A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins

<div><p>Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.</p></div

Significant Heterogeneity and Slow Dynamics of the Unfolded Ubiquitin Detected by the Line Confocal Method of Single-Molecule Fluorescence Spectroscopy

The conformation and dynamics of the unfolded state of ubiquitin doubly labeled regiospecifically with Alexa488 and Alexa647 were investigated using single-molecule fluorescence spectroscopy. The line confocal fluorescence detection system combined with the rapid sample flow enabled the characterization of unfolded proteins at the improved structural and temporal resolutions compared to the conventional single-molecule methods. In the initial stage of the current investigation, however, the single-molecule Förster resonance energy transfer (sm-FRET) data of the labeled ubiquitin were flawed by artifacts caused by the adsorption of samples to the surfaces of the fused-silica flow chip and the sample delivery system. The covalent coating of 2-methacryloyloxyethyl phosphorylcholine polymer to the flow chip surface was found to suppress the artifacts. The sm-FRET measurements based on the coated flow chip demonstrated that the histogram of the sm-FRET efficiencies of ubiquitin at the native condition were narrowly distributed, which is comparable to the probability density function (PDF) expected from the shot noise, demonstrating the structural homogeneity of the native state. In contrast, the histogram of the sm-FRET efficiencies of the unfolded ubiquitin obtained at a time resolution of 100 μs was distributed significantly more broadly than the PDF expected from the shot noise, demonstrating the heterogeneity of the unfolded state conformation. The variety of the sm-FRET efficiencies of the unfolded state remained even after evaluating the moving average of traces with a window size of 1 ms, suggesting that conformational averaging of the heterogeneous conformations mostly occurs in the time domain slower than 1 ms. Local structural heterogeneity around the labeled fluorophores was inferred as the cause of the structural heterogeneity. The heterogeneity and slow dynamics revealed by the line confocal tracking of sm-FRET might be common properties of the unfolded proteins

List of constructs used.

<p>List of constructs used.</p

Position-specific incorporation of fluorescent amino acids by the cell-free system.

<p>RNA templates encoding variants of KvAP, pro-TNF, or Syb2 were translated in the cell-free system supplemented with BODIPYFL-AF conjugated tRNA with or without the addition of Sf21microsomes (microsomes). The non-natural amino acids were incorporated into introduced TAG codon. The resulting proteins were subjected to SDS-PAGE (10-15% polyacrylamide) and detected with excitation at 488 nm and emission at 530 nm. The amber codon in the different constructs was substituted for the codon at the position corresponding to the listed amino acids (except for M6 in pro-TNF where the TAG codon was inserted as an additional codon after the M6 codon) and marked by a star in the diagram.</p

Reconstitution of the AspT aspartate transporter synthesized in the cell-free system with Sf21 microsomes.

<p>(A) Cell-free synthesized AspT purified with cobalt affinity resin under non-denaturing conditions was solubilized with 1.5% DDM, and eluted with 0.01% DDM and 250 mM imidazole. The purified AspT protein (arrow head) was subjected to SDS-PAGE. (B) L-aspartate transport activity of proteoliposomes containing purified AspT protein. Proteoliposomes loaded with 100 mM L-aspartate were resuspended in 50 mM phosphate buffer (pH 7) (8.3 µg protein/ml). At 0 min, L-[³H] aspartate was added into the buffer (2.5 mM final concentration). After the rate of influx and efflux of L-[³H] aspartate was equal (at steady state), non-radiolabelled L-aspartate was added to a final concentration of 15 mM at 7.5 min (solid line, arrow indicating time of addition of unlabelled substrate). The broken line corresponds to the same experiment performed without addition of unlabelled L-aspartate. (C) Comparison of the initial uptake rates for L-aspartate into <i>E. coli</i> expressing AspT (hatched bar) and into microsomes containing cell-free synthesized AspT (solid bar). The transport activity at 1 min was regarded as the initial uptake rate.</p

Production of different membrane proteins using the cell-free system.

<p>After synthesis of the membrane proteins in the cell-free system in the presence of FluoroTect Green_Lys tRNA, the membrane fraction was precipitated and separated by SDS/PAGE. (A) 10% polyacrylamide gel; lane 1, AtKC1 (plant, K⁺ channel, 6 transmembrane segments (TMS)); lane 2, KAT1 (plant, K⁺ channel, 6 TMS); lane 3, KAT2 (plant, K⁺ channel, 6 TMS); lane 4, GORK (plant, K⁺ channel, 6 TMS); lane 5, AKT1 (plant, K⁺ channel, 6 TMS); lane 6, NhaA (bacteria, Na⁺/H⁺ antiporter, 10 TMS); Lane 7, AspT (bacteria, aspartate: alanine antiporter, 10 TMS); lane 8, MsbA (bacteria, ABC transporter, 12 TMS). (B) 15% polyacrylamide gel; lane 9, Sec61 β(human, 1 TMS).</p

Incorporation of fluorescent non-natural amino acids into NhaA.

<p>(A) Five version of NhaA (1–5) with introduced amber (TAG) codons (or Gly-Gly-TAG, introduced changes in bold) that enable incorporation of fluorescent non-natural amino acids into the N-terminal, middle or C-terminal regions of the protein (top panel) were expressed in the insect cell-free translation and translocation system in the presence of different fluorescent non-natural amino acids. The valine (V388) at the C-terminal end of NhaA was fused to additional sequences in the linker of the vectors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112874#pone-0112874-t001" target="_blank">Table 1</a>). Labeled NhaA protein was subjected to SDS-PAGE and fluorescent images were taken at the indicated excitation and emission wavelengths (bottom panel). The structure of the different fluorescent non-natural amino acids used in the experiments is shown above the gel images. (B) Incorporation of fluorescent non-natural amino acids into N-terminal or C-terminal positions in NhaA at an introduced four-base codon (CGGG) (top panel). NhaA was synthesized using the cell-free translation and translocation system in the presence of BODIPYFL-AF, subjected to SDS-PAGE and fluorescent images taken at the indicated excitation and emission wavelengths (bottom panel). (C) Double-labeling of NhaA by incorporation of BODIPY558-AF at an amber codon introduced into the N-terminal region and BODIPYFL-AF at the CGGG four-base codon introduced into the C-terminal region (top panel). NhaA was synthesized using the cell-free translation and translocation system in the presence of one or both fluorescent amino acids. Images of the SDS-PAGE were taken at the two different excitation and emission wavelengths indicated (bottom panel).</p

Flow chart for the preparation of microsomes from Sf21 cultured insect cells.

<p>Fewer centrifugation and wash steps were required compared to the conventional protocol for the preparation of rough microsomes from dog pancreas and the composition of the buffer was simplified. Single-tube reactions consisting of Sf21microsomes, cell-free translationally active lysates from cultured insect cells and mRNA were performed to synthesize membrane proteins <i>in vitro</i>.</p

Assessment of posttranslational modification of secreted proteins and membrane proteins synthesized using the cell-free system containing Sf21 microsomes.

<p>(A) Cleavage of the signal peptide of β-lactamase in microsomes. Translocation reactions containing <i>E. coli</i> β-lactamase labeled with FluoroTect Green_Lys tRNA (12.5 µl) were mixed with 1 µl of sterilized water (lane 2), with 0.5 µl of 200 µg/ml proteinase K and 0.5 µl of 20% (v/v) Triton X-100 (lane 3) or 0.5 µl of 200 mg/ml proteinase K (lane 4). The samples were incubated for 1 h at 4°C. Equal volumes (6 µl) of the samples were separated by SDS-PAGE on 15% gels. The open circle marks the mature protein without the signal peptide. Lane 1 contains β-lactamase synthesized without microsomes as a control. (B) The N-terminal signal peptide sequence of β-lactamase is required for its translocation across the microsomal membrane. The <i>E. coli</i> β-lactamase lacking its signal peptide (Δsp-β-lactamase) and the wild type (β-lactamase) were labeled with FluoroTect Green_Lys tRNA (12.5 µl). The translocation reaction was performed as in panel A. (C) N-linked glycosylation of membrane proteins in the microsomes. <i>Aeropyrum pernix</i> voltage-dependent K⁺ channel (KvAP), human pro-tumor necrosis factor (pro-TNF) and human synaptobrevin II (Syb2) were labelled with ³⁵S-methionine during translation in the presence (+) or absence (-) of Sf21 microsomes. Dots indicate the glycosylated form of the proteins. (D) Confirmation of N-linked glycosylation of Syb2 labeled with ³⁵S-methionine. One µl of 10% (v/v) Triton X-100 and 1 µl of 500 mU/ml glycopeptidase F (GpF) were added to the translation mixture (12.5 µl), followed by incubation for 5 min at 37°C. The dot indicates the glycosylated protein.</p