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Intracellular lipid surveillance by small G protein geranylgeranylation

Abstract

Imbalances in lipid homeostasis can have deleterious effects on health1,2. Yet how cells sense metabolic demand due to lipid depletion and respond by increasing nutrient absorption remains unclear. Here we describe a mechanism for intracellular lipid surveillance in Caenorhabditis elegans that involves transcriptional inactivation of the nuclear hormone receptor NHR-49 through its cytosolic sequestration to endocytic vesicles via geranylgeranyl conjugation to the small G protein RAB-11.1. Defective de novo isoprenoid synthesis caused by lipid depletion limits RAB-11.1 geranylgeranylation, which promotes nuclear translocation of NHR-49 and activation of rab-11.2 transcription to enhance transporter residency at the plasma membrane. Thus, we identify a critical lipid sensed by the cell, its conjugated G protein, and the nuclear receptor whose dynamic interactions enable cells to sense metabolic demand due to lipid depletion and respond by increasing nutrient absorption and lipid metabolism.

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Fig. 1: Transcriptional inactivation of NHR-49 via cytosolic sequestration to endocytic transport vesicles.
Fig. 2: Adaptation to loss of RAB-11.1 by rab-11.2 activation.
Fig. 3: Lipid depletion activates rab-11.2 transcription through NHR-49.
Fig. 4: Geranylgeranyl synthesis and conjugation to RAB-11.1 are required for NHR-49 cytosolic inactivation.

Data availability

All data generated and analysed during this study are included in this article and its Supplementary Information and are also available from the authors upon request. Transcriptomic data files that support the findings of this study in C. elegans have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession GSE189106Source data are provided with this paper.

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Acknowledgements

We thank A. Ghazi for the NHR-49::GFP (AGP33a) worm strain; M. Zerial for the PEPT-1::DsRed worm strain (MZE1); SunyBiotech for generation and validation of the rab-11.2(syb2999) (PHX2999) and GFP::RAB-11.1 (PHX4435) worm strains; the Caenorhabditis Genetics Center (CGC) for worm strains; A. Lemoff and the UT Southwestern Medical Center Mass Spectrometry Core for LC-MS/MS support; and M. Buszczak and M. Douglas for critical review of the manuscript. We acknowledge S. Schmid, K. Dean, R. Debose-Boyd, D. Russell and J. Goldstein for critical discussions. This work has been funded by the Clayton Foundation for Research, the Welch Foundation (I-2061-20210327), the NIH (R00AG042495, R01AG061338, and R56AG070167) and the Cancer Prevention Research Institute of Texas (RR150089). Schematics were created with BioRender.com.

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization: A.W. and P.M.D. Methodology: A.W., L.T., N.W., S.L.B.A., R.S.F., I.D., C.D.C., J.G.M. and P.M.D. Investigation: A.W., L.T., N.W., S.L.B.A., R.S.F., S.T.B., P.M., M.M. and K.R.Z. Writing, review and editing: A.W., S.L.B.A. and P.M.D. Funding acquisition, resources and supervision: P.M.D.

Corresponding author

Correspondence to Peter M. Douglas.

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The authors declare no competing interests.

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Nature thanks Peter Espenshade and the other, anonymous, reviewers for their contribution to the peer review of this work. Peer review reports are available.

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Extended data figures and tables

Extended Data Fig. 1 RAB-11.1-dependent cytosolic inactivation of NHR-49 in nutrient-rich conditions.

a, Representative micrograph of day 3 adult intestinal epithelia in transgenic worm expressing apical marker, ACT-5::GFP. Immunostaining for DAPI marks chromosomal DNA within nuclei. Reference Fig. 1b. Scale = 10 µm. n = 4 trials. b, Representative micrographs of Oil-Red-O-stained day 1 adult worms fed ad libitum (control) or starved for 24 h during late larval stages of development. n = 3 trials. c,d, Representative micrographs (c) and relative mean fluorescence by flow cytometry (d) of intestinal lipid droplet marker, DHS-3::GFP, in day 2 adults fed ad libitum (control, black) or starved for 24 h (grey). Scale = 25 µm. Mean ± SEM, ****p < 0.0001 by two-tailed unpaired t-test. n = 3 trials (circles). e,f, Fluorescence micrographs (e) and relative mean fluorescence by flow cytometry (f) of the acs-2p::GFP transcriptional reporter in day 1 adults fed ad libitum (control) or starved for 24 h. Scale = 200 µm. Mean ± SEM, ***p < 0.0001 by two-tailed unpaired t-test. n = 3 trials (circles). g, Gene Ontology (GO) term analysis from DAVID database. Plot displays the most over-represented biological processes (based on FDR) from proteins enriched by NHR-49::GFP co-immunoprecipitation and detected by liquid chromatography tandem mass spectrometry (LC-MS/MS). h, Representative confocal micrographs of NHR-49::GFP localization in day 1 adult intestinal epithelia in empty vector, EV, control or rab-11.1 RNAi conditions. Scale = 10 µm. i,j, Fluorescence micrographs (i) and relative mean fluorescence by flow cytometry (j) of the acs-2p::GFP reporter in L4 larvae on EV RNAi (black) or RNAi for the respective Rab GTPases (green). Scale = 200 µm. Mean ± SEM, ***p = 0.0003 by one-way ANOVA with Dunnett’s multiple comparisons test. n = 3 trials (circles).

Source data

Extended Data Fig. 2 Adaptation to loss of RAB-11.1 through rab-11.2 transcriptional induction.

a, Phenotype enrichment analysis (top 20) of genes differentially regulated (p < 0.05) upon rab-11.1 RNAi. b, Amino acid sequence alignment for RAB-11.1 and RAB-11.2. c,d, Fluorescence micrographs (c) and relative mean fluorescence by flow cytometry (d) of the rab-11.2p::YFP transcriptional reporter in day 1 adults fed ad libitum (control, black) or starved for 24 h (grey). Scale = 200 µm. Mean ± SEM, ***p < 0.0001 by two-tailed unpaired t-test. n = 5 trials (circles). e, Schematic displays the rab-11.2 gene and the respective rab-11.2(syb2999) mutation (731 base pair, bp, deletion). Blue box = exon, blue line = intron. f-i, Representative micrographs (f,h) and relative mean fluorescence by flow cytometry (g,i) of apical transporters, PGP-3::mCherry (f,g) and PEPT-1::DsRed (h,i), in L4 larvae and day 3 adults on EV (black) or rab-11.1 (green) RNAi. Scale = 25 µm. Mean ± SEM, ****p < 0.0001 (g) and **p = 0.0058, ****p < 0.0001 (i) by two-way ANOVA with Sidak’s multiple comparisons test, ns = no significance. n = 3 trials for each timepoint (circles). j, Confocal micrographs of phenotypes representative of TRITC-BSA/FM4–64 absorption efficiency classification categories. Scale = 25 µm. k, Categorization of FM4–64 absorption efficiency following its dietary supplementation in day 2 adult wild type and rab-11.2(syb2999) worms on EV or rab-11.1 RNAi. Shown is the relative ratio of animals which absorbed 0–25% (dark grey), 25–75% (medium grey), or 75–100% (light grey) of ingested FM4–64 into the intestinal epithelium. Mean ± SEM, ****p < 0.0001 by Chi-square test. n = 64 animals per condition over 2 trials. l, Representative micrographs of Oil-Red-O-stained day 5 adult wild type, WT, and rab-11.2(syb2999) worms on EV or rab-11.1 RNAi. n = 3 trials.

Source data

Extended Data Fig. 3 Lipid depletion activates rab-11.2 through NHR-49.

a,b, Fluorescence micrographs of the rab-11.2p::YFP reporter in L4 larvae on empty vector, EV, control RNAi or RNAi for transcriptional and enzymatic effectors of various metabolic regulatory pathways (a) or critical enzymes in different lipid metabolism pathways (b). Reference Fig. 3a, b. Scale = 200 µm. c, Representative confocal micrographs of intestinal GFP::RAB transgene association with early, late, or recycling endocytic vesicles in day 2 adults on EV or acs-1 RNAi. Scale = 25 µm. n = 3 trials. d-i, Representative micrographs (d,f,h) and relative mean fluorescence by flow cytometry (e,g,i) of lipid droplet marker, DHS-3::GFP, (d,e) or apical transporters, PEPT-1::DsRed (f,g) and PGP-3::mCherry (h,i), in the intestines of day 3 adults on EV (black) or acs-1 (blue) RNAi. Scale = 25 µm. Mean ± SEM, **p = 0.0045 (e), *p = 0.0173 (g), and **p = 0.0031 (i) by two-tailed unpaired t-test. n = 4 trials in e and 3 trials in g and i (cirlces). j, Representative confocal micrographs of NHR-49::GFP localization in day 1 adults on EV or acs-1 RNAi. Scale = 10 µm. k,l, Relative mean fluorescence by flow cytometry (k) and fluorescence micrographs (l) of the acs-2p::GFP reporter in day 1 adults on EV (black) or acs-1 (blue) RNAi. Mean ± SEM, *p = 0.0360 by two-tailed unpaired test. n = 3 trials (circles). Scale = 200 µm. m, Relative mean fluorescence by flow cytometry of the rab-11.2p::YFP reporter in wild type or nhr-49(nr2041) loss-of-function mutant day 1 adults on EV (black) or acs-1 (blue) RNAi. Mean ± SEM, **p = 0.0032, ***p = 0.0006, ns = no significance by two-way ANOVA with Tukey’s multiple comparisons test. n = 3 trials (circles). n, Fluorescence of the rab-11.2p::YFP reporter by flow cytometry of wild type and nhr-49(et13) gain-of-function mutant day 1 adults on EV (black) or nhr-49 (red) RNAi. Mean ± SEM, ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. From left to right, n = 633, 989, 499, and 553 animals (dots) over 3 trials.

Source data

Extended Data Fig. 4 Inhibition of geranylgeranyl pyrophosphate synthesis or transfer to Rab GTPases is sufficient for pathway activation.

a,b, Fluorescence micrographs (a) and relative fluorescence by flow cytometry (b) of the rab-11.2p::YFP transcriptional reporter in day 1 adults on empty vector, EV, RNAi (black) or RNAi for enzymes involved in mitochondrial acyl-CoA import (cpt-2/cpt-3) and citrate export (K11H3.3) (grey). Scale = 200 µm. Mean ± SEM, ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. n = 999 (EV), 211 (cpt-2), 1399 (cpt-3) and 1546 (K11H3.3) animals (dots) across 3 trials. c, Relative mean DHS-3::GFP fluorescence by flow cytometry of day 3 adults on EV RNAi (black) or RNAi for enzymes in different lipid metabolism pathways (blue). Reference Fig. 3a, b. Mean ± SEM, *p = 0.0143, ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test, ns = no significance. n = 3 trials (circles). d, Fluorescence micrographs of the rab-11.2p::YFP reporter in day 1 adults on EV RNAi or RNAi for enzymes involved in prenol lipid synthesis and transfer to small G proteins. Reference Fig. 4a, b. Scale = 200 µm. e, Relative fold change in transcript abundance of the farnesyl and geranylgeranyl transferase holoenzyme subunits involved in small G protein prenylation upon rab-11.1 RNAi. Reads per kilobase million, RPKM, standardized relative to EV RNAi control. Mean ± SEM, ***p = 0.0005 and 0.0004 by two-way ANOVA with Sidak’s multiple comparisons test. n = 3 biological replicates (circles). f, Heatmap displays the fold change in transcript abundance of Rab GTPases in primary human microvascular endothelial cells treated with either atorvastatin or atorvastatin + mevalonate in relation to vehicle (DMSO) control. Scale corresponds to relative fold change. Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test, *p = 0.0169 for RAB11A, *p = 0.0450 for RAB38, and **p = 0.0078 for RAB13 for transcripts differentially regulated by atorvastatin treatment in a mevalonate-dependent fashion. Reference Supplementary Table 1 for summary of statistics. n = 3 replicates. g, Relative mean fluorescence by flow cytometry of the rab-11.2p::YFP reporter in wild type or nhr-49(nr2041) day 1 adults on EV (black), hmgr-1 (orange), or ggtb-1 (red) RNAi. Reference Fig. 4c. Mean ± SEM, *p = 0.0125, ***p = 0.0004 and 0.0009, ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. n = 5 trials for EV and 4 trials for hmgr-1 and ggtb-1 RNAi (circles). h,i, Representative micrographs of NHR-49::GFP localization (h) and percent of intestinal epithelia with nuclear-localized NHR-49::GFP (i) in L4 larvae on EV (black), hmgr-1 (orange), or ggtb-1 (red) RNAi. Scale = 25 µm. Median with quartiles, ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. n = 50 animals per condition over 3 trials. j,k, Fluorescence micrographs (j) and relative mean fluorescence by flow cytometry (k) of the acs-2p::GFP reporter in day 1 adults on EV (black), hmgr-1 (orange), or ggtb-1 (red) RNAi. Scale = 200 µm. Mean ± SEM, ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. n = 4 trials (circles).

Source data

Extended Data Fig. 5 Ectopic geranylgeraniol supplementation restores vesicular trafficking and absorptive defects in a GGTB-1-dependent manner.

a, Representative micrographs of intestinal GFP::RAB-11.1 localization in day 2 adults on empty vector, EV, acs-1, hmgr-1, or ggtb-1 RNAi ± 1mM GGOH. Scale = 25 µm. n = 3 trials. b,c, Representative micrographs (b) and relative mean fluorescence by flow cytometry (c) of PEPT-1::DsRed expression in day 4 adults on EV (black), hmgr-1 (orange), or ggtb-1 (red) RNAi ± 1mM GGOH. Scale = 25 µm. Mean ± SEM, **p = 0.0029, ***p = 0.0008 and 0.0002, ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test, ns = no significance. n = 3 trials (circles). d,e, TRITC-BSA absorption into the intestinal epithelium following its dietary supplementation in day 3 adults cultured at 25 °C on EV (black), hmgr-1 (orange), or ggtb-1 (red) RNAi ± 1mM GGOH. Representative micrographs (d) and proportion which absorbed 75–100% TRITC-BSA (e). Scale = 25 µm. Mean ± SEM, ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. n = 5 trials for EV and 4 trials for hmgr-1 and ggtb-1 RNAi (circles). f,g, Representative micrographs (f) and relative mean fluorescence by flow cytometry (g) of DHS-3::GFP in day 3 adults on EV (black), hmgr-1 (orange), or ggtb-1 (red) RNAi ± 1mM GGOH. Scale = 25 µm. Mean ± SEM, ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. n = 5 trials for EV and 4 trials for hmgr-1 and ggtb-1 RNAi (circles). h, Representative micrographs of Oil-Red-O-stained day 5 adults cultured at 25 °C on EV, hmgr-1, or ggtb-1 RNAi ± 1mM GGOH. n = 3 trials. i,j, Western blot analysis of GFP::RAB transgene prenylation in EV (black) and hmgr-1 (orange) RNAi conditions. Unprenylated (‘U’, top band) and prenylated (‘P’, bottom band) GFP::RABs with tubulin loading control (i) and proportion of total GFP::RAB signal in prenylated form (j). For gel source data, see Supplementary Fig. 1. Mean ± SEM, ****p < 0.0001 by two-way ANOVA with Sidak’s multiple comparisons test. n = 3 trials (circles). k, Quantification of geranylgeranylated GFP::RAB-11.1 band intensity relative to total GFP::RAB-11.1 signal from western blot analysis of GFP::RAB-11.1 mobility in EV (black), acs-1 (blue), and hmgr-1 (orange) RNAi conditions ± 1mM GGOH. Reference Fig. 4e. Mean ± SEM, *p = 0.0133, ***p = 0.0001, ****p < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. n = 3 trials (circles). l,m, Representative micrographs (l) and relative mean fluorescence by flow cytometry (m) of PEPT-1::DsRed in day 4 adults on EV (black) or acs-1 (blue) RNAi ± 1mM GGOH. Scale = 25 µm. Mean ± SEM *p = 0.0283 and 0.0489 by two-way ANOVA with Tukey’s multiple comparisons test. n = 2 trials (circles). n,o, Representative micrographs (n) and relative mean fluorescence by flow cytometry (o) of DHS-3::GFP in day 3 adults on EV (black) or acs-1 (blue) RNAi ± 1mM GGOH. Scale = 25 µm. Mean ± SEM, *p = 0.0217, **p = 0.0033 by two-way ANOVA with Tukey’s multiple comparisons test. n = 4 trials (circles).

Source data

Extended Data Fig. 6 Geranylgeranyl availability and conjugation to RAB-11.1 are required for NHR-49 cytosolic inactivation.

a, Percent of intestinal epithelia with nuclear-localized NHR-49::GFP in day 1 adults on empty vector, EV, hmgr-1, or ggtb-1 RNAi supplemented with vehicle control (white) or 1mM squalene (grey), oleic acid (light grey), or geranylgeraniol (GGOH) (dark grey). Median with quartiles, ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test, ns = no significance. n = 90 animals per condition over 3 trials. b,c, Relative mean fluorescence by flow cytometry (b) and fluorescence micrographs (c) of the rab-11.2p::YFP reporter in L4 larvae on EV, hmgr-1, or ggtb-1 RNAi supplemented with vehicle control (white) or 1mM squalene (grey), oleic acid (light grey), or GGOH (dark grey). Mean ± SEM, ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. n = 4 trials (circles). Scale= 200 µm. d, Relative mean fluorescence by flow cytometry of the acs-2p::GFP reporter in day 1 adults on EV (black), hmgr-1 (orange), or ggtb-1 (red) RNAi ± 1mM GGOH. Mean ± SEM, **p = 0.0023, 0.0050 and ***p = 0.006, 0.004 by two-way ANOVA with Tukey’s multiple comparisons test. n = 3 trials (circles). e, Percent of intestinal epithelia with nuclear-localized NHR-49::GFP in day 1 adults on EV (black) or acs-1 (blue) RNAi ± 1mM GGOH. Median with quartiles, ****p < 0.0001 by two-way ANOVA with Tukey’s multiple comparisons test. n = 55 animals per condition over 3 trials. f-h, Fluorescence micrographs (f) and relative mean fluorescence by flow cytometry (g,h) of the rab-11.2p::YFP (f,g) or acs-2p::GFP (h) reporters in day 1 adults on EV (black) or acs-1 (blue) RNAi ± 1mM GGOH. Scale = 200 µm. Mean ± SEM, ****p < 0.0001 (g) and *p = 0.0222, **p = 0.0030 (h) by two-way ANOVA with Tukey’s multiple comparisons test. n = 3 trials (circles). i, Fluorescence micrographs of the rab-11.2p::YFP reporter in L4 larvae on EV (black) or rab-11.1 (green) RNAi ± 1mM GGOH. Scale = 200 µm. j, Relative mean fluorescence by flow cytometry of the acs-2p::GFP reporter in L4 larvae on EV (black) or rab-11.1 (green) RNAi ± 1mM GGOH. Mean ± SEM, p = 0.0014, ns = no significance by two-way ANOVA with Tukey’s multiple comparisons test. n = 3 trials (circles). k, Representative confocal micrographs of mRuby2::RAB-11.1WT (wild type, top) or mRuby2::RAB-11.1C208A,C209A (prenylation motif mutant, bottom) localization within day 1 adult intestinal epithelia. Scale = 25 µm. l, Relative mean fluorescence by flow cytometry of day 1 adult transgenic worms expressing mRuby2::RAB-11.1WT (red) or mRuby2::RAB-11.1C208A,C209A (blue) compared to non-transgenic control (grey). Mean ± SEM, **p = 0.0024, ***p = 0.0005 by one-way ANOVA with Tukey’s multiple comparisons test. n = 3 trials (circles).

Source data

Extended Data Fig. 7 Model of intracellular lipid surveillance pathway.

In conditions of ample resources (blue, left), NHR-49 is sequestered to endocytic vesicles in a RAB-11.1 geranylgeranylation-dependent manner. Upon lipid depletion (red, right), NHR-49 translocates to the nucleus and activates a transcriptional program to restore lipid homeostasis and nutrient absorption.

Extended Data Table 1 Enrichment status of endocytosis (KEGG pathway cel04144) proteins in NHR-49::GFP immunoprecipitation (IP)/LC-MS/MS dataset (relative to non-transgenic control)

Supplementary information

Supplementary Information

This file contains Supplementary Figs. 1, 2, Supplementary Tables 1–3 and Discussion.

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Supplementary Data 1

Peptide enrichment in NHR-49–GFP and N2 non-transgenic control immunoprecipitation analysis by liquid chromatography tandem mass spectrometry.

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Watterson, A., Tatge, L., Wajahat, N. et al. Intracellular lipid surveillance by small G protein geranylgeranylation. Nature 605, 736–740 (2022). https://doi.org/10.1038/s41586-022-04729-7

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