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
. 2017 Feb 15;36(4):441-457.
doi: 10.15252/embj.201694866. Epub 2016 Dec 21.

COPI-TRAPPII activates Rab18 and regulates its lipid droplet association

Chunman Li  1   2 , Xiaomin Luo  2 , Shan Zhao  2 , Gavin Ky Siu  2 , Yongheng Liang  3 , Hsiao Chang Chan  2   4 , Ayano Satoh  5 , Sidney Sb Yu  6   4
Affiliations

COPI-TRAPPII activates Rab18 and regulates its lipid droplet association

Chunman Li et al. EMBO J. .

Abstract

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII-specific subunits by various methods including siRNA depletion and CRISPR-Cas9-mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII-deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.

Keywords: COPI; TRAPPII; Rab18; TRAPPC10; TRAPPC9; lipid droplets.

PubMed Disclaimer

Figures

Figure 1
Figure 1. The TRAPPII complex binds preferentially to the nucleotide‐free form of Rab18

  1. Lysates from 293T cells transfected with the indicated DNA constructs were incubated with 1 mM Mg2+ plus 0.1 mM GTPγS, GDP, or 2 mM EDTA and immunoprecipitated with anti‐Myc antibody. Rab18‐HA co‐immunoprecipitated with Myc‐TRAPPC9 when it was nucleotide‐free (+EDTA).

  2. A mutant of Rab18 unable to bind nucleotide, N122I, was co‐immunoprecipitated with Myc‐TRAPPC9 by anti‐Myc antibody.

  3. HA‐tagged Rab1 and Rab18, but not Rab2, could co‐immunoprecipitate endogenous TRAPPII complex but not TRAPPIII complex.

  4. GST‐Rab18 could pull down endogenous TRAPPII complex in an in vitro binding assay. GST‐Rab18 was pre‐loaded with GTPγS or GDP or stripped of nucleotide by EDTA. Lysates from 293T cells were subjected to GST‐Rab18 pull‐down. The presence of various TRAPP subunits was detected by immunoblotting. TRAPPII complex, but not TRAPPIII complex, was co‐isolated with GST‐Rab18, most strongly with Rab18 in nucleotide‐free state.

Figure 2
Figure 2. TRAPPII complex stimulates guanine nucleotide exchange on Rab1 and Rab18
  1. A

    TRAPPII and TRAPPIII complexes were immunoprecipitated by antibodies specifically recognizing TRAPPC9 or TRAPPC12, and the presence of various co‐precipitating TRAPP subunits was detected by immunoblotting.

  2. B–D

    TRAPP‐stimulated guanine nucleotide exchange on purified Rab18, Rab1, and Rab2 GST fusion proteins measured by release of [3H]GDP that was pre‐loaded onto the Rab proteins.

  3. E–G

    Nucleotide exchange reactions measured by time‐dependent binding of [35S]GTPγS. Immunoprecipitates using the indicated antibodies were included in the assay as the GEF to be tested. In the [3H]GDP‐release experiments, the reactions were incubated for 30 min at 37°C before the Rab proteins were subjected to nitrocellulose filter binding. In the [35S]GTPγS binding experiments, the reactions were incubated for the indicated amount of time before filter binding.

  4. H, I

    Anti‐Myc antibody immunoprecipitates from lysates of HEK293T cells transfected with Myc‐TRAPPC9 were used as the GEF for GST‐Rab1 or GST‐Rab18 in 30‐min incubations at 37°C with [35S]GTPγS.

Data information: (B–I) Error bars = SD; n = 3.
Figure 3
Figure 3. Loss of TRAPPC9 function resulted in aberrantly large lipid droplets

  1. Depleting TRAPPC9, but not TRAPPC8, increased lipid droplet size in HEK293T cells. The efficiency of siRNA depletion of TRAPPC8 and TRAPPC9 in HeLa cells is shown by immunoblotting in the top left panel. Representative fluorescence images show the status of lipid droplets in these siRNA‐treated cells after oleic acid incubation. Scale bar = 10 μm.

  2. After loading with oleic acid, human skin fibroblasts containing a R475* mutation in TRAPPC9 accumulated lipid droplets of greater sizes than skin fibroblasts similarly isolated from a human individual with TRAPPC9 wild type. Scale bar = 10 μm.

Figure EV1
Figure EV1. Deletion of TRAPPII subunits does not affect the morphologies of the indicated cellular organelle markers or inhibit the internalization of transferrin
Wild‐type, TRAPPC9‐deleted (TRAPPC9−/−), TRAPPC10‐deleted (TRAPPC10−/−), or TRAPPC9 and TRAPPC10 doubly deleted (C9−/−;C10−/−) 293T cells were stained with antibodies for the labeled proteins. The cells were counter‐stained with DAPI (blue). Scale bars = 10 μm.

  1. ER marker calnexin.

  2. ER exit site marker Sec23.

  3. ERGIC marker ERGIC‐53.

  4. Golgi marker GM130.

  5. Internalization of rhodamine‐transferrin (red) for 5 min (left panels) and 15 min (right panels).

Figure EV2
Figure EV2. Deletion of TRAPPII subunits does not affect ER‐to‐Golgi traffic as monitored by the trafficking of GFP‐FM4‐CD8 protein
Transport of FM4‐CD8 out of the ER was initiated by applying the disaggregating drug AP21998 (D/D) to a final concentration of 2 μM for the indicated time (Lavieu et al 2013). Transport of GFP‐FM4‐CD8 to the Golgi (GM130) was monitored for up to 30 min in wild‐type and TRAPPII‐deleted (TRAPPC9−/−;TRAPPC10−/−) HEK293T cells. Scale bar = 10 μm.
Figure 4
Figure 4. Recruitment of Rab18 onto small lipid droplets was defective in TRAPPII‐deleted cells

  1. HEK293T cells with the indicated genotypes were loaded with 400 μM oleic acid for 24 h before staining with Bodipy 493/503. Scale bar = 10 μm.

  2. Wild‐type or TRAPPII‐deleted cells were loaded with oleic acid and stained with Bodipy 493/503 (green) and endogenous Rab18 by immunofluorescence (red). Scale bar = 10 μm.

  3. DsRed‐Rab18 was transfected into wild‐type (top panels and insets) or TRAPPII‐deleted HEK293T cells (bottom panels and insets) that were loaded with oleic acid and stained with Bodipy 493/503 (green). Scale bar = 10 μm.

  4. Homogenates of wild‐type or TRAPPII‐deleted cells were subjected to sucrose gradient fractionation. Fractions 1 and 2 were enriched with LDs. The presence of Rab18, TRAPPC9, TRAPPC2, LD marker TIP47, and ER marker calnexin was detected by SDS–PAGE followed by immunoblotting.

Figure EV3
Figure EV3. The ability of Rab18 to be associated with LD surface varies among different cell lines

  1. Endogenous Rab18 is enriched in the perinuclear region and colocalized largely with Golgi marker GM130 in cells grown in growth medium. Scale bars = 10 μm.

  2. The cells were incubated with 400 μM of oleic acid for 24 h before staining with Rab18. Rab18 signals on the Golgi were slightly reduced and became more dispersed throughout the cytoplasm in COS and HeLa cells, but were very poorly colocalized with LDs. In Huh‐7 cells, Rab18 signals redistributed to highly punctate structures which partially decorated the LDs. Scale bars = 10 μm.

  3. Overexpression of DsRed‐Rab18 slightly increased its association with LDs in HeLa but not in COS cells even after 48 h of oleic acid loading. Scale bars = 10 μm.

  4. Statistical quantification of the percentage of LDs decorated with Rab18 in each of the indicated cell lines. Gray bars = endogenous Rab18; red bars = overexpressed DsRed‐Rab18. Total number of LD fluorescence dots counted: n = 834, 1041, 670, and 383, for Huh‐7, COS, HeLa, and HEK293T, respectively, in the endogenous Rab18 group (gray bars). N = 860, 1119, and 469 for COS, HeLa, and HEK293T, respectively, in the DsRed‐Rab18‐transfected group (red bars). In each cell sample, the LD fluorescence dots were derived from at least 10 different cells.

Figure 5
Figure 5. Release of NEFA at various time points by 293T cells with the indicated genotypes or treatments
Control siRNA depletion (siCtrl) was conducted with siRNA oligos specific to a firefly luciferase sequence; siCOPB = depletion of β‐COP; siRab18 = depletion of Rab18. C9C10KO = HEK293T cells with TRAPPC9 and TRAPPC10 genes deleted. N = 3, error bars = SD.
Figure 6
Figure 6. Interaction between COPI and Rab18 is TRAPPII‐dependent
  1. A

    Myc‐TRAPPC9 directly interacted with FLAG‐γ‐COP. The presence of FLAG‐γ‐COP in immunoprecipitations of Myc‐TRAPPC9 and Myc‐TRAPPC10 from cell lysates of the indicated genetic backgrounds was investigated by immunoblotting.

  2. B, C

    Co‐immunoprecipitation between FLAG‐γ‐COP and HA‐Rab18 in HEK293T cells with indicated genetic backgrounds. The presence of FLAG‐γ‐COP in immunoprecipitates of HA‐Rab18 (and reciprocal IP in C) was investigated by immunoblotting.

  3. D

    The interaction between endogenous COPI and Rab18 was TRAPPII‐dependent. Endogenous Rab18 was immunoprecipitated by anti‐Rab18 antibody from lysates of indicated genetic background. The presence of γ‐COP and various TRAPP subunits was detected by their specific antibodies as indicated.

  4. E

    Co‐immunoprecipitation between TRAPPII and HA‐Rab18 in control (siFFL) and γ‐COP (si‐γ‐COP) siRNA‐depleted cells. TRAPPII was precipitated with transfected Myc‐TRAPPC10, and the presence of HA‐Rab18 was determined by immunoblotting.

  5. F

    Co‐immunoprecipitation between FLAG‐γ‐COP and TRAPPII in control (siFFL) and Rab18 (si‐Rab18) siRNA‐depleted cells. TRAPPII was precipitated with Myc‐TRAPPC10, and the presence of FLAG‐γ‐COP was determined by immunoblotting. The molecular weight of endogenous Rab18 overlaps with IgG light chain, and therefore, the amount of endogenous Rab18 precipitated with TRAPPII cannot be determined in this experiment.

Figure 7
Figure 7. Differential effect of COPI inhibition by siRNA depletion and BFA on Rab18 recruitment onto LD surface

  1. Huh‐7 cells were depleted with control siRNA specific to firefly luciferase sequence (top panels) or with siRNA specific to γ‐COP (bottom panels). Endogenous Rab18 (red) and LD (green) were stained and visualized. Scale bar = 10 μm.

  2. Huh‐7 cells were treated with or without BFA for 6 h before staining for Rab18 (red) and LD (green). Scale bar = 10 μm.

  3. TRAPPC9 were detected on Golgi but not LD surface. Huh‐7 cells were permeabilized with digitonin before fixation and staining. LDs were labeled with Bodipy 493/503 and pseudocolored in white. TRAPPC9 (red) and LD surface marker ADRP (green) were detected by their respective antibodies. Scale bar = 10 μm.

Figure 8
Figure 8. Time‐dependent relocations of COPI, TRAPPII, and Rab18 onto LD surface upon oleic acid incubation
Huh‐7 cells were first serum‐starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493/503, TRAPPC9, Rab18, and γ‐COP.

  1. In the merged images, Bodipy 493/503 was pseudocolored in green, TRAPPC9 in red, and ADRP in blue. In the colocalization between TRAPPC9 and ADRP on the right side, the ADRP signal was re‐colored to green for easy visualization. Scale bar = 10 μm.

  2. Bodipy 493/503 was pseudocolored in green, Rab18 in red, and γ‐COP in blue. In the colocalization between Rab18 and γ‐COP on the right side, the γ‐COP signal was re‐colored to green for easy visualization. Scale bar = 10 μm.

Figure EV4
Figure EV4. TRAPPC9 localization on LD surface was investigated as a function of time after oleic acid incubation
Huh‐7 cells were first serum‐starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493/503, TRAPPC9, and ADRP. In the merge images, Bodipy 493/503 was pseudocolored in green, TRAPPC9 in red, and ADRP in blue. In the colocalization between TRAPPC9 and ADRP on the right side, the ADRP signal was re‐colored to green for easy visualization and overlapped signals around the LDs became yellow. Scale bar = 10 μm.
Figure EV5
Figure EV5. COPI and Rab18 localizations were investigated as a function of time after oleic acid incubation
Huh‐7 cells were first serum‐starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493/503, Rab18, and γ‐COP. In the merged images, Bodipy 493/503 was pseudocolored in green, Rab18 in red, and γ‐COP in blue. In the colocalization between Rab18 and γ‐COP on the right side, the γ‐COP signal was re‐colored to green for easy visualization. Scale bar = 10 μm.
Figure EV6
Figure EV6. Subcellular localization of Rab3GAP1 and GM130 in response to oleic acid incubation and TRAPPII deletion

  1. Rab3GAP1 and GM130 were stained after Huh‐7 cells were serum‐starved (top panels), and then incubated with oleic acid for 8 and 24 h. Bodipy 493/503 was pseudocolored in green, Rab3GAP1 in red, and GM130 in blue. Rab3GAP1 signals remained perinuclear and significantly colocalized with Golgi marker GM130 regardless of oleic acid incubation. Scale bar = 10 μm.

  2. Rab3GAP1 and GM130 were stained after HEK293T cells of indicated genetic background were incubated with oleic acid for 24 h. TRAPPII deletion did not change the perinuclear signal of Rab3GAP1. Rab3GAP1 was not associated with LDs in any of the cells shown. Scale bar = 10 μm.

Figure 9
Figure 9. Recruitment of Rab18 onto LD is dependent on COPI
Huh‐7 cells were first starved with serum‐free medium and then incubated with oleic acid for 12 h. Condition 1 (control): The cells were not treated with BFA at any time point (left column). Condition 2: The cells were incubated with both 5 μg/ml of BFA and oleic acid incubation for 6 h and then in oleic acid alone for 10 h (middle column). Condition 3: The cells were treated with 5 μg/ml of BFA starting at 12 h after oleic acid incubation (right column). Scale bar = 10 μm.
Figure 10
Figure 10. Hypothesis of how COPI and TRAPPII activate Rab18 and promote the association of Rab18 on LD surface
COPI relocates from Golgi to LD surface or relocates to the surface of ER tubules via retrograde transport. The interaction between COPI and TRAPPII allows the GEF activity of TRAPPII to activate Rab18. Rab18‐GTP becomes LD‐associated and promotes the formation of ER–LD bridge so that lipid metabolizing enzymes can access the content of LD.
See this image and copyright information in PMC

Comment in

  • TRAPPing Rab18 in lipid droplets.
    Zappa F, Venditti R, De Matteis MA. Zappa F, et al. EMBO J. 2017 Feb 15;36(4):394-396. doi: 10.15252/embj.201696287. Epub 2017 Jan 27. EMBO J. 2017. PMID: 28130247 Free PMC article.

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aligianis IA, Johnson CA, Gissen P, Chen D, Hampshire D, Hoffmann K, Maina EN, Morgan NV, Tee L, Morton J, Ainsworth JR, Horn D, Rosser E, Cole TRP, Stolte‐Dijkstra I, Fieggen K, Clayton‐Smith J, Megarbane A, Shield JP, Newbury‐Ecob R et al (2005) Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome. Nat Genet 37: 221–224 - PubMed
    1. Barrowman J, Bhandari D, Reinisch K, Ferro‐Novick S (2010) TRAPP complexes in membrane traffic: convergence through a common Rab. Nat Rev Mol Cell Biol 11: 759–763 - PubMed
    1. Bassik Michael C, Kampmann M, Lebbink Robert J, Wang S, Hein Marco Y, Poser I, Weibezahn J, Horlbeck Max A, Chen S, Mann M, Hyman Anthony A, LeProust Emily M, McManus Michael T, Weissman Jonathan S (2013) A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility. Cell 152: 909–922 - PMC - PubMed
    1. Beller M, Sztalryd C, Southall N, Bell M, Jackle H, Auld DS, Oliver B (2008) COPI complex is a regulator of lipid homeostasis. PLoS Biol 6: e292 - PMC - PubMed
    1. Bem D, Yoshimura S‐I, Nunes‐Bastos R, Bond Frances F, Kurian Manju A, Rahman F, Handley Mark TW, Hadzhiev Y, Masood I, Straatman‐Iwanowska Ania A, Cullinane Andrew R, McNeill A, Pasha Shanaz S, Kirby Gail A, Foster K, Ahmed Z, Morton Jenny E, Williams D, Graham John M, Dobyns William B et al (2011) Loss‐of‐function mutations in RAB18 cause Warburg micro syndrome. Am J Hum Genet 88: 499–507 - PMC - PubMed

Publication types

MeSH terms

Substances