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

ACBD3 is required for FAPP2 transferring glucosylceramide through maintaining the Golgi integrity Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 doi:10.1093/jmcb/mjy030 Journal of Molecular Cell Biology (2019), 11(2), 107–117 j 107 Published online May 10, 2018 Article ACBD3 is required for FAPP2 transferring glucosylceramide through maintaining the Golgi integrity 1,† 1,† 1 1 1 1 Jing Liao , Yuxiang Guan , Wei Chen , Can Shi , Dongdong Yao , Fengsong Wang , 2 2 1, Sin Man Lam , Guanghou Shui , and Xinwang Cao School of Life Sciences, Anhui Medical University, Hefei 230032, China State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China These authors contributed equally to this work. * Correspondence to: Xinwang Cao, E-mail: caoxw@ahmu.edu.cn Edited by Feng Liu 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 contain- ing 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 import- ance of the integrated Golgi complex for the transfer of GlcCer and complex GSL synthesis. Keywords: FAPP2, ACBD3, Golgi fragmentation, glucosylceramide, glycosphingolipids the importance of the Golgi integrity for the synthesis of complex Introduction GSLs. In addition, the molecular determinants of FAPP2-mediated Complex glycosphingolipids (GSLs) function in various cell bio- GlcCer transport remain poorly understood. logical processes, including cell growth, cell signaling, cell differ- Golgi membranes of mammalian cells are organized into entiation, autophagy, cell death, cell migration, immune response, ordered stacks of cisternae (Klumperman, 2011). The morph- and inflammation (Lingwood, 2011; D’Angelo et al., 2013a; ology is maintained by Golgi structural proteins, microtubule Hannun and Obeid, 2018; Ogretmen, 2018). Glucosylceramide and microtubule-associated motor protein, and the proteins in (GlcCer) is an important precursor of GSLs synthesized in Golgi the Golgi transport machinery (Haase and Rabouille, 2015). complex, which is transported via vesicle trafficking to the Golgi Morphological changes of the Golgi apparatus, for example, its cisternae to synthesize monosialodihexosylganglioside (GM3)or fragmentation, normally are linked to a pre-clinical feature of via Golgi-associated phosphatidylinositol four-phosphate adaptor some neurodegenerative diseases such as Alzheimer’s disease protein FAPP2 to the trans-Golgi network (TGN) to produce globo- (AD) (Joshi et al., 2014) and amyotrophic lateral sclerosis (ALS) triaosylceramide (Gb3)(D’Angelo et al., 2013b). It is apparent that (Maruyama et al., 2010). For mammalian cells, Golgi fragmenta- the transfer of GlcCer between different Golgi subcompartments is tion also occurs during mitosis (Sutterlin et al., 2002), cell apop- required for complex GSL biosynthesis (D’Angelo et al., 2007, tosis (Chiu et al., 2002; Lane et al., 2002), and DNA damage 2013b; Halter et al., 2007). However, we still lack evidence to show (Farber-Katz et al., 2014). Previous studies showed that Golgi fragmentation may affect modification, trafficking, and activa- Received April 3, 2018. Revised April 25, 2018. Accepted May 7, 2018. tion of the amyloid precursor protein (APP) and its processing © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. enzyme (Choy et al., 2012; Joshi et al., 2014). In a similar light, Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 108 j Liao et al. we postulate that Golgi morphological changes can affect traf- Moreover, our quantitative analysis of fluorescence images ficking of other proteins or lipids important for the cellular indicated that most of ACBD3 and FAPP2 were colocalized physiological processes. (Pearson’s coefficient = 0.714, Figure 1F and G). Here, Golgi protein acyl-coenzyme A binding domain contain- ing 3 (ACBD3) is identified as a FAPP2-interacting partner. ACBD3 knockdown triggers Golgi fragmentation ACBD3 is previously known as the Golgi complex-associated Previous study by Sohda et al. (2001) has shown that over- protein (GCP60)(Sohda et al., 2001) and peripheral-type benzo- expression of C-terminal domain of ACBD3 caused impairment diazepine receptor (PBR)-associated protein 7 (PAP7)(Li et al., of the Golgi structure. Here, we further examined the function of 2001). It is consisted of 528 amino acids, with an N-terminal ACBD3 in the maintenance of Golgi morphology through knock- ACBP (acyl-CoA binding protein) domain, a middle GOLD (Golgi ing down its expression by transfecting siRNA. First, we tested dynamics) domain, and a C-terminal coil-coiled domain (Neess the efficiency of siRNAs targeting ACBD3 and found that siRNA1 et al., 2015). Accumulated evidence suggested that ACBD3 per- and siRNAmix (a mixture of four ACBD3 siRNAs) had a similar forms the biological function in steroidogenesis, neurogenesis, effect (Figure 2A). Thus, the siRNA1 was used in the further embryogenesis, and cancer (Fan et al., 2010). In the present experiments. Next, we examined the effect of ACBD3 knockdown study, we demonstrate that ACBD3 knockdown triggers Golgi on Golgi morphology. As shown in Figure 2B, ACBD3 knockdown fragmentation, which further leads to FAPP2 dispersed distribu- led to a significant change in Golgi morphology from the normal tion throughout the cytoplasm and its decreased localization at perinuclear ribbons into punctate structures, dispersed through- TGN. Our lipidomic analysis indicates that ACBD3 depletion out the cytoplasm. We further quantified Golgi dispersal by causes aberrant sphingolipid metabolism. In addition, we find measuring the Golgi area per cell with ImageJ and determined that expression of siRNA-resistant ACBD3 in knockdown cells their distribution relative to the mean of Golgi area in control can rescue the defects in Golgi morphology and the alterations cells. Compared with control cells, ACBD3 knockdown signifi- of GSL levels. These findings indicate the importance of the cantly increased relative Golgi area (Figure 2C). Under electron integrity of Golgi complex maintained by ACBD3 for FAPP2 trans- microscope (EM), the Golgi in ACBD3-knockdown HeLa cell was ferring GlcCer and cellular GSL homeostasis. seen to be severely fragmented in the red boxed area, with swollen cisternae and disorganized stacks (Figure 2E), while the Results Golgi in control cell exhibited typically highly organized Golgi ACBD3 is a novel interacting partner of FAPP2 stacks (Figure 2D). Protein interactions are involved in almost every cellular physiological process. To get an insight into the mechanism of ACBD3 depletion causes FAPP2 dispersal in the cytoplasm and FAPP2-mediated transfer of GlcCer, we used the Matchmaker less TGN localization Gold Yeast Two-Hybrid System (Clontech) to identify proteins ACBD3 is required for Golgi recruitment of phosphatidylinosi- that interact with FAPP2. In order to generate baits for yeast tol 4-kinase IIIβ (PI4KB), which is one of the kinases responsible two-hybrid screening, full-length FAPP2 and its deletion mutants for the production of phosphatidylinositol-4-phosphate (PI4P) on (Figure 1A) were inserted into a BD vector, respectively. These the Golgi (Sasaki et al., 2012). However, the depletion of PI4P baits were then tested for their autoactivation. We found that on the Golgi does not damage the Golgi morphology (Dippold only PRDGLTP deletion mutant could be used for further screen- et al., 2009; Ng et al., 2013). It is known that TGN localization of ing. Interestingly, Golgi-resident protein ACBD3 was found FAPP2 is PI4P dependent (Vieira et al., 2005). Therefore, we next among some positive molecules from the screening (Figure 1B). tried to examine the effect of ACBD3 depletion on FAPP2 distri- To further confirm the interaction between ACBD3 and FAPP2, bution in the cell. GFP-tagged FAPP2 was transfected into GFP-tagged ACBD3 was co-expressed with 3× FLAG-tagged FAPP2 ACBD3-knockdown cells. We found that FAPP2 dispersed in cyto- in 293T cells, and then a co-immunoprecipitation assay was per- plasm following Golgi fragmentation, neither enriched in the formed. Consistent with our yeast two-hybrid screening results, region of TGN as in control cell nor localized at cis-Golgi ACBD3 was co-immunoprecipitated with FAPP2 (Figure 1D). In (Figure 3A and Supplementary Figure S1). Furthermore, our order to define the region in ACBD3 required for its interacting quantitative analysis of co-localization also showed that, com- with FAPP2, we made various GFP-tagged deletions of ACBD3 pared with control cells, less FAPP2 was localized to the (Figure 1C) and co-transfected them with 3× FLAG-FAPP2 into impaired TGN in ACBD3-knockdown cells (Figure 3B–D). We then 293T cells, respectively. The co-immunoprecipitation assay determined relative FAPP2 distribution in ACBD3 depleted cells showed that ACBD3 N-terminal region (1–180 amino acids) con- and control cells, as to determine the relative Golgi area before, taining ACBP domain is essential for its interaction with FAPP2 and found that FAPP2 distribution in the ACBD3-knockdown cell (Figure 1E). was increased significantly (Figure 3E). To examine whether ACBD3 colocalizes with FAPP2, we co- transfected GFP-ACBD3 and mCherry-FAPP2 into HeLa cells, and ACBD3 knockdown leads to abnormal GSL metabolism then carried out a fluorescence microscope assay. Both ACBD3 Previous study showed that FAPP2 localization at the TGN is and FAPP2 were detected in the perinuclear area, in line with determined by its pleckstrin homology (PH) domain binding to their Golgi localization reported previously (Sohda et al., 2001). PI4P enriched in the TGN, though the interaction between the Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 ACBD3-maintained Golgi integrity for FAPP2-mediated GlcCer transfer j 109 Figure 1 ACBD3 is an interacting partner of FAPP2.(A) Schematic representation of full-length FAPP2 and its deletion mutants. FAPP2 con- tains 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 coeffi- cient = 0.714). Scale bar, 10 μm. (G) The plot of fluorescence intensity along the white dashed line in the merged image in F. PH domain and the small GTPase ARF1 facilitates FAPP2 local- the synthesis of complex GSLs. As shown in Figure 4A, GlcCer ization to the Golgi complex (Godi et al., 2004). Recently, transferring to the TGN lumen is a critical step for GSL biosyn- D’Angelo et al. (2013b) proposed that FAPP2 locates at the TGN thesis, because the further glycosylation reactions for complex dynamically, due to the fact that GlcCer-bound FAPP2 has an GSL synthesis occur there. GlcCer can be transported to the increased affinity for PI4P, which is consistent with its function- Golgi cisternae via vesicle (blue arrow) or to TGN via FAPP2 (red ing as a glycolipid transfer protein to transfer GlcCer to TGN for arrow) (D’Angelo et al., 2013b). Next, we wanted to examine Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 110 j Liao et al. 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. whether the dispersed FAPP2 by ACBD3 knockdown affects GSL (Sph). However, no appreciable change was noted for the level biosynthesis. of ceramide (Cer) (Figure 4B). To this end, we performed lipidomics analysis to compare GSL levels in ACBD3-knockdown cells and control cells. As shown in Defects by ACBD3 knockdown can be rescued by expression Figure 4B, a significant increase of GlcCer in ACBD3-knockdown of ACBD3 cells was observed. The similar changes were also observed In order to address the issue whether defects of the Golgi among different GlcCer species (Figure 4C) and GlcCer species morphology and GSL biosynthesis by ACBD3 knockdown could be with the same number of C=C (Figure 4D) and the same carbon rescued by expressing ACBD3, we constructed siRNA-resistant chain length (Figure 4E). ACBD3 knockdown also resulted in the GFP-ACBD3-4M (Supplementary Figure S3), a silent mutant with increase of total sphingomyelin (SM) (Figure 4B), which could be four mutated bases, and transfected it into ACBD3-knockdown observed among some species of SM as well (Supplementary cells. We found that the Golgi area in these GFP-ACBD3-4M- Figure S2A). However, due to ACBD3 knockdown, the total levels transfected cells (indicated by white arrow) was smaller than that of lactosylceramides (LacCer), GM3, and Gb3 were reduced in the non-transfected cells (indicated by white arrowhead) remarkably (Figure 4B), and the levels of some species were (Figure 5A). We next transfected GFP-ACBD3(1–180), the deletion also decreased, although to a different degree (Supplementary mutant required for ACBD3 interaction with FAPP2, into ACBD3- Figure S2B and C), compared with the control cells. We also knockdown cells (indicated by white arrow). However, the deletion observed ACBD3 knockdown increased the levels of sphingosine mutant dispersed throughout the whole cell, not localized to the Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 ACBD3-maintained Golgi integrity for FAPP2-mediated GlcCer transfer j 111 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 mea- sured 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. Golgi complex as the wild-type, and could not rescue disrupted GFP-ACBD3-4M and GFP-ACBD3(171–528) in ACBD3-knockdown Golgi morphology effectively (Figure 5B). Interestingly, when GFP- cells significantly decreased GlcCer levels and increased the ACBD3(171–528), a deletion mutant from GFP-ACBD3-4M, contain- levels of LacCer, GM3, and Gb3. However, no appreciable res- ing the region responsible for the interaction with the Golgi cued effect was observed for those cells expressing GFP-ACBD3 integral protein Giantin (Sohda et al., 2001), was expressed in (1–180) (Figure 6A). Together, these data indicate that GFP- ACBD3-knockdown cells (indicated by white arrow), it could res- ACBD3-4M and GFP-ACBD3(171–528) rescue the defects of GSL cue the defected Golgi morphology significantly (Figure 5C). Our metabolism by ACBD3 knockdown more effectively than GFP- statistical analysis for the relative Golgi area of the rescued cells ACBD3(1–180), consistent with their rescued effect on defects indicated that GFP-ACBD3-4Mand GFP-ACBD3(171–528)had bet- of Golgi morphology and FAPP2 distribution caused by ACBD3 ter rescuing effects than GFP-ACBD3(1–180)(Figure 5E). depletion. We next tested whether the dispersed FAPP2 by ACBD3 deple- tion could go back to the recovered Golgi complex in the res- Discussion cued experiments. Interestingly, as shown in Figure 5E–H, the In this study, we showed that ACBD3 knockdown caused dispersed FAPP2 by ACBD3 knockdown was re-localized to the Golgi fragmentation, which further affected the TGN localization rescued Golgi complex to some degree in the ACBD3-knockdown of FAPP2 and GSL homeostasis. As shown in Figure 6B, ACBD3 cells expressing GFP-ACBD3-4M or GFP-ACBD3(171–528). knockdown decreased the TGN localization of FAPP2, and thus We further utilized lipidomics analysis to compare the GSL inhibited GlcCer transport mediated by FAPP2. In addition, Golgi levels of ACBD3-knockdown cells and the rescued cells trans- fragmentation by ACBD3 depletion interrupted vesicle transport fected with GFP-ACBD3(1–180), GFP-ACBD3(171–528), and of GlcCer and resulted in the loss of some GSL synthetases in GFP-ACBD3-4M, respectively. We found that the expression of the lumen of TGN. Therefore, Golgi fragmentation caused by Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 112 j Liao et al. 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. ACBD3 knockdown attenuated both vesicle transport of GlcCer levels might result from FAPP2 dispersal in the cytoplasm and and GlcCer transport by FAPP2. We concluded that the Golgi impaired its efficiency to transfer GlcCer. The increase of GlcCer integrity maintained by ACBD3 is necessary for FAPP2 transfer- content resulted in a substantial increase in the levels of SM ring GlcCer for further GSL biosynthesis. and Sph, which may be attributed to GlcCer accumulation. The ACBD3 is required for maintaining Golgi integrity. The previ- decreased levels of LacCer in ACBD3-knockdown cells could ous report by Sohda et al. (2001) indicated that ACBD3 is result from less FAPP2 localization to TGN, which lowered involved in the maintenance of Golgi structure through the inter- FAPP2-mediated GlcCer transfer to TGN. As described before, action of its C-terminal domain with Golgi integral protein gian- the levels of PI4P of TGN are critical for FAPP2 localization, tin. Most recently, Yue et al. (2017) reported that ACBD3 which is produced by PI4KB recruited to the Golgi by ACBD3, maintains Golgi structure through organizing the Golgi stacking thus ACBD3 depletion lowers the PI4P levels in TGN. LacCer proteins and a Rab33b-GAP at the medial-Golgi. Here, we locates at upstream of synthesis pathway of GM3 and Gb3 showed that ACBD3 depletion triggered Golgi fragmentation, (Figure 3F), thus, the decreased level of LacCer will reduce the which further supports these reports. levels of its downstream products. In addition, Golgi fragmenta- Golgi integrity is required for processing, sorting proteins and tion caused by ACBD3 knockdown could release the enzymes lipids from ER, and transporting them to their destination (Wang responsible for the synthesis of GM3 and Gb3 into the cyto- et al., 2008; Choy et al., 2012; Joshi et al., 2014). However, the plasm. This could be another reason for the decrease in Gb3 importance of Golgi integrity for FAPP2 transferring GlcCer has not and GM3 synthesis in ACBD3-knockdown cells. been studied. In this study, we examined the structural and func- ACBD3 performs many other functions, including steroidogen- tional defects of the Golgi caused by ACBD3 knockdown and esis (Papadopoulos et al., 2007; Fan et al., 2010), apoptosis found that ACBD3 is involved in transferring GlcCer through main- (Sbodio et al., 2006; Sbodio and Machamer, 2007), neurogenesis taining the Golgi integrity and determining FAPP2 TGN localization. (Cheah et al., 2006; Zhou et al., 2007), and embryogenesis (Zhou Our lipidomics data indicated that ACBD3 knockdown causes et al., 2007). In addition, ACBD3 recruits the protein phosphatase abnormal GSL metabolism. A pronounced increase of GlcCer PPM1L to ER-Golgi membrane contact sites (Shinoda et al., 2012), Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 ACBD3-maintained Golgi integrity for FAPP2-mediated GlcCer transfer j 113 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. Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 114 j Liao et al. Figure 6 The effects of expression of ACBD3 and its deletion mutants in ACBD3-knockdown cells on GSL metabolic defects by ACBD3 knock- down. (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. to dephosphorylate the ceramide transport protein CERT and RT-PCR was performed using SuperScript reverse transcriptase regulate ceramide transport (Saito et al., 2008). The interaction (Invitrogen) with a poly-dT oligo as a primer. The cDNA obtained between ACBD3 and FAPP2 may facilitate the recruitment of was used as a template for the further PCR reaction and the fol- FAPP2 to ER-Golgi membrane contact sites and translocate GlcCer lowing primers were used: forward primer: 5′-ATGGAGGGGGTGC to the lumen of ER. GlcCer in the ER is then transported to the TGTACAAGTGGAC-3′; reverse primer: 5′-CACTTTATTCCCTGAAG Golgi cisternae via vesicle trafficking for GSL biosynthesis (Halter TTAGG-3′. The purified PCR product was inserted into pEGFP-C2 et al., 2007; D’Angelo et al., 2013a). Since GFP-ACBD3(171–528) (Clontech) or mCherry-C2 (a gift from Dr Zhen Dou, University of had a better rescuing effect on defects of the Golgi integrity and Science and Technology of China) at restriction sites of BglII GSL biosynthesis by ACBD3 knockdown than GFP-ACBD3(1–180), and KpnI following the standard protocol. The plasmid of we deduced that the Golgi integrity is also necessary for GlcCer GFP-tagged ACBD3 was a gift from Dr Carolyn Machamer (Johns transfer at ER-Golgi membrane contact sites. Certainly, this specu- Hopkins University). Site-directed mutagenesis to create lative hypothesis remains to be verified experimentally. Taken siRNA-resistant ACBD3 was performed using Mut Express II fast together, this study has revealed the importance of ACBD3 in mutagenesis kits (Vazyme Biotech Co. Ltd) according to the maintaining the Golgi morphological integrity and as a cofactor manufacturer’s instructions. The ACBD3 target sequence is that functions in the FAPP2-mediated transfer of GlcCer for com- siRNA1 (GGAUGCAGAUUCCGUGAUU, nucleotides 1168–1185), plex GSL biosynthesis. as shown in Supplementary Figure S3. Primers for all plasmids used in this paper are listed in the table of Supplementary Materials and methods material. All constructs were verified by sequencing. Cell growth and DNA transfection HeLa (CCL-2) and 293T cells were cultured according to ATCC guidelines. Briefly, cells were grown in a monolayer at 37°Cin Antibodies 5%CO and maintained in DMEM supplemented with 10% fetal Anti-ACBD3 mouse monoclonal antibody (ab57568), anti- bovine serum and 100 units/ml penicillin and 100 μg/ml strep- GM130 rabbit monoclonal antibody (ab52649), and anti-Giantin tomycin sulfate. For DNA transfection, cells with appropriate rabbit polyclonal antibody (ab24586) were from Abcam. Anti- confluence were transfected with the indicated plasmids in TGN46 sheep polyclonal antibody (AHP500G) was purchased from the experiments of this paper using Lipofectamine 3000 BIO-RAD. Anti-FLAG rabbit antibody (F7425)and anti-α-tubulin (Invitrogen) and OPTI-MEM (Invitrogen) following the manufac- antibody (DM1A) were from Sigma-Aldrich. Anti-GFP rabbit anti- turer’s instructions. body was from Cell Signaling Technology. For immunofluores- cence, AlexaFluor (488, 594,and 647)-conjugated secondary RT-PCR and plasmid construction antibodies were from Invitrogen. For immunoblotting, the horse- RT-PCR was used to fish human FAPP2 DNA. Briefly, total RNA radish peroxidase-conjugated anti-rabbit or anti-mouse antibodies was extracted from HeLa cell using RNeasy Mini Kit (Qiagen). were from Invitrogen. Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 ACBD3-maintained Golgi integrity for FAPP2-mediated GlcCer transfer j 115 RNA interference BD-PRDGLTP was taken as a bait to screen Mate & Plate™ The target sequences for ACBD3 siRNAs are listed as follows: Library—Human Testis by yeast mating. Sequence analysis was siRNA1: GGAUGCAGAUUCCGUGAUUTT; siRNA2: GUAUAGAAACCA carried out for those positive prey inserts. UGGAGUUTT; siRNA3: GCAUAUGGGAAGUAACAUUTT; siRNA4: GCAACUGUACCAAGUAAUATT, which were obtained from Immunoprecipitation Genepharma. ACBD3 siRNAs were transfected into HeLa cells with For immunoprecipitation, 293T cells with appropriate conflu- appropriate confluence using the lipofectamine 3000. For rescue ence were transfected with indicated plasmids. After 24 h, they experiments, after 48 htransfected with ACBD3 siRNA, rescue plas- were lysed in buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, mids GFP-ACBD3-4M, GFP-ACBD3(171–528), or GFP-ACBD3(1–180) 0.5% NP-40) supplemented with protease inhibitor cocktail were transfected into the cells. Twenty-four hours later, the rescued (Roche) and phosphatase inhibitor cocktail (Roche). After centri- cells were used for the further experiment. fugation, the supernatant was incubated with FLAG-M2 (Sigma) resin at 4°C for 4 h with gentle rotation. The FLAG-M2 resin was Lipid extraction then spun down and washed extensively before being resolved Lipid was extracted from ∼10 cells using a modified version by SDS-PAGE and western blotting with the indicated antibodies of the Bligh and Dyer’s method as described previously (Lam (Chu et al., 2011). et al., 2016). Briefly, cells were incubated in 750 μl of chloro- form: methanol 1:2 (v/v) with 10% deionized water for 30 min. Immunofluorescence and imaging At the end of the incubation, 350 μl of deionized water and HeLa cells were grown on coverslips before immunofluores- 250 μl of chloroform were added. The samples were then centri- cence. Where indicated, cells were transfected with the indicated fuged and the lower organic phase containing lipids was plasmids. For immunofluorescence, cells were fixed with 4% for- extracted into a clean tube. Lipid extraction was carried out maldehyde in phosphate buffered saline (PBS) for 10 min at twice and the lipid extracts were pooled into a single tube and room temperature and permeabilized with 0.1% Triton X-100 in dried in the SpeedVac under OH mode. Samples were stored at PBS for 10 min. After blocking with PBS with 0.05% Tween-20 −80°C until further analysis. (PBST) containing 1% bovine serum albumin (Sigma) for 30 min, the fixed cells were incubated with primary antibodies for 1 hat Liquid chromatography/mass spectrometry room temperature, followed by second antibodies conjugated Polar lipids were analyzed using an Exion UPLC system with appropriate fluorescence for another 1 h before three times coupled with a triple quadrupole/ion trap mass spectrometer washing with PBST. The DNA was stained with 4′,6-diamidino-2- (6500 Plus Qtrap; SCIEX) as described previously (Lam et al., phenylindole (DAPI). Samples were observed under DeltaVision 2014). Separation of individual lipid classes of polar lipids by microscope system (Applied Precision). Images were captured by normal phase (NP)-high pressure liquid chromatography (HPLC) DeltaVision softWoRx software and processed by deconvolution was carried out using a Phenomenex Luna 3μ-silica column and z-stack projection. After deconvolution, the images were (internal diameter 150 × 2.0 mm) with the indicated conditions exported as 24-bit RGB images and processed with Adobe for mobile phase A (chloroform:methanol:ammonium hydroxide, Photoshop (Bao et al., 2018). 89.5:10:0.5) and mobile phase B (chloroform:methanol:ammonium hydroxide:water, 55:39:0.5:5.5). Multiple reaction monitoring (MRM) Measurement of Golgi area and FAPP2 distribution transitions were set up for comparative analysis of various polar Immunofluorescent images of cells stained with a Golgi lipids. Individual lipid species were quantified by referencing to marker Giantin or transfected with GFP-FAPP2 were measured spiked internal standards. PC-14:0/14:0,PE-14:0/14:0,PS34:1-d31, by splitting the RGB image into the gray image, adjusting PA-17:0/17:0,PG-14:0/14:0,Cer d18:1/17:0,SMd18:1/12:0,GluCer threshold, and setting measurements of their area using ImageJ. d18:1/8:0,GalCer d18:1/8:0,LacCer d18:1/8:0,Sph d17:1 were obtained from Avanti Polar Lipids. Dioctanoyl phosphatidylinosi- tol (PI) (16:0-PI) was obtained from Echelon Biosciences, Inc. Electron microscope Gb3-C17:0 was obtained from Matreya LCC and GM3 d18:1/17:0 The treated HeLa cells were fixed with 2% glutaraldehyde in was synthesized in-house. Free cholesterol was further analyzed 0.1 M sodium cacodylate buffer (pH 7.2) for 30 min and then using HPLC/APCI/MS/MS as previously described with corre- harvested by scraping gently with a plastic scraper. The cells sponding d6-cholesterol (CDN isotopes) as internal standards were pelleted by centrifugation at 800 g for 10 min and fixed for (Shui et al., 2011). a further 2 hin 2% glutaraldehyde in 0.1 M sodium cacodylate buffer. Following washing in buffer, the cells were post-fixed for Yeast two-hybrid screening 1 hin 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. The Matchmaker Gold Yeast Two-Hybrid System (Clontech) The cells were then stained for 1 h with 1% uranyl acetate in was used to search for binding partners of FAPP2 according to water. After dehydration with a graded series of ethanol (50%, the standard protocol provided by the manufacturer. Briefly, full- 70%, 90%, 95%, and 100%), samples were embedded in Epon length FAPP2 and its deletion mutants were inserted into a BD resin. Sections were cut using LEICA EM UC7 ultramicrotome and vector, respectively. After testing these baits for autoactivation, stained for 6 min in 0.16% lead citrate in 0.1 M NaOH followed Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 116 j Liao et al. Dippold, H.C., Ng, M.M., Farber-Katz, S.E., et al. (2009). GOLPH3 bridges by three washes in water. Samples were viewed on a HITACHI phosphatidylinositol-4- phosphate and actomyosin to stretch and shape HT7700 EM. the Golgi to promote budding. Cell 139, 337–351. Fan, J., Liu, J., Culty, M., et al. (2010). Acyl-coenzyme A binding domain con- taining 3 (ACBD3; PAP7; GCP60): an emerging signaling molecule. Prog. Statistical analysis Lipid Res. 49, 218–234. Statistical calculations were performed using GraphPad Prism Farber-Katz, S.E., Dippold, H.C., Buschman, M.D., et al. (2014). DNA damage software (version 6.04). All data were obtained from independent triggers Golgi dispersal via DNA-PK and GOLPH3. 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Sohda, M., Misumi, Y., Yamamoto, A., et al. (2001). Identification and charac- Yue, X., Bao, M., Christiano, R., et al. (2017). ACBD3 functions as a scaffold terization of a novel Golgi protein, GCP60, that interacts with the integral to organize the Golgi stacking proteins and a Rab33b-GAP. FEBS Lett. 591, membrane protein giantin. J. Biol. Chem. 276, 45298–45306. 2793–2802. Sutterlin, C., Hsu, P., Mallabiabarrena, A., et al. (2002). Fragmentation and Zhou, Y., Atkins, J.B., Rompani, S.B., et al. (2007). The mammalian Golgi reg- dispersal of the pericentriolar Golgi complex is required for entry into ulates numb signaling in asymmetric cell division by releasing ACBD3 dur- mitosis in mammalian cells. Cell 109, 359–369. ing mitosis. Cell 129, 163–178. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Cell Biology Oxford University Press

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

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© The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
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Abstract

Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 doi:10.1093/jmcb/mjy030 Journal of Molecular Cell Biology (2019), 11(2), 107–117 j 107 Published online May 10, 2018 Article ACBD3 is required for FAPP2 transferring glucosylceramide through maintaining the Golgi integrity 1,† 1,† 1 1 1 1 Jing Liao , Yuxiang Guan , Wei Chen , Can Shi , Dongdong Yao , Fengsong Wang , 2 2 1, Sin Man Lam , Guanghou Shui , and Xinwang Cao School of Life Sciences, Anhui Medical University, Hefei 230032, China State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China These authors contributed equally to this work. * Correspondence to: Xinwang Cao, E-mail: caoxw@ahmu.edu.cn Edited by Feng Liu 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 contain- ing 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 import- ance of the integrated Golgi complex for the transfer of GlcCer and complex GSL synthesis. Keywords: FAPP2, ACBD3, Golgi fragmentation, glucosylceramide, glycosphingolipids the importance of the Golgi integrity for the synthesis of complex Introduction GSLs. In addition, the molecular determinants of FAPP2-mediated Complex glycosphingolipids (GSLs) function in various cell bio- GlcCer transport remain poorly understood. logical processes, including cell growth, cell signaling, cell differ- Golgi membranes of mammalian cells are organized into entiation, autophagy, cell death, cell migration, immune response, ordered stacks of cisternae (Klumperman, 2011). The morph- and inflammation (Lingwood, 2011; D’Angelo et al., 2013a; ology is maintained by Golgi structural proteins, microtubule Hannun and Obeid, 2018; Ogretmen, 2018). Glucosylceramide and microtubule-associated motor protein, and the proteins in (GlcCer) is an important precursor of GSLs synthesized in Golgi the Golgi transport machinery (Haase and Rabouille, 2015). complex, which is transported via vesicle trafficking to the Golgi Morphological changes of the Golgi apparatus, for example, its cisternae to synthesize monosialodihexosylganglioside (GM3)or fragmentation, normally are linked to a pre-clinical feature of via Golgi-associated phosphatidylinositol four-phosphate adaptor some neurodegenerative diseases such as Alzheimer’s disease protein FAPP2 to the trans-Golgi network (TGN) to produce globo- (AD) (Joshi et al., 2014) and amyotrophic lateral sclerosis (ALS) triaosylceramide (Gb3)(D’Angelo et al., 2013b). It is apparent that (Maruyama et al., 2010). For mammalian cells, Golgi fragmenta- the transfer of GlcCer between different Golgi subcompartments is tion also occurs during mitosis (Sutterlin et al., 2002), cell apop- required for complex GSL biosynthesis (D’Angelo et al., 2007, tosis (Chiu et al., 2002; Lane et al., 2002), and DNA damage 2013b; Halter et al., 2007). However, we still lack evidence to show (Farber-Katz et al., 2014). Previous studies showed that Golgi fragmentation may affect modification, trafficking, and activa- Received April 3, 2018. Revised April 25, 2018. Accepted May 7, 2018. tion of the amyloid precursor protein (APP) and its processing © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. enzyme (Choy et al., 2012; Joshi et al., 2014). In a similar light, Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 108 j Liao et al. we postulate that Golgi morphological changes can affect traf- Moreover, our quantitative analysis of fluorescence images ficking of other proteins or lipids important for the cellular indicated that most of ACBD3 and FAPP2 were colocalized physiological processes. (Pearson’s coefficient = 0.714, Figure 1F and G). Here, Golgi protein acyl-coenzyme A binding domain contain- ing 3 (ACBD3) is identified as a FAPP2-interacting partner. ACBD3 knockdown triggers Golgi fragmentation ACBD3 is previously known as the Golgi complex-associated Previous study by Sohda et al. (2001) has shown that over- protein (GCP60)(Sohda et al., 2001) and peripheral-type benzo- expression of C-terminal domain of ACBD3 caused impairment diazepine receptor (PBR)-associated protein 7 (PAP7)(Li et al., of the Golgi structure. Here, we further examined the function of 2001). It is consisted of 528 amino acids, with an N-terminal ACBD3 in the maintenance of Golgi morphology through knock- ACBP (acyl-CoA binding protein) domain, a middle GOLD (Golgi ing down its expression by transfecting siRNA. First, we tested dynamics) domain, and a C-terminal coil-coiled domain (Neess the efficiency of siRNAs targeting ACBD3 and found that siRNA1 et al., 2015). Accumulated evidence suggested that ACBD3 per- and siRNAmix (a mixture of four ACBD3 siRNAs) had a similar forms the biological function in steroidogenesis, neurogenesis, effect (Figure 2A). Thus, the siRNA1 was used in the further embryogenesis, and cancer (Fan et al., 2010). In the present experiments. Next, we examined the effect of ACBD3 knockdown study, we demonstrate that ACBD3 knockdown triggers Golgi on Golgi morphology. As shown in Figure 2B, ACBD3 knockdown fragmentation, which further leads to FAPP2 dispersed distribu- led to a significant change in Golgi morphology from the normal tion throughout the cytoplasm and its decreased localization at perinuclear ribbons into punctate structures, dispersed through- TGN. Our lipidomic analysis indicates that ACBD3 depletion out the cytoplasm. We further quantified Golgi dispersal by causes aberrant sphingolipid metabolism. In addition, we find measuring the Golgi area per cell with ImageJ and determined that expression of siRNA-resistant ACBD3 in knockdown cells their distribution relative to the mean of Golgi area in control can rescue the defects in Golgi morphology and the alterations cells. Compared with control cells, ACBD3 knockdown signifi- of GSL levels. These findings indicate the importance of the cantly increased relative Golgi area (Figure 2C). Under electron integrity of Golgi complex maintained by ACBD3 for FAPP2 trans- microscope (EM), the Golgi in ACBD3-knockdown HeLa cell was ferring GlcCer and cellular GSL homeostasis. seen to be severely fragmented in the red boxed area, with swollen cisternae and disorganized stacks (Figure 2E), while the Results Golgi in control cell exhibited typically highly organized Golgi ACBD3 is a novel interacting partner of FAPP2 stacks (Figure 2D). Protein interactions are involved in almost every cellular physiological process. To get an insight into the mechanism of ACBD3 depletion causes FAPP2 dispersal in the cytoplasm and FAPP2-mediated transfer of GlcCer, we used the Matchmaker less TGN localization Gold Yeast Two-Hybrid System (Clontech) to identify proteins ACBD3 is required for Golgi recruitment of phosphatidylinosi- that interact with FAPP2. In order to generate baits for yeast tol 4-kinase IIIβ (PI4KB), which is one of the kinases responsible two-hybrid screening, full-length FAPP2 and its deletion mutants for the production of phosphatidylinositol-4-phosphate (PI4P) on (Figure 1A) were inserted into a BD vector, respectively. These the Golgi (Sasaki et al., 2012). However, the depletion of PI4P baits were then tested for their autoactivation. We found that on the Golgi does not damage the Golgi morphology (Dippold only PRDGLTP deletion mutant could be used for further screen- et al., 2009; Ng et al., 2013). It is known that TGN localization of ing. Interestingly, Golgi-resident protein ACBD3 was found FAPP2 is PI4P dependent (Vieira et al., 2005). Therefore, we next among some positive molecules from the screening (Figure 1B). tried to examine the effect of ACBD3 depletion on FAPP2 distri- To further confirm the interaction between ACBD3 and FAPP2, bution in the cell. GFP-tagged FAPP2 was transfected into GFP-tagged ACBD3 was co-expressed with 3× FLAG-tagged FAPP2 ACBD3-knockdown cells. We found that FAPP2 dispersed in cyto- in 293T cells, and then a co-immunoprecipitation assay was per- plasm following Golgi fragmentation, neither enriched in the formed. Consistent with our yeast two-hybrid screening results, region of TGN as in control cell nor localized at cis-Golgi ACBD3 was co-immunoprecipitated with FAPP2 (Figure 1D). In (Figure 3A and Supplementary Figure S1). Furthermore, our order to define the region in ACBD3 required for its interacting quantitative analysis of co-localization also showed that, com- with FAPP2, we made various GFP-tagged deletions of ACBD3 pared with control cells, less FAPP2 was localized to the (Figure 1C) and co-transfected them with 3× FLAG-FAPP2 into impaired TGN in ACBD3-knockdown cells (Figure 3B–D). We then 293T cells, respectively. The co-immunoprecipitation assay determined relative FAPP2 distribution in ACBD3 depleted cells showed that ACBD3 N-terminal region (1–180 amino acids) con- and control cells, as to determine the relative Golgi area before, taining ACBP domain is essential for its interaction with FAPP2 and found that FAPP2 distribution in the ACBD3-knockdown cell (Figure 1E). was increased significantly (Figure 3E). To examine whether ACBD3 colocalizes with FAPP2, we co- transfected GFP-ACBD3 and mCherry-FAPP2 into HeLa cells, and ACBD3 knockdown leads to abnormal GSL metabolism then carried out a fluorescence microscope assay. Both ACBD3 Previous study showed that FAPP2 localization at the TGN is and FAPP2 were detected in the perinuclear area, in line with determined by its pleckstrin homology (PH) domain binding to their Golgi localization reported previously (Sohda et al., 2001). PI4P enriched in the TGN, though the interaction between the Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 ACBD3-maintained Golgi integrity for FAPP2-mediated GlcCer transfer j 109 Figure 1 ACBD3 is an interacting partner of FAPP2.(A) Schematic representation of full-length FAPP2 and its deletion mutants. FAPP2 con- tains 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 coeffi- cient = 0.714). Scale bar, 10 μm. (G) The plot of fluorescence intensity along the white dashed line in the merged image in F. PH domain and the small GTPase ARF1 facilitates FAPP2 local- the synthesis of complex GSLs. As shown in Figure 4A, GlcCer ization to the Golgi complex (Godi et al., 2004). Recently, transferring to the TGN lumen is a critical step for GSL biosyn- D’Angelo et al. (2013b) proposed that FAPP2 locates at the TGN thesis, because the further glycosylation reactions for complex dynamically, due to the fact that GlcCer-bound FAPP2 has an GSL synthesis occur there. GlcCer can be transported to the increased affinity for PI4P, which is consistent with its function- Golgi cisternae via vesicle (blue arrow) or to TGN via FAPP2 (red ing as a glycolipid transfer protein to transfer GlcCer to TGN for arrow) (D’Angelo et al., 2013b). Next, we wanted to examine Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 110 j Liao et al. 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. whether the dispersed FAPP2 by ACBD3 knockdown affects GSL (Sph). However, no appreciable change was noted for the level biosynthesis. of ceramide (Cer) (Figure 4B). To this end, we performed lipidomics analysis to compare GSL levels in ACBD3-knockdown cells and control cells. As shown in Defects by ACBD3 knockdown can be rescued by expression Figure 4B, a significant increase of GlcCer in ACBD3-knockdown of ACBD3 cells was observed. The similar changes were also observed In order to address the issue whether defects of the Golgi among different GlcCer species (Figure 4C) and GlcCer species morphology and GSL biosynthesis by ACBD3 knockdown could be with the same number of C=C (Figure 4D) and the same carbon rescued by expressing ACBD3, we constructed siRNA-resistant chain length (Figure 4E). ACBD3 knockdown also resulted in the GFP-ACBD3-4M (Supplementary Figure S3), a silent mutant with increase of total sphingomyelin (SM) (Figure 4B), which could be four mutated bases, and transfected it into ACBD3-knockdown observed among some species of SM as well (Supplementary cells. We found that the Golgi area in these GFP-ACBD3-4M- Figure S2A). However, due to ACBD3 knockdown, the total levels transfected cells (indicated by white arrow) was smaller than that of lactosylceramides (LacCer), GM3, and Gb3 were reduced in the non-transfected cells (indicated by white arrowhead) remarkably (Figure 4B), and the levels of some species were (Figure 5A). We next transfected GFP-ACBD3(1–180), the deletion also decreased, although to a different degree (Supplementary mutant required for ACBD3 interaction with FAPP2, into ACBD3- Figure S2B and C), compared with the control cells. We also knockdown cells (indicated by white arrow). However, the deletion observed ACBD3 knockdown increased the levels of sphingosine mutant dispersed throughout the whole cell, not localized to the Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 ACBD3-maintained Golgi integrity for FAPP2-mediated GlcCer transfer j 111 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 mea- sured 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. Golgi complex as the wild-type, and could not rescue disrupted GFP-ACBD3-4M and GFP-ACBD3(171–528) in ACBD3-knockdown Golgi morphology effectively (Figure 5B). Interestingly, when GFP- cells significantly decreased GlcCer levels and increased the ACBD3(171–528), a deletion mutant from GFP-ACBD3-4M, contain- levels of LacCer, GM3, and Gb3. However, no appreciable res- ing the region responsible for the interaction with the Golgi cued effect was observed for those cells expressing GFP-ACBD3 integral protein Giantin (Sohda et al., 2001), was expressed in (1–180) (Figure 6A). Together, these data indicate that GFP- ACBD3-knockdown cells (indicated by white arrow), it could res- ACBD3-4M and GFP-ACBD3(171–528) rescue the defects of GSL cue the defected Golgi morphology significantly (Figure 5C). Our metabolism by ACBD3 knockdown more effectively than GFP- statistical analysis for the relative Golgi area of the rescued cells ACBD3(1–180), consistent with their rescued effect on defects indicated that GFP-ACBD3-4Mand GFP-ACBD3(171–528)had bet- of Golgi morphology and FAPP2 distribution caused by ACBD3 ter rescuing effects than GFP-ACBD3(1–180)(Figure 5E). depletion. We next tested whether the dispersed FAPP2 by ACBD3 deple- tion could go back to the recovered Golgi complex in the res- Discussion cued experiments. Interestingly, as shown in Figure 5E–H, the In this study, we showed that ACBD3 knockdown caused dispersed FAPP2 by ACBD3 knockdown was re-localized to the Golgi fragmentation, which further affected the TGN localization rescued Golgi complex to some degree in the ACBD3-knockdown of FAPP2 and GSL homeostasis. As shown in Figure 6B, ACBD3 cells expressing GFP-ACBD3-4M or GFP-ACBD3(171–528). knockdown decreased the TGN localization of FAPP2, and thus We further utilized lipidomics analysis to compare the GSL inhibited GlcCer transport mediated by FAPP2. In addition, Golgi levels of ACBD3-knockdown cells and the rescued cells trans- fragmentation by ACBD3 depletion interrupted vesicle transport fected with GFP-ACBD3(1–180), GFP-ACBD3(171–528), and of GlcCer and resulted in the loss of some GSL synthetases in GFP-ACBD3-4M, respectively. We found that the expression of the lumen of TGN. Therefore, Golgi fragmentation caused by Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 112 j Liao et al. 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. ACBD3 knockdown attenuated both vesicle transport of GlcCer levels might result from FAPP2 dispersal in the cytoplasm and and GlcCer transport by FAPP2. We concluded that the Golgi impaired its efficiency to transfer GlcCer. The increase of GlcCer integrity maintained by ACBD3 is necessary for FAPP2 transfer- content resulted in a substantial increase in the levels of SM ring GlcCer for further GSL biosynthesis. and Sph, which may be attributed to GlcCer accumulation. The ACBD3 is required for maintaining Golgi integrity. The previ- decreased levels of LacCer in ACBD3-knockdown cells could ous report by Sohda et al. (2001) indicated that ACBD3 is result from less FAPP2 localization to TGN, which lowered involved in the maintenance of Golgi structure through the inter- FAPP2-mediated GlcCer transfer to TGN. As described before, action of its C-terminal domain with Golgi integral protein gian- the levels of PI4P of TGN are critical for FAPP2 localization, tin. Most recently, Yue et al. (2017) reported that ACBD3 which is produced by PI4KB recruited to the Golgi by ACBD3, maintains Golgi structure through organizing the Golgi stacking thus ACBD3 depletion lowers the PI4P levels in TGN. LacCer proteins and a Rab33b-GAP at the medial-Golgi. Here, we locates at upstream of synthesis pathway of GM3 and Gb3 showed that ACBD3 depletion triggered Golgi fragmentation, (Figure 3F), thus, the decreased level of LacCer will reduce the which further supports these reports. levels of its downstream products. In addition, Golgi fragmenta- Golgi integrity is required for processing, sorting proteins and tion caused by ACBD3 knockdown could release the enzymes lipids from ER, and transporting them to their destination (Wang responsible for the synthesis of GM3 and Gb3 into the cyto- et al., 2008; Choy et al., 2012; Joshi et al., 2014). However, the plasm. This could be another reason for the decrease in Gb3 importance of Golgi integrity for FAPP2 transferring GlcCer has not and GM3 synthesis in ACBD3-knockdown cells. been studied. In this study, we examined the structural and func- ACBD3 performs many other functions, including steroidogen- tional defects of the Golgi caused by ACBD3 knockdown and esis (Papadopoulos et al., 2007; Fan et al., 2010), apoptosis found that ACBD3 is involved in transferring GlcCer through main- (Sbodio et al., 2006; Sbodio and Machamer, 2007), neurogenesis taining the Golgi integrity and determining FAPP2 TGN localization. (Cheah et al., 2006; Zhou et al., 2007), and embryogenesis (Zhou Our lipidomics data indicated that ACBD3 knockdown causes et al., 2007). In addition, ACBD3 recruits the protein phosphatase abnormal GSL metabolism. A pronounced increase of GlcCer PPM1L to ER-Golgi membrane contact sites (Shinoda et al., 2012), Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 ACBD3-maintained Golgi integrity for FAPP2-mediated GlcCer transfer j 113 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. Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 114 j Liao et al. Figure 6 The effects of expression of ACBD3 and its deletion mutants in ACBD3-knockdown cells on GSL metabolic defects by ACBD3 knock- down. (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. to dephosphorylate the ceramide transport protein CERT and RT-PCR was performed using SuperScript reverse transcriptase regulate ceramide transport (Saito et al., 2008). The interaction (Invitrogen) with a poly-dT oligo as a primer. The cDNA obtained between ACBD3 and FAPP2 may facilitate the recruitment of was used as a template for the further PCR reaction and the fol- FAPP2 to ER-Golgi membrane contact sites and translocate GlcCer lowing primers were used: forward primer: 5′-ATGGAGGGGGTGC to the lumen of ER. GlcCer in the ER is then transported to the TGTACAAGTGGAC-3′; reverse primer: 5′-CACTTTATTCCCTGAAG Golgi cisternae via vesicle trafficking for GSL biosynthesis (Halter TTAGG-3′. The purified PCR product was inserted into pEGFP-C2 et al., 2007; D’Angelo et al., 2013a). Since GFP-ACBD3(171–528) (Clontech) or mCherry-C2 (a gift from Dr Zhen Dou, University of had a better rescuing effect on defects of the Golgi integrity and Science and Technology of China) at restriction sites of BglII GSL biosynthesis by ACBD3 knockdown than GFP-ACBD3(1–180), and KpnI following the standard protocol. The plasmid of we deduced that the Golgi integrity is also necessary for GlcCer GFP-tagged ACBD3 was a gift from Dr Carolyn Machamer (Johns transfer at ER-Golgi membrane contact sites. Certainly, this specu- Hopkins University). Site-directed mutagenesis to create lative hypothesis remains to be verified experimentally. Taken siRNA-resistant ACBD3 was performed using Mut Express II fast together, this study has revealed the importance of ACBD3 in mutagenesis kits (Vazyme Biotech Co. Ltd) according to the maintaining the Golgi morphological integrity and as a cofactor manufacturer’s instructions. The ACBD3 target sequence is that functions in the FAPP2-mediated transfer of GlcCer for com- siRNA1 (GGAUGCAGAUUCCGUGAUU, nucleotides 1168–1185), plex GSL biosynthesis. as shown in Supplementary Figure S3. Primers for all plasmids used in this paper are listed in the table of Supplementary Materials and methods material. All constructs were verified by sequencing. Cell growth and DNA transfection HeLa (CCL-2) and 293T cells were cultured according to ATCC guidelines. Briefly, cells were grown in a monolayer at 37°Cin Antibodies 5%CO and maintained in DMEM supplemented with 10% fetal Anti-ACBD3 mouse monoclonal antibody (ab57568), anti- bovine serum and 100 units/ml penicillin and 100 μg/ml strep- GM130 rabbit monoclonal antibody (ab52649), and anti-Giantin tomycin sulfate. For DNA transfection, cells with appropriate rabbit polyclonal antibody (ab24586) were from Abcam. Anti- confluence were transfected with the indicated plasmids in TGN46 sheep polyclonal antibody (AHP500G) was purchased from the experiments of this paper using Lipofectamine 3000 BIO-RAD. Anti-FLAG rabbit antibody (F7425)and anti-α-tubulin (Invitrogen) and OPTI-MEM (Invitrogen) following the manufac- antibody (DM1A) were from Sigma-Aldrich. Anti-GFP rabbit anti- turer’s instructions. body was from Cell Signaling Technology. For immunofluores- cence, AlexaFluor (488, 594,and 647)-conjugated secondary RT-PCR and plasmid construction antibodies were from Invitrogen. For immunoblotting, the horse- RT-PCR was used to fish human FAPP2 DNA. Briefly, total RNA radish peroxidase-conjugated anti-rabbit or anti-mouse antibodies was extracted from HeLa cell using RNeasy Mini Kit (Qiagen). were from Invitrogen. Downloaded from https://academic.oup.com/jmcb/article-abstract/11/2/107/4994671 by Ed 'DeepDyve' Gillespie user on 05 March 2019 ACBD3-maintained Golgi integrity for FAPP2-mediated GlcCer transfer j 115 RNA interference BD-PRDGLTP was taken as a bait to screen Mate & Plate™ The target sequences for ACBD3 siRNAs are listed as follows: Library—Human Testis by yeast mating. Sequence analysis was siRNA1: GGAUGCAGAUUCCGUGAUUTT; siRNA2: GUAUAGAAACCA carried out for those positive prey inserts. UGGAGUUTT; siRNA3: GCAUAUGGGAAGUAACAUUTT; siRNA4: GCAACUGUACCAAGUAAUATT, which were obtained from Immunoprecipitation Genepharma. ACBD3 siRNAs were transfected into HeLa cells with For immunoprecipitation, 293T cells with appropriate conflu- appropriate confluence using the lipofectamine 3000. For rescue ence were transfected with indicated plasmids. After 24 h, they experiments, after 48 htransfected with ACBD3 siRNA, rescue plas- were lysed in buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, mids GFP-ACBD3-4M, GFP-ACBD3(171–528), or GFP-ACBD3(1–180) 0.5% NP-40) supplemented with protease inhibitor cocktail were transfected into the cells. Twenty-four hours later, the rescued (Roche) and phosphatase inhibitor cocktail (Roche). After centri- cells were used for the further experiment. fugation, the supernatant was incubated with FLAG-M2 (Sigma) resin at 4°C for 4 h with gentle rotation. The FLAG-M2 resin was Lipid extraction then spun down and washed extensively before being resolved Lipid was extracted from ∼10 cells using a modified version by SDS-PAGE and western blotting with the indicated antibodies of the Bligh and Dyer’s method as described previously (Lam (Chu et al., 2011). et al., 2016). Briefly, cells were incubated in 750 μl of chloro- form: methanol 1:2 (v/v) with 10% deionized water for 30 min. Immunofluorescence and imaging At the end of the incubation, 350 μl of deionized water and HeLa cells were grown on coverslips before immunofluores- 250 μl of chloroform were added. The samples were then centri- cence. Where indicated, cells were transfected with the indicated fuged and the lower organic phase containing lipids was plasmids. For immunofluorescence, cells were fixed with 4% for- extracted into a clean tube. Lipid extraction was carried out maldehyde in phosphate buffered saline (PBS) for 10 min at twice and the lipid extracts were pooled into a single tube and room temperature and permeabilized with 0.1% Triton X-100 in dried in the SpeedVac under OH mode. Samples were stored at PBS for 10 min. After blocking with PBS with 0.05% Tween-20 −80°C until further analysis. (PBST) containing 1% bovine serum albumin (Sigma) for 30 min, the fixed cells were incubated with primary antibodies for 1 hat Liquid chromatography/mass spectrometry room temperature, followed by second antibodies conjugated Polar lipids were analyzed using an Exion UPLC system with appropriate fluorescence for another 1 h before three times coupled with a triple quadrupole/ion trap mass spectrometer washing with PBST. The DNA was stained with 4′,6-diamidino-2- (6500 Plus Qtrap; SCIEX) as described previously (Lam et al., phenylindole (DAPI). Samples were observed under DeltaVision 2014). Separation of individual lipid classes of polar lipids by microscope system (Applied Precision). Images were captured by normal phase (NP)-high pressure liquid chromatography (HPLC) DeltaVision softWoRx software and processed by deconvolution was carried out using a Phenomenex Luna 3μ-silica column and z-stack projection. After deconvolution, the images were (internal diameter 150 × 2.0 mm) with the indicated conditions exported as 24-bit RGB images and processed with Adobe for mobile phase A (chloroform:methanol:ammonium hydroxide, Photoshop (Bao et al., 2018). 89.5:10:0.5) and mobile phase B (chloroform:methanol:ammonium hydroxide:water, 55:39:0.5:5.5). Multiple reaction monitoring (MRM) Measurement of Golgi area and FAPP2 distribution transitions were set up for comparative analysis of various polar Immunofluorescent images of cells stained with a Golgi lipids. Individual lipid species were quantified by referencing to marker Giantin or transfected with GFP-FAPP2 were measured spiked internal standards. PC-14:0/14:0,PE-14:0/14:0,PS34:1-d31, by splitting the RGB image into the gray image, adjusting PA-17:0/17:0,PG-14:0/14:0,Cer d18:1/17:0,SMd18:1/12:0,GluCer threshold, and setting measurements of their area using ImageJ. d18:1/8:0,GalCer d18:1/8:0,LacCer d18:1/8:0,Sph d17:1 were obtained from Avanti Polar Lipids. Dioctanoyl phosphatidylinosi- tol (PI) (16:0-PI) was obtained from Echelon Biosciences, Inc. Electron microscope Gb3-C17:0 was obtained from Matreya LCC and GM3 d18:1/17:0 The treated HeLa cells were fixed with 2% glutaraldehyde in was synthesized in-house. Free cholesterol was further analyzed 0.1 M sodium cacodylate buffer (pH 7.2) for 30 min and then using HPLC/APCI/MS/MS as previously described with corre- harvested by scraping gently with a plastic scraper. The cells sponding d6-cholesterol (CDN isotopes) as internal standards were pelleted by centrifugation at 800 g for 10 min and fixed for (Shui et al., 2011). a further 2 hin 2% glutaraldehyde in 0.1 M sodium cacodylate buffer. Following washing in buffer, the cells were post-fixed for Yeast two-hybrid screening 1 hin 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. The Matchmaker Gold Yeast Two-Hybrid System (Clontech) The cells were then stained for 1 h with 1% uranyl acetate in was used to search for binding partners of FAPP2 according to water. After dehydration with a graded series of ethanol (50%, the standard protocol provided by the manufacturer. Briefly, full- 70%, 90%, 95%, and 100%), samples were embedded in Epon length FAPP2 and its deletion mutants were inserted into a BD resin. Sections were cut using LEICA EM UC7 ultramicrotome and vector, respectively. 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Journal of Molecular Cell BiologyOxford University Press

Published: Feb 1, 2019

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