Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jan 20;12(1):108-119.
doi: 10.1021/acssynbio.2c00406. Epub 2022 Nov 29.

Reconstitution of a Reversible Membrane Switch via Prenylation by One-Pot Cell-Free Expression

Lei Kai  1   2 , Sonal  1   3 , Tamara Heermann  1 , Petra Schwille  1
Affiliations

Reconstitution of a Reversible Membrane Switch via Prenylation by One-Pot Cell-Free Expression

Lei Kai et al. ACS Synth Biol. .

Abstract

Reversible membrane targeting of proteins is one of the key regulators of cellular interaction networks, for example, for signaling and polarization. So-called "membrane switches" are thus highly attractive targets for the design of minimal cells but have so far been tricky to reconstitute in vitro. Here, we introduce cell-free prenylated protein synthesis (CFpPS), which enables the synthesis and membrane targeting of proteins in a single reaction mix including the prenylation machinery. CFpPS can confer membrane affinity to any protein via addition of a 4-peptide motif to its C-terminus and offers robust production of prenylated proteins not only in their soluble forms but also in the direct vicinity of biomimetic membranes. Thus, CFpPS enabled us to reconstitute the prenylated polarity hub Cdc42 and its regulatory protein in vitro, implementing a key membrane switch. We propose CFpPS to be a versatile and effective platform for engineering complex features, such as polarity induction, in synthetic cells.

Keywords: Cdc42; cell-free protein synthesis; prenylation; reversible membrane switch; synthetic biology; synthetic cell.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Schematic illustration of CFpPS and its application for the reconstitution of a reversible membrane switch. (a) Schematic of the reversible membrane switch process of Cdc42 via prenylation and regulator protein RhoGDI. (b) Co-translational prenylation was achieved by introducing prenyltransferase-enriched extract, the isoprenoid prenyl donor and the plasmid carrying the template of a target CAAX protein to the normal CFPS system. Newly expressed and prenylated proteins can either be directly incorporated into biomimetic membranes such as SLBs or solubilized with amphipathic reagents, such as detergents or nanodiscs. Prenylation efficiency can be monitored in real time through confocal microscopy with fluorescent fusion proteins or detected via in-gel fluorescence using fluorescent prenyl donors. Upon introduction of RhoGDI, a membrane switchable system could be established and monitored in real time.
Figure 2
Figure 2
Establishment of CFpPS for geranylgeranylation. (a) Schematic illustration of the chimeric proteins GST-CAAXCdc42 that are geranylgeranylated via purified GGTase-I- or GGTase-I-enriched extracts. (b) Titration of the NBD-FPP with purified GGTase-I using in-gel fluorescence. The last lane showed the competition assay performed by adding the unlabeled analogue—GGPP—at a concentration fivefold that of the highest tested for the NBD-modified analogue. Concentration (μM) of lipid donor in each reaction is stated above the corresponding gel lane. (c) Titration of GGTase-I-enriched extracts using in-gel fluorescence with10 μM GST-CAAXCdc42 and 80 μM NBD-FPP. Extract concentration is shown as percentage volume of the GGTase-I-enriched extract included in the standard E. coli CFPS. (d) Schematic depicting the expression and solubilization of prenylated CAAX proteins in CFpPS systems with or without solubilizing additives. (e) Prenylated GST-CAAXKrasB or GST-CAAXCdc42 demonstrates low solubility after co-translational prenylation in CFpPS extracts lacking solubilizing additives. 20/80 μM NBD-GPP/FPP were used and prenylated proteins were measured using in-gel fluorescence in the supernatant and the pellet fractions after centrifugation at 20,000g. Measurements were normalized to the mean total protein amount in both the pellet and soluble fractions for each protein. Symbols represent intensity measured in three independent replicates. (f) Concentration optimization of the GGTase-I-enriched extract in the CFpPS system using in-gel fluorescence. Extract concentration is shown as percentage volume of the enriched extract included in the standard E. coli CFPS. (g) In-gel fluorescence analysis for optimizing the concentration of NBD-modified lipid donor in the CFpPS system. (h) Screening of detergents for soluble expression of geranylgeranylated GST-CAAXCdc42. Respective control reactions were performed without any detergent. (i) Nanodisc titration for the soluble expression of GST-CAAXCdc42 in the CFpPS system. Fluorescence intensities of the protein band for each fraction were measured through in-gel fluorescence. Each image (b,c,f,g) includes a representative gel imaged in fluorescence mode to visualize NBD (upper) and colorimetric mode to visualize Coomassie staining (lower). In all graphs, intensity is normalized to the highest average value measured in a dataset. In all graphs (b,c,f,g), mean values from three independent replicates are shown as black dots, while the gray shading represents standard deviation, n = 3.
Figure 3
Figure 3
Prenylated mCherry-CAAXCdc42 produced using the CFpPS system binds to biomimetic membranes. (a) Schematic illustration of the membrane targeting of mCherry-CAAXCdc42, as studied on SLBs using confocal microscopy. (b) Orthogonal views of the SLB membrane (upper, green), mCherry-CAAXCdc42 (middle, magenta), and a merge of both channels (lower) at different time points after prenylation was initiated in the CFpPS reaction by adding GGPP. The SLB composition is 80% DOPC, 19.95% DOPS, and 0.05% Atto-488 PE. All scale bars are 10 μm. (c) Normalized intensities of corresponding images from (b). Intensities of mCherry-Cdc42 were normalized to maximum and minimum intensities recorded in the z-stack during the time-lapsed experiments; intensities of the membrane channel were normalized to maximum and minimum intensities recorded in the z-stack at each time point. (d) Time series of mCherry-CAAXCdc42 intensity on the membrane (pink) and in solution (black). Intensities were normalized to maximum and minimum intensities measured during a time-lapse experiment. Solid lines represent the mean intensity measured over a 75-pixel by 75-pixel region, and gray shading represents the standard deviation. Data are representative of three independent replicates.
Figure 4
Figure 4
Reconstitution of the reversible membrane-binding of mCherry-Cdc42 produced by the CFpPS system. (a) Schematic illustration of the reconstitution of Cdc42’s membrane targeting and RhoGDI-dependent membrane extraction on SLBs, as visualized by confocal microscopy. (b) Representative images of orthogonal views of the SLB membrane (upper, green), mCherry-Cdc42 (middle, magenta), and a merge of both channels (lower) at different time points. Time was measured from the addition of GGPP during membrane targeting and from the initiation of time-lapse imaging for the extractions process. All scale bars are 10 μm. (c) Normalized intensities of corresponding images from (b). Intensities of mCherry-Cdc42 were normalized to maximum and minimum intensities recorded in the z-stack during the time-lapsed experiments; intensities of the membrane channel were normalized to maximum and minimum intensities recorded in the z-stack at each time point. During extraction, the same normalization was performed for the membrane channel; while the intensities of mCherry-Cdc42 were normalized to the maximum intensity recorded in the z-stack at 3 min before the addition of RhoGDI. (d,e) Time series of mCherry-Cdc42 intensity on the membrane (pink) and in solution (black) during membrane targeting (d) and membrane extraction (e). Intensities were normalized to maximum and minimum intensities measured during a time-lapse experiment. Solid lines represent the mean intensity measured over a 75-pixel by 75-pixel region, and gray shading represents the standard deviation. Data are representative of three independent replicates. The lipid composition of SLBs used in this figure is 80% DOPC, 19.95% DOPS, and 0.05% Atto-488 PE.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Ganzinger K. A.; Schwille P. More from less - bottom-up reconstitution of cell biology. J. Cell Sci. 2019, 132, jcs227488.10.1242/jcs.227488. - DOI - PubMed
    1. Dzieciol A. J.; Mann S. Designs for life: protocell models in the laboratory. Chem. Soc. Rev. 2012, 41, 79–85. 10.1039/c1cs15211d. - DOI - PubMed
    1. Joyce G. F.; Szostak J. W. Protocells and RNA Self-Replication. Cold Spring Harbor Perspect. Biol. 2018, 10, a034801.10.1101/cshperspect.a034801. - DOI - PMC - PubMed
    1. Altenburg W. J.; Yewdall N. A.; Vervoort D. F. M.; van Stevendaal M. H. M. E.; Mason A. F.; van Hest J. C. M. Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells. Nat. Commun. 2020, 11, 6282.10.1038/s41467-020-20124-0. - DOI - PMC - PubMed
    1. Blanken D.; Foschepoth D.; Serrão A. C.; Danelon C. Genetically controlled membrane synthesis in liposomes. Nat. Commun. 2020, 11, 4317.10.1038/s41467-020-17863-5. - DOI - PMC - PubMed

Publication types