016
    • This vector is stable in E. coli, and can produce proteins via the standard T7 polymerase promoter system (Studier and Moffatt, 1986). The 240 bp sequence incorporating the 77 amino acid MTP of the F1-ATPase γ subunit (Lee et al., 2012) was chemically synthesized with a NotI and AscI flanking region permitting ligation into pCW441, generating pRA1. Klebsiella oxytcoa Nif genes were codon optimized for eukaryotic expression and commercially synthesized (Geneart).
    [Show abstract] [Hide abstract] ABSTRACT: The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world’s increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.
    Full-text · Article · Mar 2017
    • Taken together, our results indicate that the core signaling components involved differs in the presence of exogenous ABA compared with stress-induced endogenous ABA in both leaves and roots. In addition to the type of specificity indicated above, it should be noted that there are tissue-, cell-, subcellularand even physiological process-specific responses by ABA core components [3, 7, 18,[50][51][52] , and such variations in the expression and affinity of receptor and PP2C Fig. 7Endogenous ABA content of maize seedlings after stress. a Mean ABA content of maize roots after treatment with 20 % PEG6000 for 0 (control), 0.5, 1, 2, or 3 h.
    [Show abstract] [Hide abstract] ABSTRACT: Background The different actions of abscisic acid (ABA) in the aboveground and belowground parts of plants suggest the existence of a distinct perception mechanism between these organs. Although characterization of the soluble ABA receptors PYR1/PYL/RCAR as well as core signaling components has greatly advanced our understanding of ABA perception, signal transduction, and responses, the environment-dependent organ-specific sensitivity of plants to ABA is less well understood. Results By performing real-time quantitative PCR assays, we comprehensively compared transcriptional differences of core ABA signaling components in response to ABA or osmotic/dehydration stress between maize (Zea mays L.) roots and leaves. Our results demonstrated up-regulation of the transcript levels of ZmPYLs homologous to dimeric-type Arabidopsis ABA receptors by ABA in maize primary roots, whereas those of ZmPYLs homologous to monomeric-type Arabidopsis ABA receptors were down-regulated. However, this trend was reversed in the leaves of plants treated with ABA via the root medium. Although the mRNA levels of ZmPYL1-3 increased significantly in roots subjected to polyethylene glycol (PEG)-induced osmotic stress, ZmPYL4-11 transcripts were either maintained at a stable level or increased only slightly. In detached leaves subjected to dehydration, the transcripts of ZmPYL1-3 together with ZmPYL5, ZmPYL6, ZmPYL10 and ZmPYL11 were decreased, whereas those of ZmPYL4, ZmPYL7 and ZmPYL8 were significantly increased. Our results also showed that all of the evaluated transcripts of PP2Cs and SnRK2 were quickly up-regulated in roots by ABA or osmotic stress; conversely they were either up-regulated or maintained at a constant level in leaves, depending on the isoforms within each family. Conclusions There is a distinct profile of PYR/PYL/RCAR ABA receptor gene expression between maize roots and leaves, suggesting that monomeric-type ABA receptors are mainly involved in the transmission of ABA signals in roots but that dimeric-type ABA receptors primarily carry out this function in leaves. Given that ZmPYL1 and ZmPYL4 exhibit similar transcript abundance under normal conditions, our findings may represent a novel mechanism for species-specific regulation of PYR/PYL/RCAR ABA receptor gene expression. A difference in the preference for core signaling components in the presence of exogenous ABA versus stress-induced endogenous ABA was observed in both leaves and roots. It appears that core ABA signaling components perform their osmotic/dehydration stress response functions in a stress intensity-, duration-, species-, organ-, and isoform-specific manner, leading to plasticity in response to adverse conditions and, thus, acclimation to life on land. These results deepen our understanding of the diverse biological effects of ABA between plant leaves and roots in response to abiotic stress at the stimulus-perception level. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0764-x) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2016
    • Most OEM proteins [5, 23] and a few IEM Proteins [24][25][26]are encoded in the nucleus as a mature form without a transit peptide. A series of elegant studies have established that a subset of OEM proteins are co-translationally recognized in the cytosol at their TMD and a positively charged flanking region at its C terminus by an ankyrin repeat protein, which directs its client proteins specifically to the chloroplast OEM [27][28][29]. Insertion of these proteins involves the core TOC component Toc75 [30], which forms a transmembrane β-barrel [31, 32]. Toc75 itself is unique among the OEM proteins in that it is synthesized in the cytosol as a larger precursor with an N-terminal extension of 100–140 residues called t75 (also termed tp75) [3, 33].
    [Show abstract] [Hide abstract] ABSTRACT: PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.
    Full-text · Article · Dec 2016