Alicia Kight

Deletion of the Chloroplast-Localized Thylakoid Formation1 Gene Product in Arabidopsis Leads to Deficient Thylakoid Formation and Variegated Leaves

Development of thylakoid membranes depends upon the transport of membrane vesicles from the chloroplast inner envelope and subsequent fusion of vesicles within the interior of the plastid. The Arabidopsis (Arabidopsis thaliana) Thylakoid formation1 (Thf1) gene product is shown here to control an important step required for the normal organization of these vesicles into mature thylakoid stacks and ultimately for leaf development. The Arabidopsis Thf1 gene encodes an imported chloroplast protein, as shown by in vitro import and localization of a Thf1-green fluorescent protein fusion product in transgenic plants. This gene is conserved in oxygenic photoautotrophs ranging from cyanobacteria to flowering land plants. Transcript levels for Thf1 are induced in the light and decrease under dark conditions, paralleling profiles of light-regulated nuclear genes involved in chloroplast function. Disruption of the Thf1 gene via T-DNA insertion results in plants that are severely stunted with variegated leaf patterns. Nongreen sectors of variegated leaves lacking Thf1 expression contain plastids that accumulate membrane vesicles on the interior and lack organized thylakoid structures. Green sectors of Thf1-disrupted leaves contain some chloroplasts that form organized thylakoid membranes, indicating that an inefficient compensatory mechanism supports thylakoid formation in the absence of Thf1. Genetic complementation of a Thf1 knockout line confirms the role of this gene in chloroplast and leaf development. Transgenic plants expressing the Thf1 gene in antisense orientation are stunted with altered thylakoid organization, especially in young seedlings. The data indicate that the Thf1 gene product plays a crucial role in a dynamic process of vesicle-mediated thylakoid membrane biogenesis.

Regulation of the GTPase cycle in post-translational signal recognition particle-based protein targeting involves cpSRP43.

The chloroplast signal recognition particle consists of a conserved 54-kDa GTPase and a novel 43-kDa chromodomain protein (cpSRP43) that together bind light-harvesting chlorophyll a/b-binding protein (LHCP) to form a soluble targeting complex that is subsequently directed to the thylakoid membrane. Homology-based modeling of cpSRP43 indicates the presence of two previously identified chromodomains along with a third N-terminal chromodomain. Chromodomain deletion constructs were used to examine the role of each chromodomain in mediating distinct steps in the LHCP localization mechanism. The C-terminal chromodomain is completely dispensable for LHCP targeting/integration in vitro. The central chromodomain is essential for both targeting complex formation and integration because of its role in binding the M domain of cpSRP54. The N-terminal chromodomain (CD1) is unnecessary for targeting complex formation but is required for integration. This correlates with the ability of CD1 along with the ankyrin repeat region of cpSRP43 to regulate the GTPase cycle of the cpSRP-receptor complex.

A Dynamic cpSRP43-Albino3 Interaction Mediates Translocase Regulation of Chloroplast Signal Recognition Particle (cpSRP)-targeting Components

The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). Here, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm. Furthermore, we show Alb3-Cterm dissociates a cpSRP·LHCP targeting complex in vitro and stimulates GTP hydrolysis by cpSRP54 and cpFtsY in a strictly cpSRP43-dependent manner. These results support a model in which interactions between the ankyrin region of cpSRP43 and the C terminus of Alb3 promote distinct membrane-localized events, including LHCP release from cpSRP and release of targeting components from Alb3.

ATP Stimulates Signal Recognition Particle (SRP)/FtsY-supported Protein Integration in Chloroplasts

The signal recognition particle (SRP) and its receptor (FtsY in prokaryotes) are essential for cotranslational protein targeting to the endoplasmic reticulum in eukaryotes and the cytoplasmic membrane in prokaryotes. An SRP/FtsY-like protein targeting/integration pathway in chloroplasts mediates the posttranslational integration of the light-harvesting chlorophyll a/b-binding protein (LHCP) into thylakoid membranes. GTP, chloroplast SRP (cpSRP), and chloroplast FtsY (cpFtsY) are required for LHCP integration into thylakoid membranes. Here, we report the reconstitution of the LHCP integration reaction with purified recombinant proteins and salt-washed thylakoids. Our data demonstrate that cpSRP and cpFtsY are the only soluble protein components required for LHCP integration. In addition, our studies reveal that ATP, though not absolutely required, remarkably stimulates LHCP integration into salt-washed thylakoids. ATP stimulates LHCP integration by a mechanism independent of the thylakoidal pH gradient (ΔpH) and exerts no detectable effect on the formation of the soluble LHCP-cpSRP-targeting complex. Taken together, our results indicate the participation of a thylakoid ATP-binding protein in LHCP integration.

The Membrane-binding Motif of the Chloroplast Signal Recognition Particle Receptor (cpFtsY) Regulates GTPase Activity

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally. Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpSRP-dependent protein targeting. This membrane-binding motif is both necessary and sufficient to direct cpFtsY and fused passenger proteins to thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may be functionally replaced by the corresponding region from E. coli, confirming that the membrane-binding motif is conserved among organellar and prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding correlates with liposome-induced GTP hydrolysis stimulation. Mutations that debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP hydrolysis, suggesting that negative regulation is provided by the intact membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD structural studies of the N-terminal region and the analogous region in the E. coli SRP receptor revealed a conformational change in secondary structure that takes place upon lipid binding. These studies suggest that the cpFtsY membrane-binding motif plays a critical role in the intramolecular communication that regulates cpSRP receptor functions at the membrane.

Preclinical characterization of an anti-methamphetamine monoclonal antibody for human use

Ch-mAb7F9, a human-mouse chimeric monoclonal antibody (mAb) designed to bind (+)-methamphetamine (METH) with high affinity and specificity, was produced as a treatment medication for METH abuse. In these studies, we present the preclinical characterization that provided predictive evidence that ch-mAb7F9 may be safe and effective in humans. In vitro ligand binding studies showed that ch-mAb7F9 is specific for and only binds its target ligands (METH, (+)-amphetamine, and 3,4-methylenedioxy-N-methylamphetamine) with high affinity. It did not bind endogenous neurotransmitters or other medications and was not bound by protein C1q, thus it is unlikely to stimulate in vivo complement-dependent cytotoxicity. Isothermal titration calorimetry potency studies showed that METH binding by ch-mAb7F9 is efficient. Pharmacokinetic studies of METH given after ch-mAb7F9 doses in rats demonstrated the in vivo application of these in vitro METH-binding characteristics. While METH had little effect on ch-mAb7F9 disposition, ch-mAb7F9 substantially altered METH disposition, dramatically reducing the volume of distribution and clearance of METH. The elimination half-life of METH was increased by ch-mAb7F9, but it was still very fast compared with the elimination of ch-mAb7F9. Importantly, the rapid elimination of unbound METH combined with previous knowledge of mAb:target ligand binding dynamics suggested that ch-mAb7F9 binding capacity regenerates over time. This finding has substantial therapeutic implications regarding the METH doses against which ch-mAb7F9 will be effective, on the duration of ch-mAb7F9 effects, and on the safety of ch-mAb7F9 in METH users who use METH while taking ch-mAb7F9. These results helped to support initiation of a Phase 1a study of ch-mAb7F9.

Identification and characterization of native proteins of Escherichia coli BL-21 that display affinity towards Immobilized Metal Affinity Chromatography and Hydrophobic Interaction Chromatography Matrices.

The purpose of this study was to identify and characterize Escherichia coli proteins which display affinity towards both Immobilized Metal Affinity Chromatography (IMAC) and Hydrophobic Interaction Chromatography (HIC). Co(II) IMAC was chosen as the primary capture step, followed by HIC employing different concentrations of salt to promote adsorption. Results provided insight on this rather small pool of E. coli proteins. Nine out of the ten have isoelectric values less than six, and half are considered nonessential. These data indicate that the combination of IMAC and HIC could be developed as a potent method for the purification of recombinant proteins by judicious choice of the salt concentration used to promote HIC, the development of E. coli strain(s) deficient in certain genomic proteins, and the design of an IMAC-HIC affinity tail for recombinant protein isolation based on the very proteins deleted from the genome.

Assembly of Chloroplast Signal Recognition Particle Involves Structural Rearrangement in cpSRP43

Signal recognition particle in chloroplasts (cpSRP) exhibits the unusual ability to bind and target full-length proteins to the thylakoid membrane. Unlike cytosolic SRPs in prokaryotes and eukaryotes, cpSRP lacks an RNA moiety and functions as a heterodimer composed of a conserved 54-kDa guanosine triphosphatase (cpSRP54) and a unique 43-kDa subunit (cpSRP43). Assembly of the cpSRP heterodimer is a prerequisite for post-translational targeting activities and takes place through interactions between chromatin modifier domain 2 (CD2) of cpSRP43 and a unique 10-amino-acid region in cpSRP54 (cpSRP54(pep)). We have used multidimensional NMR spectroscopy and other biophysical methods to examine the assembly and structure of the cpSRP43-cpSRP54 interface. Our data show that CD2 of cpSRP43 binds to cpSRP54(pep) in a 1:1 stoichiometry with an apparent K(d) of approximately 1.06 muM. Steady-state fluorescence and far-UV circular dichroism data suggest that the CD2-cpSRP54(pep) interaction causes significant conformational changes in both CD2 and the peptide. Comparison of the three-dimensional solution structures of CD2 alone and in complex with cpSRP54(pep) shows that significant structural changes are induced in CD2 in order to establish a binding interface contributed mostly by residues in the N-terminal segment of CD2 (Phe5-Val10) and an arginine doublet (Arg536 and Arg537) in the cpSRP54 peptide. Taken together, our results provide new insights into the mechanism of cpSRP assembly and the structural forces that stabilize the functionally critical cpSRP43-cpSRP54 interaction.

Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates

Background: Targeting of proteins requires a signal recognition particle (SRP) and multiple protein interactions. Results: We observed a decrease in the structural dynamics of cpSRP43 and an increase in substrate affinity upon its binding to cpSRP54. Conclusion: Changes in domain dynamics induced by cpSRP subunit interactions mediate substrate affinity. Significance: Relating structure and dynamics of SRP proteins allows for a better understanding of vectorial targeting within cells. Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP.

Response to Falk and Sinning: The C Terminus of Alb3 Interacts with the Chromodomains 2 and 3 of cpSRP43

This is a response to a letter by Falk and Sinning (1) We recently identified the ankyrin region of cpSRP43 as the primary domain responsible for binding Alb3-Cterm during light-harvesting chlorophyll-binding protein (LHCP) targeting, an interaction shown to facilitate cpSRP43-dependent stimulation of cpSRP GTPases by Alb3-Cterm (2). Falk et al. (3), using only protein interaction assays, report that CD2CD3 of cpSRP43 forms the Alb3-Cterm binding interface, which appears inconsistent with the fact that CD3 is not required for LHCP integration (4) and that CD2 is not required for LHCP integration by a cpSRP54/cpFtsY-independent pathway that relies on cpSRP43/Alb3 (5). We suggested that buffer choice, including the use of glycerol, may play a role in why Falk et al. (3) observed μm rather than nm affinity for cpSRP43 constructs (2). The use of high concentrations of glycerol in isothermal titration calorimetry (ITC) is known to cause experimental artifacts (6). Our control experiments clearly show that the use of glycerol, even at 5% v/v, causes significant background heat changes (Fig. 1). In their letter, Sinning and Falk report a 13 μm affinity even in the absence of glycerol, suggesting glycerol may not be the primary cause for the reported differences. Although species-specific differences in Alb3-Cterm could explain the observed affinity differences, comparing GTPase stimulation by Arabidopsis and Pisum sativum Alb3-Cterm does not support this possibility (Fig. 2). FIGURE 1. Isothermal titration calorimetry investigation of the influence of glycerol in various buffers in buffer to buffer experiments. ITC was conducted by injecting a specific buffer/glycerol formulation into a sample well containing the same buffer/glycerol ... FIGURE 2. Comparison of the ability of Pisum sativum and Arabidopsis thaliana Alb3-Cterm peptide to stimulate cpSRP43-dependent GTP hydrolysis by the cpSRP GTPases. The effect of Alb3-Cterm on the GTP hydrolysis activity of cpSRP54 and cpFtsY was examined in the ... Published reports (2, 4, 5) supporting the physiological relevance of high affinity protein interactions still suggest that the low affinity of cpSRP43 for Alb3-Cterm reported by Falk et al. (3) stems from assay conditions unfavorable for observing the primary targeting interaction that takes place between Alb3-Cterm and the Ank region of cpSRP43. Importantly, buffers used by Falk et al. (3) in ITC and size-exclusion experiments do not support LHCP integration (Fig. 3). In addition, Ank-containing cpSRP43 constructs, including those that lack CD2 and/or CD3, are able to prevent binding of radiolabeled cpSRP43 to Alb3 in salt-washed thylakoids whereas chromodomains do not (Fig. 4). FIGURE 3. Buffer influence on LHCP integration. Salt-washed thylakoids in IBM were incubated with 5 mm ATP, 1 mm GTP, 12.5 μl of radiolabeled pLHCP translation product, and recombinant cpSRP43, cpSRP54, and cpFtsY (2). The final volume was brought to 150 ... FIGURE 4. Competition for cpSRP43 binding to the C terminus of Alb3 in salt-washed thylakoids. Salt-washed thylakoids (equivalent to 75 μg of chlorophyll) and 8 nmol of recombinant cpSRP43 construct as indicated were incubated for 15 min at 25 °C ...