Membrane-Bound Protein Scaffolding in Diverse Hosts Using

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Membrane-bound protein scaffolding in diverse hosts using thylakoid protein CURT1A James B. Y. H. Behrendorff, Omar A. Sandoval-Ibañez, Anurag Sharma, and Mathias Pribil ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00418 • Publication Date (Web): 18 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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ACS Synthetic Biology

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Membrane-bound protein scaffolding in diverse hosts using thylakoid protein CURT1A James B. Y. H. Behrendorff, Omar A. Sandoval-Ibañez, Anurag Sharma, Mathias Pribil* Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark *Corresponding author, email: [email protected]

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ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Membrane-bound protein scaffolding in diverse hosts using thylakoid protein CURT1A James B. Y. H. Behrendorff, Omar A. Sandoval-Ibañez, Anurag Sharma, Mathias Pribil* Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark *Corresponding author, email: [email protected] ORCID James B. Y. H. Behrendorff: 0000-0003-4130-6252 Omar A. Sandoval-Ibañez: 0000-0002-4513-1704 Anurag Sharma: 0000-0001-5345-8830 Mathias Pribil: 0000-0002-9174-9548

Abstract Protein scaffolding is a useful strategy for controlling the spatial arrangement of cellular components via protein-protein interactions. Protein scaffolding has primarily been used to co-localise soluble proteins in the cytoplasm, but many proteins require membrane association for proper function. Scaffolding at select membrane domains would provide an additional level of control over the distribution of proteins within a cell and could aid in exploiting numerous metabolic pathways that contain membrane-associated enzymes. We developed and characterised a membrane-bound protein scaffolding module based on the thylakoid protein CURT1A. This scaffolding module forms homo-oligomers in the membrane, causing proteins fused to CURT1A to cluster together at membrane surfaces. It is functional in diverse expression hosts and can scaffold proteins at thylakoid membranes in chloroplasts, endoplasmic reticulum in higher plants and Saccharomyces cerevisiae, and the inner membrane of Escherichia coli.

Keywords: protein scaffolding, enzyme, chloroplast, membrane targeting, protein expression

Synthetic protein scaffolding is an emerging approach for enhancing biocatalysis through enzyme colocalization1, the creation of proteinaceous microcompartments akin to synthetic organelles2, and for designing synthetic signalling responses3. Many enzymes such as eukaryotic cytochrome P450 enzymes4 and fatty acid elongases5 require membrane association for optimal performance, and some metabolic pathways with highly lipophilic substrates or products, such as saponins6 or prostaglandins7, may benefit from membrane association. Proteins can be targeted to membrane surfaces with hydrophobic signal peptides or via post-translational modifications such as prenylation8 or fatty acylation9, but these membrane targeting mechanisms are not necessarily transferable between different host systems. Additionally, simply targeting proteins to a common membrane surface does not bring them into sufficiently close proximity to yield the benefits of true scaffolding approaches where multi-enzyme


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metabolic complexes (metabolons) are formed10; protein-protein interactions that result in oligomers are required to create a scaffolding effect. In naturally-occurring membrane-associated metabolons, interacting enzymes are typically defined by distances of less than 10 nm11. We sought to develop a modular membrane anchor that promotes protein scaffolding at membrane surfaces and can be reliably reused and repurposed in diverse host systems. We identified the Curvature Thylakoid1A (CURT1A) protein12 from thylakoid membranes of Arabidopsis thaliana as a suitable candidate for developing a membrane-bound protein scaffolding module. CURT1A is a small (11.2 kDa) integral membrane protein consisting of two stroma-facing amphipathic helices linked by two transmembrane helices required for membrane targeting and insertion13. CURT1A self assembles into membrane-anchored high-molecular weight oligomers in thylakoid grana margins, where the oligomers contribute to membrane bending and regulation of the thylakoid ultrastructure12, 14. Because of its small size, and capacity for oligomerization, we hypothesised that CURT1A would be useful as a membranetargeting scaffolding module that can be fused to soluble or membrane-associated proteins. We characterised the scaffolding properties of CURT1A in four systems: in higher plants with and without targeting to chloroplasts, and in Saccharomyces cerevisiae and Escherichia coli.

Results and Discussion Mechanisms for processing protein-encoded membrane targeting information can differ between organelles and between groups of organisms15, 16. Consequently, a membrane-targeting peptide that functions in one system may not function in another. To identify scaffolding modules that integrate into membranes in diverse hosts,, we designed a set of five reporter proteins where a fluorescent protein (mCitrine17) was fused to variants of mature CURT1A (i.e. lacking the native chloroplast transit peptide) via a GGGGS flexible linker (Figure 1). mCitrine was attached to the CURT1A N-terminus (CURT_fluoA) or Cterminus (CURT_fluoB), or to the N- or C-termini of the CURT1A transmembrane domains (CURT_fluoC and CURT_fluoD). A double fusion construct with mCitrine at the N-terminus and mApple18 at the C-terminus of CURT1A (CURT_fluoE) was also constructed, and we tested whether these proteins retain the membrane integration and scaffolding properties of CURT1A in thylakoid membranes, and whether these properties can also be exploited in heterologous environments. We examined the expression and membrane localization of these five reporter constructs and found that Cterminal fusions to CURT1A (i.e. the CURT_fluoB reporter design) resulted in expression and membrane integration in all four host systems that we tested. We verified that a large enzyme, β-glucuronidase (GUS, 71 kDa), can be targeted to membranes via CURT1A fusion without loss of function, and that CURT1A drives protein scaffolding in native and non-native membranes. Scaffolding was assessed with three approaches: 1) identification of protein complexes via two-dimensional blue-native polyacrylamide gel electrophoresis (2D-BN-PAGE), 2) chemical crosslinking of protein complexes with bis(sulfosuccinimidyl)suberate (BS3; a membrane-impermeable crosslinking reagent with an 11.4 Å spacer arm), and 3) identifying localised fusion protein accumulation with fluorescence microscopy.



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Scaffolding in chloroplast thylakoid membranes: CURT1A fusion proteins retain native CURT1A scaffoldforming properties Expression and membrane integration: For chloroplast targeting, we added the chloroplast transit peptide of RuBisCO small subunit 1A from A. thaliana to the N-terminus of all CURT_fluo constructs (CTPCURT_fluoA-E). When expressed in tobacco leaves via Agrobacterium-mediated transient transfection, CTPCURT_fluoA accumulated in chloroplasts to greater concentrations than CTP-CURT_fluoB, but the difference was not statistically significant (n = 3, Student’s t-test p = 0.15) (Figures 2A, S1). CTP-CURT_fluoE accumulated less than CTP-CURT_fluoB (n = 3, Student’s t-test p < 0.05). Weak expression of CTPCURT_fluoC and CTP-CURT_fluoD was observable with fluorescence microscopy (Figure S1) but the total extractable fluorescence was not significantly different from negative controls (n = 3, one-way ANOVA p = 0.08). CTP-CURT_fluoC and CTP-CURT_fluoD were not investigated further due to their low expression levels. We tested the strength of membrane integration by treating membranes with alkaline and chaotropic salts, which promote dissociation of loosely integrated membrane proteins. CTP-CURT_fluoA, CTP-CURT_fluoB, and CTP-CURT_fluoE all integrated into thylakoid membranes as tightly as native CURT1A12, with only minor quantities of overexpressed protein released after sodium thiocyanate treatment (Figure S2A). The mCitrine reporter used in these studies has a mass of 27 kDa — smaller than many useful enzymes. We fused a GUS reporter enzyme (71 kDa) to the C-terminus of CURT1A (CTP-CURT_GUS, i.e. in the same arrangement as CTP-CURT_fluoB) to test whether CURT1A can still localize to membranes when fused to a much larger soluble protein, and to test whether a soluble enzyme can still fold and function correctly when fused to CURT1A. Fusing GUS to the C-terminus of CURT1A resulted in a 45-fold enrichment of GUS activity in the membrane fraction (Figure 2B, n = 3, Student’s t-test p < 0.005). GUS activity from membrane bound CURT-GUS activity was similar to activity measured from the soluble GUS enzyme in the stromal fraction, indicating a high capacity for incorporating CURT-fused enzymes into thylakoid membranes (activity normalised to total protein concentration, Student’s t-test p = 0.42). CTP-CURT_fluoB oligomerization in thylakoid membranes: Solubilised thylakoid membranes were separated via 2D-BN-PAGE. Immunoblotting revealed that CTP-CURT_fluoB exists in a continuum of low- to highmolecular weight complexes in the thylakoid membrane, showing that CTP-CURT_fluoB retains the same capacity for complex formation as native CURT1A in A. thaliana12 (Figure 2C). When thylakoid membrane proteins were cross-linked with BS3, we detected monomers, homodimers, homotrimers, and homotetramers of CTP-CURT_fluoB (Figure 2D). The specificity of these multimers supports previous findings that CURT1A complex formation is driven by CURT1A-CURT1A interactions12. The use of BS3 as a cross-linking reagent demonstrates that the stromal-exposed components (i.e. mCitrine or the CURT1A amphipathic helices) must be within 11.4 Å of each other — a shorter distance than can be resolved with most fluorescent protein FRET pairs19. CTP-CURT_fluoA and CTP-CURT_fluoE also formed multimers in thylakoid membranes (Figure S2B), demonstrating that fusing soluble proteins to either end of CURT1A does not prevent native-like oligomerization in thylakoids.

Scaffolding in higher plants without organelle targeting: CURT1A C-terminal fusion proteins target the endoplasmic reticulum


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Untargeted expression and membrane integration: The CURT_fluo fusion proteins lacking the chloroplast transit peptide were expressed in tobacco leaves via Agrobacterium-mediated transient transfection. CURT_fluoA expression was greater than CURT_fluoB and CURT_fluoE (n = 3, Student’s t-test p < 0.005 and p < 0.05, respectively) (Figure 3A). As was the case with chloroplast-targeted expression, only weak expression was detectable for the CURT_fluoC and CURT_fluoD designs and the extractable fluorescence from CURT_fluoD was indistinguishable from negative controls (n = 3, Student’s t-test p = 0.6). Only CURT_fluoB showed promise as a scaffolding module when subcellular localization was taken into consideration (Figure S3). CURT_fluoA and CURT_fluoC were present in the cytosol and nucleus, similar in appearance to soluble mCitrine expression. CURT_fluoB was absent from nuclei and formed a distinct filamentous pattern at the cell periphery. CURT_fluoD was also excluded from the nucleus, like CURT_fluoB, but had very weak expression. Co-expression of CURT_fluoB with an mOrange218-tagged Derlin120 protein revealed that CURT_fluoB integrates into the endoplasmic reticulum (ER) (Figure S4). CURT_fluoE showed a mixed phenotype: it was excluded from the nucleus but did not form the same filamentous pattern as CURT_fluoB. The expression patterns of CURT_fluoA and CURT_fluoC indicate stable expression of the fluorescent protein but a lack of ER membrane integration, meaning that proteins must be fused to the Cterminus of CURT1A for insertion into the ER (i.e. CURT1A acts as a hydrophobic ER-targeting leader sequence). This follows the paradigm that most ER signal sequences are hydrophobic motifs located at the N-terminus of the protein, and that these motifs are recognised co-translationally by the eukaryotic signal recognition particle (SRP) for trafficking to the ER21. The β-glucuronidase activity of CURT_GUS fusion proteins was enriched more than 200-fold in the microsomal membrane fraction (n = 3, Student’s t-test p < 0.0005), indicating successful folding and ERmembrane integration of CURT1A-fused β-glucuronidase (Figure 3B). CURT_fluoB oligomerization in N. benthamiana ER. Microsomes containing ER were extracted from leaves expressing CURT_fluoB. Separation of solubilised microsomes via 2D-BN-PAGE revealed a continuum of low- to high-molecular weight CURT_fluoB complexes (Figure 3C), as we had observed with chloroplasttargeted CURT_fluoB in thylakoid membranes. BS3-crosslinked dimers, trimers, and tetramers of scaffolded CURT_fluoB were also identifiable via immunoblotting after separation via SDS-PAGE (Figure 3D). These results demonstrate that proteins fused to the C-terminus of CURT1A integrate into the ER and retain the oligomer-forming characteristics of CURT1A even though the lipid composition of the ER (primarily phospholipids22) is substantially different to thylakoids (primarily digalactosyl- and monogalactosyl diacylglycerols23).

Saccharomyces cerevisiae: C-terminal fusions to CURT1A scaffold in clusters in the peripheral endoplasmic reticulum Expression and membrane integration. CURT_fluoB and CURT_fluoD expressed almost twice as strongly as the soluble mCitrine control on a per biomass basis (Figure 4A; n = 3, one-way ANOVA p < 0.05). Strains expressing CURT_fluoB and CURT_fluoD did not exhibit any observable growth penalty (Figure S5A), indicating that these anchors are useful for stable accumulation of very high concentrations of protein. Fluorescence from CURT_fluoB and CURT_fluoD accumulated with an uneven distribution suggestive of membrane integration (Figures 4B, S6). Contrastingly, CURT_fluoA, -C, and -E expression produced either



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weak, evenly distributed fluorescence (CURT_fluoC) or small fluorescent foci (CURT_fluoA, CURT_fluoE) (Figure S6). Fluorescence from CURT_fluoA, -C, and -E was only observable with fluorescence microscopy and was indistinguishable from the negative control in whole-cell measurements (Figure 4A, S6), indicating that CURT_fluoA, -C, and -E are degraded and that soluble N-terminal domains cannot be expressed fused to CURT1A in S. cerevisiae24. Co-expression of an mCherry-tagged Erg3p25 with CURT_fluoB or CURT_fluoD showed that CURT_fluoB and CURT_fluoD both localise to elements of the ER network, albeit with different distribution (Figure S7A). CURT_fluoD is distributed evenly in the nuclear and cortical ER, while CURT_fluoB localisation has minimal overlap with the nuclear ER but accumulates in large patches in the cortical ER. The erg3-mCherry fluorescent signal takes on the same shape as the CURT_fluoB fluorescent signal, suggesting a significant modification to the ER ultrastructure in regions of CURT_fluoB accumulation. The β-glucuronidase activity of CURT_GUS fusion proteins was enriched 13-fold in the membrane fraction (n = 3, Student’s t-test p < 0.0005), indicating successful folding and ER-membrane integration of CURT1Afused β-glucuronidase (Figure 4C). Furthermore, membrane bound CURT_GUS activity was more than twice that of soluble GUS activity, reinforcing the observation that CURT1A-fusion enhances protein expression in S. cerevisiae (Student’s t-test p < 0.005). CURT_fluoB oligomerization in S. cerevisiae ER. Monomers, dimers, trimers, and tetramers of CURT_fluoB were detectable in S. cerevisiae microsomes after chemical crosslinking with BS3, whereas CURT_fluoD did only formed minor quantities of cross-linkable dimers (Figure 4D). CURT1A-mediated scaffolding is possible in the ER of S. cerevisiae, primarily in cortical regions (Figure S7), but one or both of the N- and C-terminal amphipathic helices of CURT1A are necessary for efficient oligomerization. The low concentration of CURT_fluoD dimers observed in crosslinking experiments could be a consequence of crowding due to protein overexpression rather than active oligomerization, and reinforce that the formation of highmolecular weight complexes is due to CURT1A-CURT1A interactions. To test whether multiple CURT1A-fused proteins could be co-localised in complexes, we designed expression constructs to co-express CURT_fluoB with a CURT1A-mCerulean3 fusion protein, where mCerulean326 is fused to the C-terminal of CURT1A in the same manner as mCitrine in CURT_fluoB. An alternate codon usage was designed for CURT1A-mCerulean3 to avoid homologous recombination between the two CURT1A coding sequences. Co-expression of CURT_fluoB and CURT1A-mCerulean3 resulted in overlapping co-localisation of mCerulean3 and mCitrine fluorescent signals (Figure S8A) and a 66 % increase in apparent FRET (compared with co-expression of soluble mCerulean3 and mCitrine, Figure S8B, n = 3 p < 0.0005).

Escherichia coli: C-terminal fusions to CURT1A scaffold in clusters in the inner membrane Expression and membrane integration in E. coli: CURT_fluoA and CURT_fluoB both accumulate when expressed in E. coli but with distinct phenotypes. The total fluorescent signal from CURT_fluoA and CURT_fluoB in whole cells is comparable to the soluble mCitrine control on a per biomass basis (Figure 5A at 19 h; n = 3, one-way ANOVA p = 0.24) but not in absolute terms because CURT_fluoB has a negative impact on E. coli growth (Figure S5B). Accumulation of CURT_fluoB eventually halts E. coli growth but the culture remains metabolically active as evidenced by continued synthesis and accumulation of the


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CURT_fluoB protein. CURT_fluoB proteins accumulate in localised patches at the inner membrane (Figure 5B) that grow in size as the culture progresses. Contrastingly, CURT_fluoA fluorescence forms a thin stripe at the cell periphery indicating inner membrane integration but primarily on one side of the cell rather than evenly around the entire periphery as was observed elsewhere with native E. coli membrane proteins DjlC and Flk27. As CURT_fluoA protein accumulates, the fluorescent signal adopts a twisting stripe shape. This distinct phenotype may be due to CURT_fluoA having weaker oligomerization interactions than CURT_fluoB. Analysis of mCitrine in subcellular fractions by fluorescence quantification (Figure 5C) and immunoblotting (Figure 5D) revealed that CURT_fluoA and CURT_fluoB were both highly enriched in the membrane fraction along with multiple degradation products indicating protein turnover, particularly of CURT_fluoB. CURT_fluoE expressed at lower levels than CURT_fluoA or CURT_fluoB, while CURT_fluoC and CURT_fluoD were barely detectable. CURT_GUS expressed in E. coli was enriched in the membrane fraction in comparison to GUS expressed without fusion to CURT1A, but the majority of GUS and CURT_GUS activity was detected in the cytosolic fraction (Figures 6A, S9). This shows that CURT_GUS is incorporated into the E. coli inner membrane but that the rate of protein overexpression exceeds the capacity for membrane integration in this strain28. Membrane-associated CURT_GUS activity increased over time but at a slower rate than cytosolic CURT_GUS activity (Figure S9). The opportunity to accumulate high concentrations of protein at the membrane means that CURT_fluoB design may still have practical utility in E. coli despite the negative impact on growth and high protein turnover. We tested whether CURT1A can function as a membrane anchor for expressing a more challenging class of proteins, using a human liver cytochrome P450 enzyme (P450), CYP2C19, as a model. The CYP2C19 sequence used in this study has a modified N-terminal sequence required for successful expression in E. coli29. Mammalian P450s often require modifications to the hydrophobic N-terminal membrane anchor or other mutations or bespoke fermentation conditions to produce correctly-folded holoenzyme in E. coli30, 31. We tested two constructs where: 1) CURT1A was fused directly to the N-terminus of CYP2C19, and 2) the hydrophobic N-terminal anchor region of CYP2C19 (amino acids 1-25) was removed and replaced with CURT1A, and measured P450 expression in whole cells using carbon monoxide difference spectroscopy (ligand-binding assay that directly measures the concentration of correctly-folded P450 holoenzyme32). CURT1A-fused CYP2C19 expressed as a correctly folded holoenzyme both when fused directly to the C-terminus of CURT1A and when the CYP2C19 N-terminal membrane anchor was replaced with CURT1A, and the specific production of folded CYP2C19 ([P450] per biomass) was enhanced by >50 % (Figure 6B). Inner membrane protein scaffolding in E. coli: CURT_fluoA and CURT_fluoB fusion proteins have distinct scaffolding and membrane association phenotypes in E. coli. Crosslinking and immunoblot of CURT_fluoB in E. coli membranes revealed complexes with similar molecular weights to the anticipated homo-oligomers, but also additional bands corresponding to degradation products of CURT_fluoB, and potentially heterocomplexes between CURT_fluoB and native E. coli membrane proteins. Contrastingly, only monomers and dimers were observed in CURT_fluoA crosslinking experiments, along with potential degradation products and non-specific interactions (Figure 6C). Co-expression of CURT_fluoB and CURT1A_mCerulean3 resulted in co-localisation of the two CURT1A-fused proteins and produced a detectable FRET interaction, but the negative impact on growth was severe (Figure S10).



Implications for designing membrane bound protein scaffolds The position of the fused partner protein (e.g. mCitrine in this study) has a pronounced effect on CURT1A expression and oligomerization, and this differs between expression hosts. N- and C-terminal fusions expressed well and formed oligomers in the native context of thylakoid membranes, but fusion to the Cterminus of CURT1A was necessary for high expression levels and oligomerization in heterologous systems. In heterologous eukaryotic expression systems, CURT1A fusion proteins integrated into ER membranes and retained their capacity for self-recognition and oligomerization without causing observable growth defects. Additionally, overexpression of CURT_fluoD in S. cerevisiae without oligomerization showed that the CURT1A N- and C- terminal amphipathic helices are required for CURT1A-driven protein scaffolding. S. cerevisiae was the only host in which CURT_fluoD could be expressed to significant levels. Weak expression of CURT_fluoC and CURT_fluoD both in E. coli and in N. benthamiana suggests that these proteins are targeted for degradation. It is unlikely that weak expression of CURT_fluoC is due to transcriptional or translational inefficiencies because CURT_fluoA and CURT_fluoC begin with the same mCitrine coding sequence. The highly hydrophobic transmembrane helices positioned at either the N- or Cterminus in CURT_fluoC and CURT_fluoD may serve as protein turnover signals33. Phenotypes resulting from different CURT_fluo designs are particularly divergent in E. coli, which has only a single inner membrane into which CURT1A-fused proteins can integrate. Attachment of proteins to the CURT1A N-terminus results in membrane integration but apparently limited oligomerization, while fusions to the C-terminus result in large membrane-associated complexes that impede normal growth, and an apparent saturation of the membrane’s capacity for accommodating heterologous membrane proteins. Despite a strong growth impediment and evidence that CURT_fluoB is subject to degradation in E. coli, fusion to the C-terminus of CURT1A still increased membrane-targeted expression of GUS and CYP2C19 in E. coli. However, E. coli is not a preferred host for creating CURT1A-scaffolded heterocomplexes due to the severe impact on growth when two CURT1A-fused proteins were co-expressed. Overexpression of CURT1A without any fusion partner in E. coli resulted in filamentous growth, suggesting that CURT1A accumulation affects the function of native E. coli membrane proteins at new cellular poles (Figure S11). CURT1 homologues appear to have specialised functional roles at poles in filamentous cyanobacteria34 and may also contribute to chloroplast vesicle fusion in A. thaliana35. Previous studies of membrane-bound scaffolding have focused on developing systems that function in one specific membrane. For example, fusing enzymes to the twin-arginine translocase complex in thylakoid membranes allows for up to three different soluble or membrane-associated proteins to be scaffolded in heterocomplexes in a 1:1:1 ratio at non-appressed thylakoid membranes36. In another example, soluble enzymes can be scaffolded at the surface of lipid droplets by fusion to oleosin proteins and the use of synthetic cohesin-dockerin interactions37. Our work expands on the available options for membranetargeted protein scaffolding with a simple, modular unit that is deployable in a wide variety of biological contexts. Anchoring protein scaffolds to membranes provides additional spatial control over protein and metabolite distribution within the cell, particularly if the scaffold concentrates in membrane sub-domains. Natively, CURT1A occupies the thylakoid grana margin, which contains relatively little catalytic activity compared


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with the rest of the thylakoid membrane38. Expressing CURT1A-fused catalytic enzymes in the chloroplast presents an opportunity to decorate a discrete region of the thylakoid with scaffolded enzyme complexes. Similarly, CURT_fluoB expressed in S. cerevisiae defined its own membrane domain. It preferentially accumulated in large clusters in the cortical ER and distorted the membrane into a new morphology without negatively affecting growth, presenting an opportunity to create new ER-associated catalytic regions within the cell. The use of CURT1A as a modular, self-oligomerizing membrane anchor presents an exciting opportunity to engineer new membrane functions and morphologies.

Materials and Methods Complete materials and methods including full details of molecular cloning, organism cultivation, protein expression and enzymatic assays are included in Supplementary file 1. Several plasmids produced in this study are available at Addgene (www.addgene.org) (Supplementary table 1). Briefly, proteins were expressed in N. benthamiana via Agrobacterium-mediated transient transfection, in S. cerevisiae encoded on galactose-inducible expression plasmids, and in E. coli encoded on arabinose-inducible expression plasmids. Expression was measured by fluorescence detection (BioTek Synergy H1 plate reader [BioTek Instruments, Inc., USA]), and confocal microscopy (Leica SP5X confocal laser scanning microscope [Leica Microsystems GmbH, Germany]). Thylakoid membrane integration was tested with salt washing39 and analysed via immunoblotting. CURT1A-driven oligomerization was assessed using 2D-BN-PAGE and chemical crosslinking with bis(sulfosuccinimidyl)suberate (BS3).

Author contributions JBYHB designed the experiments, constructed the strains, prepared microbial subcellular fractions, performed the enzyme assays and protein expression and confocal microscopy experiments. OASI prepared chloroplast and leaf tissue fractions, and performed 2D-BN-PAGE, chemical crosslinking and immunoblotting experiments. AS contributed to microscopy experiments. MP conceived of the study, secured the funding and contributed to experimental design. All authors contributed to writing the manuscript.

Acknowledgements Project funding was provided by the Novo Nordisk Foundation (NNF15OC0016586) and the Copenhagen Plant Science Centre funded by the University of Copenhagen. Confocal laser scanning microscopy facilities were provided by the Center for Advanced Bioimaging (CAB) at the Department for Plant and Environmental Sciences, University of Copenhagen. Anurag Sharma was supported by a postdoctoral fellowship awarded by the Carlsberg Foundation. We thank our colleagues Dr Julie Zedler, Dr David Russo, and Dr Silas Mellor for their constructive feedback.

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Figure 1 Schematic representations of the CURT_fluo fusion proteins tested in this work. CURT1A is shown in green with the N- and C-terminal (N, C) and transmembrane (TM) helices indicated. mCitrine and mApple fluorescent proteins are shown in yellow and red, respectively. Molecular weights are displayed below each construct.



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Figure 2 Expression, function, and oligomerization of chloroplast-targeted CURT1A-fusion proteins. (A) Transient expression of chloroplast-targeted CTP-CURT_fluo proteins in tobacco leaves. Fusion proteins were prepared by grinding leaf tissue in an extraction buffer and quantified by measuring mCitrine fluorescence (λex 514 nm, λem 529 nm) in the extract supernatant. Fluorescence was normalised to tissue fresh weight (n = 3 technical replicates, mean ± SD). CTP-CURT_fluoA and CTP-CURT_fluoB expressed at similarly-high concentrations (Student’s t-test p = 0.15). CTPCURT_fluoE accumulated less than CTP-CURT_fluoB (Student’s t-test p < 0.05). CTP-CURT_fluoC and CTP-CURT_fluoD expression were similar to negative controls (one-way ANOVA p = 0.08). Inset: empty vector negative control and soluble mCitrine positive control. (B) Membrane enrichment of CURT1A-fused β-glucuronidase (GUS). Chloroplasttargeted GUS was expressed in tobacco leaves without () or with () fusion to the C-terminus of CURT1A. Metabolism of 4-methylumbelliferone glucuronide was measured in stromal and membrane chloroplast fractions (n = 3 technical replicates, mean ± SD, ** = p < 0.005). (C) Thylakoid membrane protein complexes from tobacco expressing either CTP-CURT_fluoB or the soluble control CTP-mCitrine were separated via two-dimensional blue native page (2D-BN-PAGE). CTP-CURT_fluoB and soluble CTP-mCitrine were identified immunoblotting with an antiGFP primary antibody. High-to-low molecular weight complexes are shown from left to right. (D) Chemical crosslinking of thylakoid-bound CTP-CURT_fluoB. BS3-treated (+) or untreated (-) membrane samples were separated via SDS-PAGE for immunoblotting with an anti-GFP primary antibody. The stromal fraction from tobacco expressing CTP-mCitrine was included for comparison. Bands corresponding to anticipated homo-oligomers in the crosslinked CTP-CURT_fluoB sample are indicated (). ACS Paragon Plus Environment

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Figure 3 Expression, function, and oligomerization of CURT1A-fusion proteins in leaves without organelle targeting. (A) Transient expression of CURT_fluo proteins in tobacco leaves. Fusion proteins were prepared by grinding leaf tissue in an extraction buffer and quantified by measuring mCitrine fluorescence (λex 514 nm, λem 529 nm) in the extract supernatant. Fluorescence was normalised to tissue fresh weight (n = 3 technical replicates, mean ± SD). CURT_fluoB expression was less than CURT_fluoA (Student’s t-test p < 0.005) and similar to CURT_fluoE (Student’s ttest p = 0.06). CURT_fluoD fluorescence was indistinguishable from negative controls (Student’s t-test p = 0.6). Inset: empty vector negative control and soluble mCitrine positive control. (B) Membrane enrichment of CURT1A-fused βglucuronidase (GUS). GUS was expressed in tobacco leaves without (, primary y-axis) or with (, secondary y-axis) fusion to the C-terminus of CURT1A. Metabolism of 4-methylumbelliferone glucuronide was measured in the cytosolic and membrane fractions (n = 3 technical replicates, mean ± SD, *** = p < 0.0005). (C) Microsomal membrane protein complexes from tobacco expressing either CURT_fluoB or the soluble mCitrine control were separated via two-dimensional blue native page (2D-BN-PAGE). CURT_fluoB and soluble mCitrine were identified immunoblotting with an anti-GFP primary antibody. High-to-low molecular weight complexes are shown from left to right. (D) Chemical crosslinking of membrane-bound CURT_fluoB. BS3-treated (+) or untreated (-) membrane samples were separated via SDS-PAGE for immunoblotting with an anti-GFP primary antibody. The cytosolic fraction from tobacco expressing mCitrine was included for comparison. Bands corresponding to anticipated homo-oligomers in the crosslinked CURT_fluoB sample are indicated ().



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Figure 4 Expression, function, and oligomerization of CURT1A-fusion proteins in S. cerevisiae. (A) Expression of CURT_fluo fusion proteins in S. cerevisiae. mCitrine fluorescence (λex 500 nm, λem 529 nm) is normalised to optical density (OD600 nm) (n = 3 biological replicates, mean ± SD) Key: pESC empty vector (), mCitrine (), CURT_fluoA (), CURT_fluoB (), CURT_fluoC (), CURT_fluoD (), CURT_fluoE (). (B) Visualization of CURT_fluoB, mCitrine, and CURT_fluoD expressed in S. cerevisiae. Samples were taken from expression cultures at 5, 12.5, and 24 h and imaged via confocal laser scanning microscopy (λex = 510 nm, λem = 525-552 nm). Fluorescence from mCitrine or CURT_fluo fusion proteins is colored yellow. Scale bar = 2 μm. (C) Membrane enrichment of CURT1A-fused β-glucuronidase (GUS). GUS was expressed in S. cerevisiae without () or with () fusion to the C-terminus of CURT1A. Metabolism of 4methylumbelliferone glucuronide was measured in the cytosolic and membrane fractions (n = 3 technical replicates, mean ± SD, *** = p < 0.0005). (D) Chemical crosslinking of microsomal CURT_fluoB, soluble mCitrine, and microsomal CURT_fluoD expressed in S. cerevisiae. BS3-treated (+) or untreated (-) membrane samples were separated via SDS-PAGE for immunoblotting with an anti-GFP primary antibody. Bands corresponding to anticipated homo-oligomers in the crosslinked CURT_fluoB sample are indicated ().


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Figure 5 Expression and membrane integration of CURT1A-fusion proteins in E. coli. (A) Expression of CURT_fluo fusion proteins in E. coli. mCitrine fluorescence (λex 500 nm, λem 529 nm) is normalised to optical density (OD600 nm) (n = 3 biological replicates, mean ± SD). Key: pBAD empty vector (), mCitrine (), CURT_fluoA (), CURT_fluoB (), CURT_fluoC (), CURT_fluoD (), CURT_fluoE (). (B) Visualization of CURT_fluoB, mCitrine, and CURT_fluoA expressed in E. coli. Samples were taken from expression cultures at 5, 12.5, and 24 h and imaged via confocal laser scanning microscopy (λex = 510 nm, λem = 525-552 nm). Fluorescence from mCitrine or CURT_fluo fusion proteins is colored yellow. Additionally, the cell envelope was stained with FM4-64 fluorescent dye (λex = 515 nm, λem = 628-694 nm, colored pink). Scale bar = 2 μm. (C) Fluorescent signal from CURT_fluo fusion proteins was analysed in 20,000 g supernatant (clarified lysate, ) and 180,000 g pellet (membranes, ) fractions of E. coli. Subcellular fractions were prepared from auto-inducing expression cultures of E. coli harvested 16 h after inoculation. CURT_fluoA and CURT_fluoB expression is compared to soluble mCitrine expression, and the empty pBAD expression vector is included as a negative control (n = 3 technical replicates, mean ± standard deviation). (D) Immunoblot of membrane (180,000 g pellet) and soluble (clarified lysate, 20,000 g supernatant) fractions. Proteins from membrane (M) and soluble (S) fractions were separated via denaturing SDS-PAGE and immunoblotted with an anti-GFP primary antibody.



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Figure 6 Function and oligomerization of CURT1A-fusion proteins in E. coli. (A) Membrane enrichment of CURT1A-fused β-glucuronidase (GUS). GUS was expressed in E. coli without () or with () fusion to the C-terminus of CURT1A. Metabolism of 4-methylumbelliferone glucuronide was measured in the cytosolic and membrane fractions harvested from auto-inducing cultures at the indicated timepoints (n = 3 technical replicates, mean ± SD, * = p < 0.05, ** = p < 0.005, *** = < 0.0005). GUS activity is expressed as RFU/min/μg protein. (B) Expression of cytochrome P450 CYP2C19 fused to CURT1A either directly at the N-terminus of CYP2C19 (CURT_2C19), or replacing the CYP2C19 N-terminal membrane anchor (CURT_Δ25-2C19). Folded P450 holoenzyme was quantified in whole E. coli cells via CO-difference spectroscopy (n = 3 biological replicates, mean ± 1 standard deviation, * = p < 0.05 [Student’s t-test]). (C) Chemical crosslinking of membrane-bound CURT_fluoB, soluble mCitrine, and membrane-bound CURT_fluoA expressed in E. coli. BS3-treated (+) or untreated (-) membrane samples were separated via SDS-PAGE for immunoblotting with an anti-GFP primary antibody. Bands corresponding to anticipated homo-oligomers in the crosslinked CURT_fluoB sample are indicated ().


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Supporting information Membrane-bound protein scaffolding in diverse hosts using thylakoid protein CURT1A Behrendorff et al.

Supporting file 1: Materials and methods, supporting tables and figures Supporting file 2: Oligonucleotide primers, construct sequences, and template DNA references

Materials and methods Supporting table 1 Plasmids from this study available at Addgene. Supporting figure S1 Chloroplast-targeted expression of CURT_fluo fusion proteins in tobacco leaves. Supporting figure S2 Chloroplast-targeted CURT_fluo fusion proteins CTP-CURT_fluoA, -B, and -E integrate into thylakoid membranes and form oligomers. Supporting figure S3 Expression of CURT_fluo fusion proteins in tobacco leaves without chloroplast targeting. Supporting figure S4 CURT_fluoB associates with the endoplasmic reticulum in tobacco leaves. Supporting figure S5 Growth of S. cerevisiae and E. coli expressing CURT_fluo fusion proteins. Supporting figure S6 Expression of CURT_fluo fusion proteins in Saccharomyces cerevisiae. Supporting figure S7 CURT_fluoB and CURT_fluoD interact with nuclear and cortical endoplasmic reticulum in Saccharomyces cerevisiae. Supporting figure S8 Colocalization of two CURT1A-fused proteins in S. cerevisiae. Supporting figure S9 β-glucuronidase expression with and without CURT1A-fusion in E. coli. Supporting figure S10 Colocalization of two CURT1A-fused proteins in E. coli. Supporting figure S11 Expression of CURT1A alone causes filamentous growth of E. coli. Supporting figure S12 Schematic examples of plasmids used in this study.


Membrane-Bound Protein Scaffolding in Diverse Hosts Using

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