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. 2019 Feb 1;11(2):107-117.
doi: 10.1093/jmcb/mjy030.

ACBD3 is required for FAPP2 transferring glucosylceramide through maintaining the Golgi integrity

Jing Liao  1 , Yuxiang Guan  1 , Wei Chen  1 , Can Shi  1 , Dongdong Yao  1 , Fengsong Wang  1 , Sin Man Lam  2 , Guanghou Shui  2 , Xinwang Cao  1
Affiliations

ACBD3 is required for FAPP2 transferring glucosylceramide through maintaining the Golgi integrity

Jing Liao et al. J Mol Cell Biol. .

Abstract

Glycosphingolipid (GSL) metabolism is involved in various physiological processes, including all major cell signaling pathways, and its dysregulation is linked to some diseases. The four-phosphate adaptor protein FAPP2-mediated glucosylceramide (GlcCer) transport for complex GSL synthesis has been studied extensively. However, the molecular machinery of FAPP2 as a GlcCer-transferring protein remains poorly defined. Here, we identify a Golgi-resident protein, acyl-coenzyme A binding domain containing 3 (ACBD3), as an interacting partner of FAPP2. We find that ACBD3 knockdown leads to dramatic Golgi fragmentation, which subsequently causes FAPP2 dispersal throughout the cytoplasm and a decreased localization at trans-Golgi network. The further quantitative lipidomic analysis indicates that ACBD3 knockdown triggers abnormal sphingolipid metabolism. Interestingly, the expression of siRNA-resistant full-length ACBD3 can rescue these defects caused by ACBD3 knockdown. These data reveal critical roles for ACBD3 in maintaining the integrity of Golgi morphology and cellular sphingolipid homeostasis and establish the importance of the integrated Golgi complex for the transfer of GlcCer and complex GSL synthesis.

Keywords: ACBD3; FAPP2; Golgi fragmentation; glucosylceramide; glycosphingolipids.

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Figures

Figure 1
Figure 1
ACBD3 is an interacting partner of FAPP2. (A) Schematic representation of full-length FAPP2 and its deletion mutants. FAPP2 contains the characteristic domains including PH, proline-rich domain (PRD), and glycolipid transfer protein-like domain (GLTP). Numbers indicate amino-acid positions. Full-length FAPP2, PH, and PRDGLTP were inserted into the BD vector for yeast two-hybrid screening. (B) Candidate molecules identified by yeast two-hybrid screening to interact with FAPP2. (C) Schematic representation of full-length ACBD3 and its deletion mutants. Numbers indicate amino-acid positions. ACBD3 possesses proline-rich domain (PR), Acyl-CoA binding region (ACB), charged amino acid-rich domain (CAR), glutamine-rich domain (QR), and Golgi dynamic domain (GOLD). GFP-tagged deletions, including 1–180, 1–327, 328–528, 171–327, and 171–528, were constructed for mapping the domain interacting with FAPP2. (D) Co-immunoprecipitation of ACBD3 with FAPP2. (E) Co-immunoprecipitation of GFP-tagged ACBD3 deletion mutants with 3× FLAG-FAPP2. (F) Co-localization of FAPP2 with ACBD3 (Pearson’s coefficient = 0.714). Scale bar, 10 μm. (G) The plot of fluorescence intensity along the white dashed line in the merged image in F.
Figure 2
Figure 2
Golgi is fragmented in ACBD3-knockdown HeLa cells. (A) Expression of ACBD3 in control and ACBD3-knockdown cells. HeLa cells were treated with either control, ACBD3 siRNA1, or ACBD3 siRNAmix and samples were probed for ACBD3 or α-tubulin as a loading control. (B) Fluorescence images of the Golgi marker Giantin (red) and DAPI (blue) staining of control and ACBD3-knockdown cells. Scale bar, 10 μm. (C) Statistal analysis of relative Golgi area. Golgi area in each ACBD3-knockdown cell was measured with ImageJ and relative to the mean value of Golgi area in control cells. For each treatment, n > 25 pooled from three experiments, ***P < 0.001, unpaired two-tailed Student’s t-test. (D and E) EM images of the Golgi regions in a control cell (D) and an ACBD3-knockdown cell (E). Scale bar, 500 nm.
Figure 3
Figure 3
ACBD3 knockdown causes FAPP2 dispersal distribution in cytoplasm. (A) Fluorescence images of the TGN marker TGN46 (red), DAPI (blue), and GFP-FAPP2 (green) staining of control and ACBD3-knockdown cells. The right pictures are the enlargement of the white boxed regions. Scale bar, 10 μm. (B and C) Plots of fluorescence intensity of GFP-FAPP2 and TGN46 along the white dashed line in a control cell (B) and an ACBD3-knockdown cell (C). (D) Statistical analysis of Pearson’s coefficient of GFP-FAPP2 vs. TGN46 in control and ACBD3-knockdown cells. For each treatment, n > 25 pooled from three experiments, **P < 0.01, unpaired two-tailed Student’s t-test. (E) Statistical analysis of relative FAPP2 distribution in control and ACBD3-knockdown cells. FAPP2-positive area in each ACBD3-knockdown cell was measured with ImageJ and relative to the mean value of FAPP2-positive area in control cells. For each treatment, n > 45 pooled from three experiments, ***P < 0.001, unpaired two-tailed Student’s t-test.
Figure 4
Figure 4
ACBD3 knockdown affects GSL metabolism. (A) Simplified schematic representation of GSL biosynthetic pathway. (B) Sphingolipid compositions in control and ACBD3-knockdown cells. (C) ACBD3 knockdown increased the levels of different species of GlcCer. (D and E) ACBD3 knockdown increased the levels of GlcCer species with the same number of the double bond (D) and the same chain length (E). Data are represented as mean ± SD (n = 6). ***P < 0.001, two-way ANOVA.
Figure 5
Figure 5
Expression of ACBD3 and its deletion mutants in ACBD3-knockdown cells rescues the Golgi morphological defects. (A–C) Fluorescence images of Giantin (red) and DAPI (blue) staining in control (ACBD3-knockdown) cells and ACBD3-knockdown cells transfected with GFP-ACBD3-4M (A), GFP-ACBD3(1–180) (B), or GFP-ACBD3(171–528) (C). Scale bar, 10 μm. (D) Statistical analysis of the relative Golgi area in ACBD3-knockdown cells and rescued cells. For each treatment, n > 37 pooled from three experiments, ***P < 0.001, unpaired two-tailed Student’s t-test. (E–G) Fluorescence images of Giantin (red) and DAPI (blue) staining of ACBD3-knockdown cells transfected with GFP-FAPP2 (E), GFP-ACBD3-4M and mCherry-FAPP2 (F), or GFP-ACBD3(171–528) and mCherry-FAPP2 (G). Scale bar, 10 μm. (H) Statistical analysis of the relative FAPP2 distribution in ACBD3-knockdown cells and rescued cells. For each treatment, n > 42 pooled from three experiments, *P < 0.05, **P < 0.01, unpaired two-tailed Student’s t-test.
Figure 6
Figure 6
The effects of expression of ACBD3 and its deletion mutants in ACBD3-knockdown cells on GSL metabolic defects by ACBD3 knockdown. (A) Comparative lipidomics analysis for levels of sphingolipids in ACBD3-knockdown cells and the cells rescued by GFP-ACBD3-4M, GFP-ACBD3(171–528), or GFP-ACBD3(1–180). Data are represented as mean ± SD (n = 6). ***P < 0.001, two-way ANOVA. (B) A proposed model depicting that the integrity of Golgi complex maintained by ACBD3 is required for FAPP2 transferring GlcCer for GSL biosynthesis.
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