DataSheet1_Expression, purification and folding of native like mitochondrial carrier proteins in lipid membranes.docx

Mitochondrial Carrier Family proteins (MCFs) are located in the mitochondrial inner membrane and play essential roles in various cellular processes. Due to the relatively low abundance of many members of the family, in vitro structure and function determination of most MCFs require over-expression and purification of recombinant versions of these proteins. In this study, we report on a new method for overexpression of MCFs in Escherichia coli (E. coli) membranes, efficient purification of native-like proteins, and their reconstitution in mitochondrial inner membrane lipid mimics. cDNAs of Uncoupling Protein 4 (UCP4), Adenine Nucleotide Translocase (ANT) and Phosphate Translocase (PiT) were subcloned into the pET26b (+) expression vector such that fusion proteins with a short N-terminal pelB leader sequence and a six-histidine tag were produced to target the proteins toward the inner membrane of E. coli and facilitate affinity purification, respectively. Utilizing a modified autoinduction method, these proteins were overexpressed and extracted from the membrane of E. coli BL21 (DE3) and two modified strains, E. coli BL21 C43 (DE3) and E. coli BL21 Lobstr (DE3), in high yields. The proteins were then purified by immobilized metal affinity chromatography as monomers. Purity, identity, and concentration of the eluted monomers were determined by semi-native SDS-PAGE, Western blotting and mass spectrometry, and a modified Lowry assay, respectively. Cleavage of the pelB leader sequence from proteins was verified by mass spectrometric analysis. The purified proteins, surrounded by a shell of bacterial membrane lipids, were then reconstituted from the mild non-denaturing octyl glucoside (OG) detergent into phospholipid liposomes. Monomeric UCP4 spontaneously self-associated to form stable tetramers in lipid membranes, which is consistent with our previous studies. However, PiT and ANT remained dominantly monomeric in both detergent and liposome milieus, as detected by a combination of spectroscopic and electrophoretic methods. Native-like helical conformations of proteins were then confirmed by circular dichroism spectroscopy. Overall, this study demonstrates that targeting mitochondrial carrier family proteins to E. coli membranes provides an effective expression system for producing this family of proteins for biophysical studies.</p

A Split-Ubiquitin Yeast Two-Hybrid Screen to Examine the Substrate Specificity of atToc159 and atToc132, Two Arabidopsis Chloroplast Preprotein Import Receptors

<div><p>Post-translational import of nucleus-encoded chloroplast pre-proteins is critical for chloroplast biogenesis, and the Toc159 family of proteins serve as receptors for the process. Toc159 shares with other members of the family (e.g. Toc132), homologous GTPase (G−) and Membrane (M−) domains, but a highly dissimilar N-terminal acidic (A−) domain. Although there is good evidence that atToc159 and atToc132 from Arabidopsis mediate the initial sorting step, preferentially recognizing photosynthetic and non-photosynthetic preproteins, respectively, relatively few chloroplast preproteins have been assigned as substrates for particular members of the Toc159 family, which has limited the proof for the hypothesis. The current study expands the number of known preprotein substrates for members of the Arabidopsis Toc159 receptor family using a split-ubiquitin membrane-based yeast two-hybrid system using the atToc159 G-domain (Toc159G), atToc132 G-domain (Toc132G) and atToc132 A- plus G-domains (Toc132AG) as baits. cDNA library screening with all three baits followed by pairwise interaction assays involving the 81 chloroplast preproteins identified show that although G-domains of the Toc159 family are sufficient for preprotein recognition, they alone do not confer specificity for preprotein subclasses. The presence of the A-domain fused to atToc132G (Toc132AG) not only positively influences its specificity for non-photosynthetic preproteins, but also negatively regulates the ability of this receptor to interact with a subset of photosynthetic preproteins. Our study not only substantiates the fact that atToc132 can serve as a receptor by directly binding to chloroplast preproteins but also proposes the existence of subsets of preproteins with different but overlapping affinities for more than one member of the Toc159 receptor family.</p></div

Direct binding of atToc159AG and atToc132AG to transit peptides (TPs) from two representative photosynthetic preproteins.

<p>(A) Schematic representation of the LHCA4(TP)-DHFR_His and FNR1(TP)-DHFR_His constructs with C-terminal histidine tags (His-Tag) used as baits in the binding reactions. The numbers refer to the amino acid numbers of the TPs from LHC4 and FNR. (B and C) Equal amounts of <i>in vitro</i> translated [³⁵S]atToc132AG, or [³⁵S]atToc159AG were incubated in the presence of GTP with the indicated amounts of immobilized hexahistidine-tagged LHCA4(TP)-DHFR_His (A) or FNR1(TP)-DHFR_His (B). Bound proteins were eluted and separated by SDS-PAGE, and detected in dried gels using a phosphorimager. Top panels of B and C show representative experiments of triplicates. Lane, IVT in each panel contains 10% of the <i>in vitro</i> translation product added to each reaction. The graphs show quantitative analysis of the triplicate binding experiments with SE bars. atToc159AG binds to LHCA4(TP)-DHFR_His and FNR1(TP)-DHFR_His at levels that are two-fold and five-fold higher than atToc132AG, respectively. The atToc132AG and atToc159AG do not significantly bind to 800 pmol of a negative control immobilized hexahistidine-tagged DHFR_His protein (D).</p

Comparative strengths of protein–protein interactions as determined by a quantitative β-galactosidase assay for prey proteins originally isolated from the cDNA library using the atToc159 G-domain as the bait.

<p>(A–D) Relative enzymatic activity of β-Gal in extracts from <i>S. cerevisiae</i> strain NMY51 that expressed atToc159G, atToc132G or atToc132AG bait and also carried NubG-Prey construct as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t002" target="_blank">2</a>. Yeast cells were grown to A₅₄₆ of ∼0.8 in SD (−Leu, −Trp) medium at 30°C, followed by measurements as described in material and methods, and were quantified according to the following formula: Activity = 1,000×OD₆₁₅/V×t×OD₅₄₆, were V is the volume of assay and t is the time of incubation, for β-Gal activity in cell extracts. The measured activity was normalised to that of mock co-transformed strains expressing atToc159G, atToc132G or atToc132AG baits and empty vector, pR3-N, for respective interactions. A relative β-Gal activity of 100% was arbitrarily assigned to the atToc159G bait containing (A–B) and atToc132G bait containing (C–D) pairwise interactions. The experiments were performed in triplicate and repeated at least twice. Error bars indicate ±SD. Values marked with asterisks are significantly different (Student’s t-test; P≤0.05). (A) Interaction in yeast co-expressing atToc159G or atToc132G bait and non-photosynthetic prey interactors identified in atToc159G screening (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t001" target="_blank">Table 1</a>). (B) Interaction in yeast co-expressing atToc159G or atToc132G bait and photosynthetic prey interactors identified in atToc159G screening (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t002" target="_blank">Table 2</a>). (C) Interaction in yeast co-expressing atToc132G or atToc132AG bait and non-photosynthetic prey interactors identified in atToc159G screening (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t001" target="_blank">Table 1</a>). (D) Interaction in yeast co-expressing atToc132G or atToc132AG bait and non-photosynthetic prey interactors identified in atToc159G screening (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t002" target="_blank">Table 2</a>).</p

Comparative strengths of protein–protein interactions as determined by a quantitative β-galactosidase assay for additional prey proteins isolated from the screen using atToc132AG as the bait.

<p>(A–D) Relative enzymatic activity of β-Gal in extracts from <i>S. cerevisiae</i> strain NMY51 that expressed atToc159G, atToc132G or atToc132AG-domain bait and also carried NubG-Prey construct as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t002" target="_blank">2</a>. Yeast cells were grown to A₅₄₆ of ∼0.8 in SD (−Leu, −Trp) medium at 30°C, followed by measurements as described in materials and methods, and quantified according to the following formula: Activity = 1,000×OD₆₁₅/V×t×OD₅₄₆, were V is the volume of assay and t is the time of incubation. For β-Gal activity in cell extracts, the measured activity was normalised to that of mock co-transformed strains containing atToc159G, atToc132G or atToc132AG bait and empty vector, pR3-N for respective interactions. A relative β-Gal activity of 100% was arbitrarily assigned to the atToc159G bait containing (A–B) and atToc132G bait containing (C–D) pairwise interactions. The experiments were performed in triplicate and repeated at least twice. Error bars indicate ±SD. Values marked with asterisks are significantly different (Student’s t-test; P≤0.05). (A) Interaction in yeast co-expressing atToc159G or atToc132G bait and additional non-photosynthetic prey interactors identified in the screen using atToc132AG as the bait (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t001" target="_blank">Table 1</a>). (B) Interaction in yeast co-expressing atToc159G or atToc132G bait and additional photosynthetic prey interactors identified in the screen using atToc132AG screening (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t002" target="_blank">Table 2</a>). (C) Interaction in yeast co-expressing atToc132G or atToc132AG bait and additional non-photosynthetic prey interactors identified in the screen using atToc132AG screening (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t001" target="_blank">Table 1</a>). (D) Interaction in yeast co-expressing atToc132G or atToc132AG bait and additional non-photosynthetic prey interactors identified in the screen using atToc132AG screening (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t002" target="_blank">Table 2</a>).</p

List of plastid proteins with a non-photosynthetic function identified as interactors with atToc159G-, atToc132G- and atToc132AG-domain bait proteins in the split-ubiquitin yeast two-hybrid screen.

1<p>cTP denotes the length of the (predicted) chloroplast transit peptide.</p>2<p>‘+’ denotes which bait proteins each prey protein interacted with.</p

Comparative strengths of protein–protein interactions as determined by a quantitative β-galactosidase assay for additional prey proteins isolated from the screen using atToc132G as the bait.

<p>(A–D) Relative enzymatic activity of β-Gal in extracts from <i>S. cerevisiae</i> strain NMY51 that expressed atToc159G, atToc132G or atToc132AG baits and also carried the NubG-Prey construct as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t002" target="_blank">2</a>. Yeast cells were grown to A₅₄₆ of ∼0.8 in SD (−Leu, −Trp) medium at 30°C, followed by measurements as described in Materials and Methods, and were quantified according to the following formula: Activity = 1,000×OD₆₁₅/V×t×OD₅₄₆, were V is the volume of assay and t is the time of incubation, for β-Gal activity in cell extracts. The measured activity was normalised to that of mock co-transformed strains containing atToc159G, atToc132G or atToc132AG bait and empty vector, pR3-N, for respective interactions. A relative β-Gal activity of 100% was arbitrarily assigned to the atToc159G bait containing (A–B) and atToc132G bait containing (C–D) pairwise interactions. The experiments were performed in triplicate and repeated at least twice. Error bars indicate ±SD. Values marked with asterisks are significantly different (Student’s t-test; P≤0.05). (A) Interaction in yeast co-expressing atToc159G or atToc132G bait and additional non-photosynthetic prey interactors identified in the screen using atToc132G as the bait (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t001" target="_blank">Table 1</a>). (B) Interaction in yeast co-expressing atToc159G or atToc132G bait and additional photosynthetic prey interactors identified in the screen using atToc132G as the bait (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t002" target="_blank">Table 2</a>). (C) Interaction in yeast co-expressing atToc132G or atToc132AG bait and additional non-photosynthetic prey interactors identified in the screen using atToc132G as the bait (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t001" target="_blank">Table 1</a>). (D) Interaction in yeast co-expressing atToc132G or atToc132AG bait and additional non-photosynthetic prey interactors identified in the screen using atToc132G as the bait (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095026#pone-0095026-t002" target="_blank">Table 2</a>).</p

Flow chart outlining the protocol used for split-ubiquitin-based screening of an Arabidopsis cDNA library for interactors of atToc159G, atToc132G and atToc132AG baits.

<p><i>S. cerevisiae</i> strain NMY51 was transformed with Toc159G, Toc132G or Toc132AG bait constructs and a split-ubiquitin yeast two-hybrid assay was performed utilizing positive, negative and empty prey plasmids. Optimization of the screening stringency was achieved through pilot screening, which involved large scale transformation of bait pre-transformed yeast with empty library vector. Selection of positive clones was conducted on the quadruple dropout media supplemented with 3-aminotrizole. The colonies that grew on the selective media were re-plated on the same stringent selective medium. Prey plasmids were re-isolated from the putative positive clones. Two degrees of selection (i.e. individual retransformation with respective baits and bait dependency test) were followed by sequencing and BLAST analysis to confirm the interactions and simultaneously identify the interactors.</p

Analysis of yeast clones expressing the functional atToc159G, atToc132G and atToc132AG bait proteins.

<p>(A) Diagrammatic representation of the domain organisation of the atToc159G, atToc132G and atToc132AG bait constructs in the yeast plasmid, pBT3-STE. The bait vector provides an upstream yeast STE2 leader sequence and yeast ubiquitin Cub (34–76 aa), LexA and VP16 genes downstream. Fusion proteins produced by this cassette are expressed constitutively by the yeast CYC1 promoter and terminator. The bait protein domains were cloned directionally (using <i>Sfi</i>I) into the position indicated. The numbers refer to the amino acid sequence of atToc159 or atToc132. (B) The split-ubiquitin membrane based yeast two-hybrid analysis confirming expression of the bait proteins. atToc159G, atToc132G or atToc132AG bait was co-expressed in the <i>S. cerevisiae</i> strain NMY51 with the positive prey construct pOst1-NubI, empty prey vector, pR3-C/pR3-N or the non-interacting negative control construct pNubG-Fe65 and assayed on quadruple selective media (SD-LWHA) plates. Strains co-expressing bait protein and positive control prey exhibit growth only on SD-LWHA selective media. (C) Quantitative β-Galactosidase activity assay. Strains co-expressing respective bait and prey constructs were used in a microtitre plate-based β-galactosidase assay using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) as a substrate. The graph shows β-galactosidase activity/ml per min measured after 60 min. The values represent the mean of three independent experiments. The assay confirmed the functionality of the bait as the atToc159G, atToc132G and atToc132AG fusions interact with positive control prey but not with the empty vectors or negative control prey.</p

Disparate Effects of Mesenchymal Stem Cells in Experimental Autoimmune Encephalomyelitis and Cuprizone-Induced Demyelination

<div><p>Mesenchymal stem cells (MSCs) are pleiotropic cells with potential therapeutic benefits for a wide range of diseases. Because of their immunomodulatory properties they have been utilized to treat autoimmune diseases such as multiple sclerosis (MS), which is characterized by demyelination. The microenvironment surrounding MSCs is thought to affect their differentiation and phenotype, which could in turn affect the efficacy. We thus sought to dissect the potential for differential impact of MSCs on central nervous system (CNS) disease in T cell mediated and non-T cell mediated settings using the MOG_35–55 experimental autoimmune encephalomyelitis (EAE) and cuprizone-mediated demyelination models, respectively. As the pathogeneses of MS and EAE are thought to be mediated by IFNγ-producing (T_H1) and IL-17A-producing (T_H17) effector CD4+ T cells, we investigated the effect of MSCs on the development of these two key pathogenic cell groups. Although MSCs suppressed the activation and effector function of T_H17 cells, they did not affect T_H1 activation, but enhanced T_H1 effector function and ultimately produced no effect on EAE. In the non- T cell mediated cuprizone model of demyelination, MSC administration had a positive effect, with an overall increase in myelin abundance in the brain of MSC-treated mice compared to controls. These results highlight the potential variability of MSCs as a biologic therapeutic tool in the treatment of autoimmune disease and the need for further investigation into the multifaceted functions of MSCs in diverse microenvironments and the mechanisms behind the diversity.</p></div