Knock-Out of ACBD3 Leads to Dispersed Golgi Structure, but Unaffected Mitochondrial Functions in HEK293 and HeLa Cells

Abstract

The Acyl-CoA-binding domain-containing protein (ACBD3) plays multiple roles across the cell. Although generally associated with the Golgi apparatus, it operates also in mitochondria. In steroidogenic cells, ACBD3 is an important part of a multiprotein complex transporting cholesterol into mitochondria. Balance in mitochondrial cholesterol is essential for proper mitochondrial protein biosynthesis, among others. We generated ACBD3 knock-out (ACBD3-KO) HEK293 and HeLa cells and characterized the impact of protein absence on mitochondria, Golgi, and lipid profile. In ACBD3-KO cells, cholesterol level and mitochondrial structure and functions are not altered, demonstrating that an alternative pathway of cholesterol transport into mitochondria exists. However, ACBD3-KO cells exhibit enlarged Golgi area with absence of stacks and ribbon-like formation, confirming the importance of ACBD3 in Golgi stacking. The glycosylation of the LAMP2 glycoprotein was not affected by the altered Golgi structure. Moreover, decreased sphingomyelins together with normal ceramides and sphingomyelin synthase activity reveal the importance of ACBD3 in ceramide transport from ER to Golgi.

Case report: A rare variant m.4135T>C in the MT-ND1 gene leads to Leber hereditary optic neuropathy and altered respiratory chain supercomplexes

“…Briefly, the samples were transported on ice (at 4°C) and mitochondria were isolated immediately by standard differential centrifugation in buffer containing 150 mM KCl, 50 mM Tris/HCl (pH 7.5), 2 mM EDTA, and 2 μg/mL aprotinin. Studied skin fibroblasts were cultivated under the standard condition in high-glucose Dulbecco’s modified Eagle medium (DMEM; Pan Biotech, Aidenbach, Germany) supplemented with 10% (v/v) fetal bovine serum (GE Healthcare, Chicago, IL, United States) and antibiotic–antimycotic solution (Biosera, Nuaile, France), as described previously ( Daňhelovská et al, 2021 ), and did not exceed passage 10. For Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), mitochondria were isolated by standard differential centrifugation ( Stiburek et al, 2005 ).…”

“…In fibroblasts, CI was detected by three different antibodies (NDUFA9, NDUFV1, and NDUFS3). The immunoblots were detected with peroxidase-conjugated secondary antibodies by chemiluminescence using G:Box (Syngene, Cambridge, United Kingdom) and analyzed by Quantity one (Bio-Rad) or ImageJ 1.48 v (Wayne Rasband, National Institutes of Health, Bethesda, Maryland, United States), as described previously ( Daňhelovská et al, 2021 ).…”

Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins

“…ACBD3 is a 528 amino acid protein containing ACBD, CAR-Q, and GOLD domains (Figure 1A) that has been reported to localise to the Golgi apparatus 24,25,32,36 . We performed highresolution Airyscan imaging of HeLa cells immunolabeled for ACBD3 and cis and trans-Golgi markers.…”

“…Loss of ACBD3 is lethal in mice, and ACBD3 is upregulated in multiple breast cancers in which high ACBD3 expression correlates with poor patient survival 22,23 . ACBD3 is a cytosolic protein mainly localised to the Golgi apparatus and is important for Golgi organisation 24,25 . ACBD3 consists of an ACBD domain, a CAR-Q domain, a 'unique region', and a GOLD domain.…”

Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins

“…ACBD3 is a cytosolic protein, mainly localized to the Golgi apparatus and important for Golgi organization ( Liao et al. , 2019 ; Daňhelovská et al. , 2021 ).…”