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Functional targeting of membrane transporters and enzymes to peroxisomes

Nature Chemical Biology volume 21, pages 1544–1553 (2025)Cite this article

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Abstract

Engineered peroxisomes hold promise as a highly versatile platform for compartmentalizing engineered metabolic pathways, insulating them from native cellular factors to prevent undesired crosstalk. However, native peroxisomes often lack the required substrates and cofactors in their lumen; accordingly, nonnative membrane proteins (MPs) must be recruited to the peroxisomal membrane to support heterologous pathways requiring these molecules. We developed a robust, modular ‘chauffeur’ strategy that enables MP folding in the endoplasmic reticulum (ER) followed by trafficking to the peroxisomal membrane through an engineered interaction in the cytosol with a transmembrane domain natively trafficked from the ER to the peroxisome. We demonstrate the modularity of this strategy by successfully redirecting multiple MP cargoes, including heterologous plant MPs, and observed increased titers for a monoterpene biosynthetic pathway. This strategy overcomes the challenges of misfolding and sorting of MPs to the peroxisome and, accordingly, expands the repertoire of pathways that can be compartmentalized into this organelle.

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Fig. 1: The engineered chauffeur strategy enables efficient and consistent trafficking of MP cargoes to the peroxisome.
Fig. 2: Engineered interactions with the transmembrane motif from Pex22p can traffic a heterologous MP from the ER to the peroxisome.
Fig. 3: Trafficked transporters retain activities upon relocation to the peroxisomal membrane.
Fig. 4: Trafficked membrane-embedded prenyltransferase to peroxisome with compartmentalized pathway producing GPP enables the efficient tailoring of bergaptol.
Fig. 5: Trafficked NST enables complete biosynthesis of geranyl β-d-glucoside in the peroxisome.

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Data availability

Plasmids generated in this study were deposited to Addgene (plasmids 239493–239551) and are available upon reasonable request. Data supporting the figures in this study were published to figshare (https://doi.org/10.6084/m9.figshare.28928222.v1)68. Source data are provided with this paper.

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Acknowledgements

This work was supported by National Science Foundation (NSF) grant MCB 1818307, NSF grant CBET 2104261 and the Center for Cellular Construction, an NSF Science and Technology Center, under grant agreement DBI-1548297. We thank members of the J.E.D. laboratory for valuable discussions and feedback throughout this project and members of the W. Zhang laboratory for assistance with LC–MS, particularly A. Del Rio Flores, Y. Ko and K. Jia.

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Author notes

  1. These authors contributed equally: Ka-Hei Siu, Victoria Lee.

Authors and Affiliations

  1. Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA

    Ka-Hei Siu, Victoria Lee & John E. Dueber

  2. UC Berkeley and UCSF Joint Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA, USA

    Victoria Lee

  3. NSF Center for Cellular Construction, University of California, San Francisco, San Francisco, CA, USA

    Victoria Lee & John E. Dueber

  4. Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    John E. Dueber

  5. QB3, University of California, Berkeley, Berkeley, CA, USA

    John E. Dueber

Authors
  1. Ka-Hei Siu
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Contributions

K.S. and J.E.D. designed the research. K.S. and V.L. conducted the chauffeuring of various cargoes, bergamottin biosynthesis and the esculin assay. K.S. additionally conducted the peptide ligand optimization assay using split fluorescent protein and biosynthesis of geranyl β-d-glucoside and 8HG. V.L. additionally conducted experiments for the MP orientation in the TEV protease assay. K.S., V.L. and J.E.D. wrote the paper and created the figures.

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Correspondence to John E. Dueber.

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The authors declare no competing interests.

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Nature Chemical Biology thanks Bei Gao, Jiazhang Lian and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The chauffeur strategy enables efficient trafficking of various MP cargoes to the peroxisome.

Venus-ePTS1 (YFP) was expressed to confirm the MP and the chauffeur localized to the peroxisome. (a) The yeast vacuolar MP, Vba2p, trafficked to peroxisomal membranes using the chauffeur strategy. In comparison, direct fusion of Vba2p to tPex22 did not result in efficient trafficking to the peroxisome; most of the Vba2p appeared to traffic to the vacuole. (b) CDT1p, a heterologous multispanning cellodextrin transporter from Neurospora crassa, trafficked to the peroxisome using the chauffeur strategy. The tPex22 direct fusion seemed to redirect CDT1p to the peroxisomal membrane; however, the total amount of CFP-fused MP successfully targeted to the peroxisome was much lower than achieved via the chauffeur strategy. Collectively, the results of trafficking Yea4p (Fig. 1b), Vba2p, and CDT1p suggest that the chauffeur strategy is a more robust method to redirect native and heterologous membrane proteins to the peroxisome. All scale bars represent 5 µm.

Source data

Extended Data Fig. 2 Activity and localization of trafficked candidate UDP-glucose transporters.

(a) Localization of non-trafficked Yea4p was compared to the ER labeled with a YFP (Venus-HDEL). In the dead SH3 peptide (dSH3) control and cases of inefficient trafficking, Yea4p co-localized with the ER-localized YFP signal as expected. (b) Esculin standard curve. Fluorescence at 460 nm plotted against esculin concentration generated the standard curve used to interpolate esculin titers for esculetin-to-esculin bioconversion assays. (c) Trafficked Yea4p activity was analyzed using the esculetin-to-esculin assay. Yea4p was trafficked via the engineered SH3 domain-SH3 peptide ligand protein-protein interaction. In the direct fusion strategies, the native protein truncations tPex22 and tPex15 were fused to either the N- or C-terminus of Yea4p, respectively. While tPex22-Yea4p and Yea4p-tPex15 resulted in weak and strong peroxisome localization (Fig. 1), respectively, low levels of activity were observed for both fusions. One-way ANOVA was used to compare the sample data between each group of 4 biological replicates. (d) NSTs trafficked to the peroxisome via the chauffeur strategy were tested for their UDP-glucose transport activities on the peroxisome membrane. Previous studies suggest that Yea4p and AtUTR1 exhibit transport activities for UDP-glucose while Hut1p and Ymd8p only exhibit marginal transport activities toward the co-factor. CDT2 is an unrelated membrane protein from Neurospora crassa that transports cellodextrins and is used as a negative control. (e) AtUTR1 trafficked with an active SH3 domain traffics some of the membrane protein to the peroxisome, while the dSH3 control results in protein that does not appear to associate with a yeast membrane. All scale bars represent 5 µm. All data shown are the mean ± s.e.m. of four biological replicates and all subfigures use the following significance: * denotes p-value < 0.05; ** p-value < 0.01; **** p-value < 0.001.

Source data

Extended Data Fig. 3 The orientation of chauffeured Yea4p was screened by a TEV protease assay.

Yea4p cargo was tracked by a CFP, and the chauffeur was fused to an RFP. (a) YFP followed by a TEV protease cleavage site was fused to the N-terminus of Yea4p. With and without TEV protease induction (with either 100 nM or 1000 nM β-estradiol), the YFP signal of this N-terminal fusion remained punctate and localized at the peroxisome, consistent with protection from the TEV protease from having a lumen-facing orientation throughout trafficking and within the peroxisome. (b) The C-terminus of Yea4p-2xSH3 was fused with a TEV protease cleavage site followed by a YFP. With this fusion and induction of TEV protease, the release of YFP to the cytoplasm was observed, indicating a cytosolic-oriented C-terminus. Based on traces of Yea4p-CFP signal residing on the ER membrane and the incomplete TEV protease cleavage at 100 nM β-estradiol, the YFP fusion may partially interfere with SH3 chauffeuring and TEV protease cleavage through steric hindrance. A higher concentration of 1000 nM β-estradiol resulted in more complete cleavage of YFP from peroxisomes. All scale bars represent 5 µm.

Source data

Extended Data Fig. 4 Trafficking of tCpPT1 using the peroxisome targeting sequence (PTS1) results in inefficient trafficking and non-functional membrane protein.

(a) Trafficking of tCpPT1 (Accession: A0A810JZE7 (https://www.uniprot.org/uniprotkb/A0A810JZE7/entry)) by a peroxisome targeting sequence (PTS1) was visualized using CFP (mTurquoise2) and co-localization with tPex22 fused to RFP (mRuby2). Using the same exposure times and contrast settings as the tCpPT1 trafficked via the chauffeur strategy (Fig. 4c), CFP fluorescence was not detectable (not shown). tCpPT1 signal with greatly enhanced contrast settings remained barely observable as very small CFP punctae that co-localized with RFP, suggesting poor expression and/or membrane protein misfolding and inefficient trafficking to the peroxisome. Scale bar represents 5 µm. (b) Bergamottin titers resulting from trafficking of tCpPT1 via the chauffeur strategy were compared to those from a previously reported method where a peroxisome targeting sequence (PTS1) is fused to the C-terminus of the membrane embedded enzyme. Colors are consistent with those used in Fig. 4c. The titers obtained using the chauffeur strategy are >20-fold higher than those achieved by PTS1 fusion from samples collected 24 hrs after addition of the bergaptol substrate, likely because import via PTS1 tag relies on soluble, cytosolic receptor proteins and has not evolved for functional membrane protein insertion into the peroxisome membrane. “Per” indicates the MP cargo was targeted via Crk SH3 domain-SH3 peptide ligand interaction; “Non-targeted” refers to the use of a non-binding SH3 peptide variant; and “PTS1” refers to the C-terminal fusion of peroxisome targeting sequence (-SKL). Data are shown as the mean ± s.e.m. of three biological replicates.

Source data

Extended Data Fig. 5 tCpPT1 localizes to the endoplasmic reticulum.

Fusion of CFP (mTurquoise2) to the N- or C-terminus of tCpPT1 results in localization to the ER. YFP (Venus) was fused to an N-terminal Ost1 signal sequence and a C-terminal HDEL retention sequence for targeting and retention in the ER. All images were taken at the same exposure settings. Controls (“No CFP” and “No YFP”) were performed to ensure there was no overlapping signal that would cause false signals from the fluorescent proteins used. Images were captured 4 h after 50-fold dilution in fresh media. All scale bars represent 5 µm.

Source data

Extended Data Fig. 6 Peroxisome-localized bergamottin production results in better host health and bergamottin titers compared to cytosolically-localized production.

(a) mTurquoise2 fluorescence of colonies used for bergamottin production in Fig. 5. As tCpPT1 was expressed as a fusion protein with mTurquoise2, the mTurquoise2 signal can be used as a proxy for protein expression. No significant differences in CpPT1 expression levels were observed with (SH3pep) versus without (dSH3pep) trafficking to the peroxisome. Two-way ANOVA was used. ns denotes not significant. Data are shown as the mean ± s.e.m. of three biological replicates. (b) A growth defect in yeast containing cytosolically-localized bergamottin biosynthesis (Cyt=pex5Δ) is observed when compared to peroxisome-compartmentalized (Per=WT) biosynthesis. OD600 values were taken at 72 h after dilution to an initial OD600 of 0.2. Two-way ANOVA was used. * denotes p-value < 0.05; *** p-value < 0.005. Data are shown as the mean ± s.e.m. of three biological replicates.

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Extended Data Fig. 7 Chromatograms of geraniol, geranyl β-D-glucoside, and extracted samples from fermentations of strains expressing the de novo biosynthetic pathway analyzed by LC-MS.

Note the higher product peak with both peroxisomal MVA pathway and peroxisome-targeted Yea4p compared to either non-targeted Yea4p or cytoplasmic MVA pathway.

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Extended Data Fig. 8 Trafficked cytochrome P450 and P450 reductase to the peroxisome retain activity in synthesis of (6E)-8-hydroxygeraniol.

(a) Schematic of C. roseus cytochrome P450 geraniol 8-hydroxylase (CrG8H) and C. roseus P450 reductase (CrCPR) trafficking to the peroxisome for 8HG biosynthesis. (b) GCMS data of 8HG titer. One-way ANOVA was used to compare the sample data between each group of 4 biological replicates. * denotes p-value < 0.05; *** p-value < 0.005; ns denotes not significant. Data are shown as the mean ± s.e.m. of four biological replicates. (c) CrG8H and (d) CrCPR were trafficked to the peroxisome using the chauffeur strategy. The p450 and p450 reductases were fused to BFP, and peroxisomes were labeled with a YFP-ePTS1. All scale bars represent 5 µm.

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Supplementary Figs. 1–3 and Table 1.

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Siu, KH., Lee, V. & Dueber, J.E. Functional targeting of membrane transporters and enzymes to peroxisomes. Nat Chem Biol 21, 1544–1553 (2025). https://doi.org/10.1038/s41589-025-01948-7

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