Saturated fatty acids increase LPI to reduce FUNDC1 dimerization and stability and mitochondrial function

Abstract

Ectopic lipid deposition and mitochondrial dysfunction are common etiologies of obesity and metabolic disorders. Excessive dietary uptake of saturated fatty acids (SFAs) causes mitochondrial dysfunction and metabolic disorders, while unsaturated fatty acids (UFAs) counterbalance these detrimental effects. It remains elusive how SFAs and UFAs differentially signal toward mitochondria for mitochondrial performance. We report here that saturated dietary fatty acids such as palmitic acid (PA), but not unsaturated oleic acid (OA), increase lysophosphatidylinositol (LPI) production to impact on the stability of the mitophagy receptor FUNDC1 and on mitochondrial quality. Mechanistically, PA shifts FUNDC1 from dimer to monomer via enhanced production of LPI. Monomeric FUNDC1 shows increased acetylation at K104 due to dissociation of HDAC3 and increased interaction with Tip60. Acetylated FUNDC1 can be further ubiquitinated by MARCH5 for proteasomal degradation. Conversely, OA antagonizes PA-induced accumulation of LPI, and FUNDC1 monomerization and degradation. A fructose-, palmitate-, and cholesterol-enriched (FPC) diet also affects FUNDC1 dimerization and promotes its degradation in a non-alcoholic steatohepatitis (NASH) mouse model. We thus uncover a signaling pathway that orchestrates lipid metabolism with mitochondrial quality.

Keywords: FUNDC1; fatty acid metabolism; membrane protein dimerization; mitochondrial quality control.

Figure 1. PA promotes FUNDC1 degradation in a manner that is antagonized by oleic acid

1. A, B

   HepG2 cells were treated for 9 h with 0.5 mM PA, or 0.5 mM OA, or 0.5 mM PA with 0.3 mM OA. An equivalent dosage of 10% BSA solution was used as a control. Mitochondrial morphology was visualized by immunofluorescence analysis using a primary antibody against TOMM20 (A). Immunoblotting analyses were performed to determine the levels of proteins involved in mitochondrial dynamics (B).

2. C–E

   HepG2 cells were treated with 0.5 mM PA or 0.5 mM OA for 9 h. Immunoblotting analyses of mitochondrial proteins were performed (C), followed by statistical analyses of the levels of FUNDC1 (D). FUNDC1 mRNA levels were compared in the same cells by qPCR analyses (E).

3. F

   HepG2 cells were treated with 0.5 mM PA for 9 h. OA was added at the indicated concentrations. Immunoblotting analyses were performed.

4. G

   HepG2 cells were treated with 0.5 mM PA for the indicated times. qPCR analyses were carried out to determine the mitochondrial mass, which was calculated from the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA).

5. H, I

   HepG2 cells were treated for 9 h with 0.5 mM PA, 0.5 mM OA, or 0.5 mM PA with 0.3 mM OA. Relative levels of mtROS were determined by flow cytometry analysis (H), and statistical analyses (I) were performed; n = 3, biological replicates.

6. J

   HepG2 cells were treated with 0.5 mM PA or 0.5 mM OA for 9 h. An equivalent dosage of 10% BSA solution was used as a control. Cells were treated with 10 μM FCCP or DMSO for 6 h before collection. Levels of mitochondrial proteins were assessed by immunoblotting.

7. K

   HepG2 cells were treated for 9 h with 0.5 mM PA, or 0.5 mM OA, or 0.5 mM PA with 0.3 mM OA. The ATP level was then detected.

Data information: In panels (D, E, G, I and K), three biological replicates were analyzed using Student's two‐tailed t‐test. (Results are presented as the means ± SEM. *P < 0.05, **P < 0.01, ns, no significance.) Source data are available online for this figure.

Figure EV1. PA, but not OA and SA, induces FUNDC1 degradation

1. A–C

   HepG2 cells were treated with 0.5 mM PA for the indicated times. Mitochondrial morphology was visualized by immunofluorescence analyses using a primary antibody against TOMM20. The proportion of cells showing different mitochondrial morphologies was determined (C). Approximately 100 cells were analyzed for each treatment. (B) The proportion of cells showing different mitochondrial morphologies was determined about Fig 1A. Approximately 100 cells were analyzed for each treatment.

2. D

   HepG2 cells were treated with 0.5 mM PA for the indicated times. Immunoblotting analyses were performed to detect the levels of proteins related to mitochondrial dynamics.

3. E

   HepG2 cells were treated with 0.5 mM PA for the indicated times. Immunoblotting analyses of mitochondrial proteins were performed.

4. F

   HepG2 cells were treated with the indicated concentration of PA for 12 h. Immunoblotting analyses were performed to detect the level of FUNDC1.

5. G, H

   HeLa cells (G) or C2C12 cells (H) were treated with 0.5 mM PA for the indicated times. FUNDC1 and ACTIN was detected by immunoblotting.

6. I

   HepG2 cells were treated for 9 h with 0.5 mM PA and/or OA at the indicated concentrations. FUNDC1 was detected by immunoblotting.

7. J

   Statistical analyses of the FUNDC1 levels shown in Fig 1F; n = 3. Three biological replicates were analyzed using Student's two‐tailed t‐test. Data are presented as the means ± SEM. *P < 0.05, ns, no significance.

8. K

   HepG2 cells were treated with 0.5 mM SA for the indicated times. Mitochondrial proteins were detected by immunoblotting.

9. L

   Statistical analyses of the levels of mitochondrial proteins shown in Fig 1C; n = 3. (Three biological replicates were analyzed using Student's two‐tailed t‐test, and data are presented as the means ± SEM. ns = no significance).

10. M, N

    Wild‐type HepG2 cells and FUNDC1 KO HepG2 cells were treated with 0.5 mM PA for 9 h. An equivalent dosage of 10% BSA solution was used as a control. Relative levels of mtROS were determined by flow cytometry analysis (J), and statistical analyses (K) were performed; n = 4. (Four biological replicates were analyzed using Student's two‐tailed t‐test, and data are presented as the means ± SEM. ns = no significance).

11. O–Q

    HepG2 cells were transfected with vector or FUNDC1‐Myc for 24 h, then treated with 0.5 mM PA for 9 h, and the OCR was checked via seahorse. (Three or four biological replicates were analyzed using Student's two‐tailed t‐test, and data are presented as the means ± SEM. ns, no significance).

Source data are available online for this figure.

Figure 2. PA promotes FUNDC1 degradation via the CDP‐DAG pathway and LPI

1. A

   Diagram showing the lipid metabolism of free fatty acids.

2. B

   Control cells, CD36 knockdown and TLR4 knockdown HepG2 cells were treated with 0.5 mM PA for 9 h, and the cell lysates were then subjected to Western blotting analysis.

3. C

   HepG2 cells were transiently transfected with siRNA specifically targeting CDS1, CDS2 or control siRNA for 48 h. Cells were then treated with BSA or PA (0.5 mM) for 9 h, and the cell lysates were then subjected to Western blotting analysis.

4. D–F

   HepG2 cells were treated for 9 h with 0.5 mM PA, 0.5 mM OA, or 0.5 mM PA with 0.3 mM OA, and then collected for lipidomics analyses, the relative level analysis about phospholipids (D), lysophospholipids (E) and lysophosphatidylinositol (LPI) (F) were performed.

5. G

   Control cells and cPLA2α knockdown HepG2 cells were treated with 0.5 mM PA for 6 h, then the cell lysates were subjected to Western blotting analysis (left), and statistical analyses (right) were performed to determine the relative level of FUNDC1.

6. H

   Control cells and cPLA2α knockdown HepG2 cells were treated with 0.5 mM PA for 6 h, and mitochondrial morphology was visualized by immunofluorescence analysis using a primary antibody against TOMM20.

7. I

   HepG2 cells were treated for 9 h with BSA, 0.5 mM PA, or 0.5 mM OA, and then collected for mitochondrial isolation, which was used for further lipidomics analyses, and relative level analysis of lysophosphatidylinositol (LPI) was performed.

Data information: In panels (D, E, F, G and I), three or four biological replicates were analyzed using Student's two‐tailed t‐test. (Results are presented as the means ± SEM. *P < 0.05, **P < 0.01, ns, no significance.) Source data are available online for this figure.

Figure EV2. PA promotes FUNDC1 degradation via the CDP‐DAG pathway

1. A, B

   HepG2 cells were transfected with pLKO.1 plasmid expressing shRNA (shCD36 or shTLR4) specifically targeting CD36 or TLR4 or the scramble control. Cells were then treated with puromycin to select stable CD36 or TLR4 knockdown cell lines. The level of CD36 or TLR4 mRNA was assessed by qPCR analyses (A). CD36 and TLR4 was detected by immunoblotting (B).

2. C

   Diagram showing the β‐oxidation of free fatty acids.

3. D

   HepG2 cells were treated with BSA or 0.5 mM PA for 9 h. The CPT1α inhibitor etomoxir (ETO) (25 μM) was added 1 h before PA treatment. Cell lysates were subjected to Western blotting.

4. E

   Control cells and CPT1α knockdown HepG2 cells were treated with 0.5 mM PA for 9 h, and the cell lysates were then subjected to Western blotting analysis.

5. F

   Diagram showing the ceramide synthesis pathway from palmitic acids.

6. G

   HepG2 cells were transfected with pLKO.1 plasmid expressing shCPT1α specifically targeting CPT1α or the scramble control. Cells were then treated with puromycin to select stable CPT1α knockdown cell lines. The level of CPT1α mRNA was assessed by qPCR analyses.

7. H

   Control cells and CPT1α knockdown HepG2 cells were treated with 0.5 mM PA for 9 h, and the cell lysates were then subjected to Western blotting analysis.

8. I

   HeLa cells were treated with BSA or 0.5 mM PA for 9 h. The inhibitor of ceramide synthases (CerS) Fumonisin B1 (FB1) (10 μM or 20 μM) was added 1 h before PA treatment. Cell lysates were subjected to Western blotting.

9. J

   HepG2 cells were treated for 9 h with 0.5 mM PA, or 0.5 mM OA, or 0.5 mM PA with 0.3 mM OA, and then collected for lipidomics analyses, Relative level analysis of triacylglycerol (TAG) was performed.

10. K

    HepG2 cells were treated for 9 h with 0.5 mM PA, or 0.5 mM OA, or 0.5 mM PA with 0.3 mM OA, and then collected for analysis the level of triacylglycerol (TAG) using triglyceride assay kit.

11. L

    Relative mRNA level of genes about peroxisomes biogenesis or peroxisomes related.

12. M

    HepG2 cells were treated with BSA or 0.5 mM PA for 9 h. The related inhibitor of phospholipase A2 family members (PAF‐AH, iPLA2, cPLA2), was added 1 h before PA treatment. Cell lysates were subjected to Western blotting.

13. N, O

    HepG2 cells were transiently transfected siRNA specifically targeting TAMM41 (1# and 2#) or control siRNA for 48 h. Cells were then treated with BSA or PA (0.5 mM) for 9 h, the level of TAMM41 mRNA was assessed by qPCR analyses (O), and the cell lysates were then subjected to Western blotting analysis (N).

14. P

    The levels of CDS1 mRNA and CDS2 mRNA were assessed by qPCR analyses. Relates to Fig 2C,

15. Q

    The level of cPLA2a mRNA was assessed by qPCR analyses. Relates to Fig 2G.

16. R

    The proportion of cells showing different mitochondrial morphologies was determined about Fig 2H. Approximately 30 cells were analyzed for each treatment.

Data information: In panels (A, G, J, K, L, O, P and Q), three or four biological replicates were analyzed using Student's two‐tailed t‐test. (Results are presented as the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance). Source data are available online for this figure.

Figure 3. PA disrupts FUNDC1 homodimerization

1. A

   HEK293T cells were transiently transfected with FUNDC1‐Flag and FUNDC1‐Myc, and IP was performed with an anti‐Myc antibody. The interaction between Flag‐ and Myc‐tagged FUNDC1 was detected through Western blotting using an anti‐Flag antibody.

2. B

   HepG2 cells and two FUNDC1 KO HepG2 cell lines (1# and 2#) were collected, then treated with 1 mM DSS for 10 min at room temperature before quenching. Homodimers of FUNDC1 were detected by Western blot using an anti‐FUNDC1 antibody.

3. C

   HEK293T cells were transfected with Flag‐FUNDC1 and wild‐type FUNDC1‐Myc, or the indicated truncations, or the vector. Pull down was performed with Ni‐NTA beads. Interactions were detected through Western blotting using an anti‐Flag antibody.

4. D

   HEK293T cells were transfected with Flag‐FUNDC1 and wild‐type FUNDC1‐Myc, or the indicated truncations, or the vector, and IP was performed with an anti‐Myc antibody. Interactions were detected through Western blotting using an anti‐Flag antibody.

5. E, F

   HepG2 cells were treated with 0.5 mM PA (E) or OA (F) for the indicated times. Cells were collected and treated with 1 mM DSS for 10 min at room temperature before quenching. Homodimers of FUNDC1 were detected through Western blotting using an anti‐FUNDC1 antibody.

6. G

   Control cells and cPLA2α knockdown HepG2 cells were treated with 0.5 mM PA for 6 h. Cells were collected and treated with 1 mM DSS for 10 min at room temperature before quenching. Homodimers of FUNDC1 were detected through Western blotting using an anti‐FUNDC1 antibody.

7. H

   HEK293T cells were transiently transfected with FUNDC1‐Flag and FUNDC1‐Myc, and cells were collected and lysed in digitonin lysis buffer. IP was performed with an anti‐Myc magnetic beads, and then reaction buffer with indicated concentration of LPI was added, the interaction was detected through Western blotting using an anti‐Flag antibody.

8. I

   HepG2 cells were treated with LPI used as the indicated concentration for 24 h. Immunoblotting analyses of mitochondrial proteins were performed.

9. J

   The mice were fed with FPC diet for 16 weeks, and the protein level of FUNDC1 in liver were detected through Western blotting.

10. K

    Primary hepatocytes were isolated from control mice or mice fed with the FPC diet for 16 weeks, and then treated with 1 mM DSS for 30 min at room temperature before quenching. Homodimers of FUNDC1 were detected through Western blotting using an anti‐FUNDC1 antibody.

Source data are available online for this figure.

Figure 4. LPI disrupts FUNDC1 homodimerization

1. Diagram showing the GXXXG motifs in FUNDC1. The amino acid sequence of the two motifs in the first transmembrane region is shown underneath. IMS, the mitochondrial intermembrane space; LIR, LC3‐interacting region; OMM, the outer mitochondrial membrane.

2. HEK293T cells were transiently transfected with FUNDC1‐Flag and different mutants (G57/61L, G61/65L, G57/61/65L) of FUNDC1‐Myc. IP was performed with an anti‐Flag antibody, and then the interaction was detected through Western blotting using an anti‐Myc antibody.

3. HEK293T cells were cotransfected or transfected individually with FUNDC1‐Flag and FUNDC1‐Myc, and cells were collected and lysed in digitonin lysis buffer. Lysates from the individually transfected cells were mixed. IP was then performed with anti‐Myc antibody or anti‐Flag antibody to check the self‐interaction of FUNDC1 in the different systems.

4. Superimposition of molecular dynamics (MD) simulations of a dimer of the first transmembrane domain of FUNDC1 (wild‐type and the GXXXG motif mutant G57/61L).

5. Superimposition of molecular dynamics (MD) simulations of a dimer of the first transmembrane domain of FUNDC1 with LPI 16:0.

Source data are available online for this figure.

Figure EV3. The homodimerization of FUNDC1

1. The reported structure of FUNDC1 in the PDB database.

2. The model of the homodimer structure of FUNDC1‐TM (Tyr47‐Tyr70) based on the I‐TASSER Server and the ClusPro Server. Monomer 1 is shown as a surface model (white) and monomer 2 is shown as a cartoon model (blue).

3. Superimposed models of the dimer of FUNDC1‐TM (Tyr47‐Tyr70) and the dimerization domain of BNIP3 (PDB ID: 2KA2). The structure of FUNDC1‐TM is shown as blue and the structure of BNIP3 is shown as yellow. The GXXXG motifs between the monomers are shown as clubbed, and the two boundaries of the membrane lipid bilayer are shown as red and blue lines.

Figure 5. PA promotes FUNDC1 degradation via acetylation

1. FUNDC1 knockout HepG2 cells were transiently transfected with wild‐type FUNDC1‐Myc for 24 h. IP was performed with anti‐IgG and a polyclonal antibody against acetylated lysine (Ace‐K). Co‐IP exogenous Myc‐tagged protein was detected by Western blotting.

2. HepG2 cells were treated with BSA or 0.5 mM PA for 6 h. IP was performed with an anti‐FUNDC1 antibody. Co‐IP endogenous FUNDC1 and total acetylated lysine (Ace‐K) were detected by Western blotting using appropriate antibodies.

3. HEK293T cells were cotransfected with plasmids containing wild‐type FUNDC1‐Myc or mutants (K100R, K104R, K108R) and empty vector or Flag‐tagged HDAC3. Pull down was performed with Ni‐NTA beads. Acetylated lysine (Ace‐K) was detected in the pull‐down fractions through Western blotting with a polyclonal antibody.

4. HEK293T cells were transiently transfected with FUNDC1‐Myc or the K104R mutant and Flag‐Tip60 or vector. Pull down was performed with Ni‐NTA beads and the level of acetylated lysine (Ace‐K) was detected with a polyclonal antibody via Western blotting.

5. HEK293T cells transiently transfected with plasmids containing wild‐type FUNDC1‐Flag or the K104R mutant were treated with or without 0.5 mM PA for 6 h. IP was performed with an anti‐Flag antibody, and the level of Co‐IP acetylated lysine (Ace‐K) was detected with a polyclonal antibody after Western blotting.

6. HEK293T cells were transiently co‐transfected with Flag‐Tip60 or vector and wild‐type FUNDC1‐Myc or the G57/61L mutant. IP was performed with an anti‐Flag antibody and the Co‐IP FUNDC1‐Myc was detected through Western blotting using an anti‐Myc antibody.

7. HEK293T cells were transiently cotransfected with HDAC3‐Flag or vector and wild‐type FUNDC1‐Myc or the G57/61L mutant. IP was performed with an anti‐Flag antibody, and the Co‐IP FUNDC1‐Myc was detected through Western blotting using an anti‐Myc antibody.

8. HEK293T cells were transfected individually with FUNDC1‐Myc or HDAC3‐Flag, and then cells were collected and lysed as follows: cells with HDAC3‐Flag were lysed in digitonin lysis buffer and cells with FUNDC1‐Myc were half lysed in digitonin lysis buffer and half lysed in NP‐40 lysis buffer. The cell lysates were mixed to get two systems (“a” and “b”): in system “a”, the NP‐40 part of FUNDC1‐Myc was mixed with half lysis of HDAC3‐Flag; in system “b”, the digitonin part of FUNDC1‐Myc was mixed with another half lysis of HDAC3‐Flag. IP was performed with an anti‐Flag antibody. The interaction was detected through Western blotting using an anti‐Myc antibody.

Source data are available online for this figure.

Figure EV4. HDAC3 and Tip60 regulate the deacetylation and acetylation of FUNDC1

1. A, B

   HEK293T cells were transiently transfected with vector and Myc‐tagged FUNDC1 (A) or Flag‐tagged FUNDC1 (B) and treated with or without 10 μM TSA and 10 mM NAM for 12 h. Pull down was then performed with Ni‐NTA beads (A) or IP was performed with an anti‐Flag antibody (B). Acetylated lysine (Ace‐K) was detected in the pull‐down or the Co‐IP fraction by Western blotting using a polyclonal antibody.

2. C

   HepG2 cells were treated with or without 10 μM TSA or 10 mM NAM for 12 h, then lysed and subjected to Western blotting.

3. D

   HEK293T cells were transfected with FUNDC1‐Myc and Flag‐tagged HDACs (HDAC1‐8) or the vector. IP was performed with an anti‐Flag antibody and the Co‐IP FUNDC1‐Myc was detected through Western blotting using an anti‐Myc antibody.

4. E

   A model of FUNDC1 in the outer mitochondrial membrane. The positions of 11 lysines are shown. Transmembrane regions are colored green.

5. F

   HEK293T cells were transfected with wild‐type FUNDC1‐Myc, or FUNDC1‐Myc with mutations of the 11 lysines, or vector. Pull down was performed with Ni‐NTA beads and levels of acetylated lysine (Ace‐K) in the pull‐down fractions were detected through Western blotting using a polyclonal antibody.

6. G

   HEK293T cells were cotransfected with a plasmid expressing Myc‐tagged FUNDC1 and empty vector or plasmids expressing Flag‐tagged HDAC1, HDAC3, and HDAC6. Pull down was performed with Ni‐NTA beads, and the level of acetylated lysine (Ace‐K) in the pull‐down fraction was detected with a polyclonal antibody after Western blotting.

7. H

   HEK293T cells were transiently transfected with FUNDC1‐Myc and different acyltransferases (Flag‐GCN5, Flag‐PCAF, Flag‐Tip60, P300‐Flag, Flag‐CBP). Pull down was performed with Ni‐NTA beads. Acetylated lysine (Ace‐K) in the pull‐down fraction was detected with a polyclonal antibody via Western blotting.

8. I

   HEK293T cells were transiently transfected with FUNDC1‐Myc and different acyltransferases (Flag‐GCN5, Flag‐PCAF, Flag‐Tip60, P300‐Flag, Flag‐CBP). IP was performed with an anti‐Flag antibody. The interaction was detected through Western blotting using an anti‐Myc antibody.

Source data are available online for this figure.

Figure 6. PA promotes FUNDC1 degradation by MARCH5 and dependent on acetylation

1. HeLa cells were transfected with FUNDC1‐Myc or FUNDC1‐G57/61L‐Myc, and then treated with 20 μM CHX for the indicated times. The stability of FUNDC1‐Myc was detected through Western blotting using an anti‐Myc antibody (top). Statistical analyses (bottom) of the Western blotting results were performed, three biological replicates were analyzed using Student's two‐tailed t‐test. Data are presented as the means ± SEM. ***P < 0.001.

2. HEK293T cells were transiently transfected with FUNDC1‐Flag and treated with BSA, PA or OA for 6 h. IP was then performed with an anti‐Flag antibody. Co‐IP endogenous ubiquitin was detected through Western blotting using an anti‐ubiquitin antibody (FK2).

3. HepG2 cells were treated with 0.5 mM PA for the indicated times. IP was performed with an anti‐FUNDC1 antibody. Co‐IP endogenous MARCH5 was detected by Western blotting.

4. HEK293T cells were transiently co‐transfected with GFP‐MARCH5‐HW or vector and wild‐type FUNDC1‐Myc or the G57/61L mutant. IP was performed with an anti‐GFP antibody and the Co‐IP FUNDC1‐Myc was detected through Western blotting using an anti‐Myc antibody.

5. HepG2 cells were treated with or without 10 μM TSA for 12 h. IP was performed with an anti‐FUNDC1 antibody. Co‐IP endogenous MARCH5 and acetylated lysine were detected by Western blotting with appropriate antibodies.

6. HEK293T cells were transiently co‐transfected with GFP‐MARCH5‐HW or vector and wild‐type FUNDC1‐Myc or the G57/61L mutant. IP was performed with an anti‐GFP antibody and the Co‐IP FUNDC1‐Myc was detected through Western blotting using an anti‐Myc antibody.

7. HEK293T cells were transiently transfected with wild‐type or K104R FUNDC1‐Flag and GFP‐MARCH5 or vector, and treated with 10 μM MG132 for 6 h before collection. IP was performed with an anti‐FLAG antibody and the ubiquitination of FUNDC1 was examined using the anti‐ubiquitin antibody FK2 after Western blotting.

8. A hypothetical model showing the molecular mechanism by which PA regulates the mitophagy receptor protein FUNDC1. Under normal conditions, FUNDC1 associates with HDAC3 and forms homodimers via the highly conserved dimerization motif GXXXG within its first transmembrane domain. FUNDC1 is deacetylated at K104 by HDAC3. PA disrupts the homodimerization of FUNDC1 via LPI 16:0 which antagonized by OA and attenuates the interaction of FUNDC1 with HDAC3, and promotes the disassociation of FUNDC1 homodimers to monomers. Furthermore, PA facilitates Tip60‐mediated acetylation of FUNDC1 at K104. Acetylated FUNDC1 can then interact with MARCH5 for ubiquitination at K119, followed by proteasomal degradation. OMM: the outer mitochondrial membrane.

Source data are available online for this figure.

Figure EV5. PA promotes FUNDC1 degradation

1. A

   HepG2 cells were treated with BSA or 0.5 mM PA for 9 h. The proteasomal inhibitor MG132 (10 μM) or the autophagic inhibitors bafilomycin A1 (BA1) (10 nM) or chloroquine (CQ) (20 μM) were added 6 h before harvesting. Cell lysates were subjected to Western blotting (top), and statistical analyses (bottom) were performed to determine the relative level of FUNDC1, three biological replicates were analyzed using Student's two‐tailed t‐test and data are presented as the means ± SEM. *P < 0.05, ns, no significance.

2. B

   HeLa cells were transiently transfected with pLKO.1 plasmid expressing different shRNAs (shMARCH5 1# and shMARCH5 2#) specifically targeting MARCH5 or scramble shRNA and then treated with puromycin to select stable MARCH5 knockdown cell lines. The cells were treated with 0.5 mM PA for 6 h and then harvested. Cell lysates were subjected to western blotting (top), and statistical analyses (bottom) were performed to determine the relative level of FUNDC1, three biological replicates were analyzed using Student's two‐tailed t‐test and data are presented as the means ± SEM. *P < 0.05, **P < 0.01, ns, no significance.

3. C

   HEK293T cells were transiently transfected with pLKO.1 plasmid expressing shRNA specifically targeting MARCH5 or scramble shRNA and FUNDC1‐Flag for 24 h. Cells were then treated with BSA or PA (0.5 mM) for 6 h. IP was performed with an anti‐Flag antibody. Co‐IP endogenous ubiquitin was detected through western blotting using an anti‐ubiquitin antibody (FK2). Scr, scramble shRNA.

4. D, E

   The model of the full‐length structure of MARCH5 (D) and FUNDC1 (E) based on the ClusPro Server.

5. F, G

   Superimposition of molecular dynamics (MD) simulations of MARCH5 and FUNDC1.

6. H

   HepG2 cells were transiently transfected with vector or Flag‐HDAC3 for 24 h, and then treated with 0.5 mM PA for the described times, the obtained lysates were then subjected to Western blotting analysis (top), and statistical analyses of the FUNDC1 levels are shown (bottom), n = 3, three biological replicates were analyzed using Student's two‐tailed t‐test, and data are presented as the means ± SEM. *P < 0.05.

7. I

   Control cells and HDAC3 knockdown HepG2 cells were treated with 0.5 mM PA for the indicated times, and the cell lysates were then subjected to Western blotting analysis (top), and statistical analyses of the FUNDC1 levels are shown (bottom), n = 3, three biological replicates were analyzed using Student's two‐tailed t‐test, and data are presented as the means ± SEM. *P < 0.05.

8. J

   Control cells and HDAC3 knockdown HepG2 cells were treated with 20 μM CHX for the indicated times, the cell lysates were then subjected to Western blotting analysis (top), and statistical analyses of the FUNDC1 levels are shown (bottom), n = 3, three biological replicates were analyzed using Student's two‐tailed t‐test, and data are presented as the means ± SEM. **P < 0.01.

Source data are available online for this figure.