Endogenous interactomes of MFN1 and MFN2 provide novel insights into interorganelle communication and autophagy

Abstract

MFN1 (mitofusin 1) and MFN2 are key players in mitochondrial fusion, endoplasmic reticulum (ER)-mitochondria juxtaposition, and macroautophagy/autophagy. However, the mechanisms by which these proteins participate in these processes are poorly understood. Here, we studied the interactomes of these two proteins by using CRISPR-Cas9 technology to insert an HA-tag at the C terminus of MFN1 and MFN2, and thus generating HeLa cell lines that endogenously expressed MFN1-HA or MFN2-HA. HA-affinity isolation followed by mass spectrometry identified potential interactors of MFN1 and MFN2. A substantial proportion of interactors were common for MFN1 and MFN2 and were regulated by nutrient deprivation. We validated novel ER and endosomal partners of MFN1 and/or MFN2 with a potential role in interorganelle communication. We characterized RAB5C (RAB5C, member RAS oncogene family) as an endosomal modulator of mitochondrial homeostasis, and SLC27A2 (solute carrier family 27 (fatty acid transporter), member 2) as a novel partner of MFN2 relevant in autophagy. We conclude that MFN proteins participate in nutrient-modulated pathways involved in organelle communication and autophagy.Abbreviations: ACTB: actin, beta; ATG2: autophagy related 2; ATG5: autophagy related 5; ATG12: autophagy related 12; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; Baf A1: bafilomycin A₁; BECN1: beclin 1, autophagy related; BFDR: Bayesian false discovery rate; Cas9: CRISPR-associated endonuclease Cas9; CRISPR: clustered regularly interspaced short palindromic repeats; DNM1L/DRP1: dynamin 1-like; ER: endoplasmic reticulum; Faa1: fatty acid activation 1; FC: fold change; FDR: false discovery rate; FIS1: fission, mitochondrial 1; GABARAP: gamma-aminobutyric acid receptor associated protein; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HA: hemagglutinin; KO: knockout; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MARCHF5: membrane associated ring-CH-type finger 5; MDVs: mitochondria-derived vesicles; MFN1: mitofusin 1; MFN2: mitofusin 2; NDFIP2: Nedd4 family interacting protein 2; OMM: outer mitochondrial membrane; OPA1: OPA1, mitochondrial dynamin like GTPase; OXPHOS: oxidative phosphorylation; PE: phosphatidylethanolamine; PINK1: PTEN induced putative kinase 1; PS: phosphatidylserine; RAB5C: RAB5C, member RAS oncogene family; S100A8: S100 calcium binding protein A8 (calgranulin A); S100A9: S100 calcium binding protein A9 (calgranulin B); SLC27A2: solute carrier family 27 (fatty acid transporter), member 2; TIMM44: translocase of inner mitochondrial membrane 44; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like kinase 1; VCL: vinculin; VDAC1: voltage-dependent anion channel 1; WT: wild type.

Keywords: Autophagosomes; endosomes; mitochondria; mitochondria-endoplasmic reticulum contact sites; mitochondrial dynamics; nutrient deprivation.

Figure 1.

Generation and characterization of MFN1-HA and MFN2-ha CRISPR-Cas9-engineered cell lines. (A) distribution of functional domains of WT and HA-tagged forms of MFN1 and MFN2 and ha-immunoblot of analyzed crispr-engineered clones. (B) protein expression levels of MFN1 and MFN2 in ha-tagged cell lines relative to ACTB expression. N = 3. (C) Protein expression levels of OPA1 and VDAC1 in ha-tagged cell lines relative to ACTB expression. N = 3. (D) HA, MFN1 and MFN2 western blots of HA-IP performed in the ha-tagged cell lines. Input, supernatant (SN) and eluate (IP) fractions are shown. (E) quantification of MFN1 and MFN2 in different HA-IP fractions. (F) Representative images of fixed WT, MFN1-HA and MFN2-ha cells stained with an antibody against TOMM20 and DAPI. Scale bar: 5 μm. (G) quantification of mitochondrial morphology in WT, MFN1-HA and MFN2-ha HeLa cells. Percentages of each category of cells (elongated, short, oval) per experiment are shown. Three independent experiments were performed and > 120 cells were counted per cell line per experiment. Data are represented as mean ± SEM. Values were analyzed for normality with Saphiro-Wilk test and compared using paired T-test.

Figure 2.

Identification of binding candidates of MFN1-HA and MFN2-ha by MS after HA-IP. (A) HA, MFN1 and MFN2 immunoblots of input, supernatant (SN) and eluate (IP) fractions of HA-IP of WT, MFN1-HA and MFN2-ha cells in basal conditions and after 1 h of EBSS treatment. Input = 0.5%, SN = 0.5%, IP = 5%. (B) dot plot diagrams of mass spectrometry results after applying SAINT express algorithm. Bait proteins are shown in red. Known interactors of MFN1 and MFN2 found enriched after SAINT express analysis are shown in green or in blue respectively. Binding candidates showing BFDR ≥ 0.05 are displayed in black. Proteins with BFDR < 0.05 appear in gray. Dotted lines inside the diagram represent the thresholds of -log BFDR and log FC to consider a protein as a high confidence interactor. (C) venn diagrams comparing the potential binding candidates identified by mass spectrometry after HA-IP of MFN1-HA and MFN2-ha under basal and starvation conditions.

Figure 3.

MFN1 and MFN2 interact with proteins from different membranous compartments of the cell. (A) dot plots generated with ProHits visualization tool of all the significant proteins localized in the endoplasmic reticulum, endosome and plasma membrane. Dot color intensity refers to the average number of unique peptides of a protein found in the 3 biological replicates of the experiment. Dot size represents the relative abundance of each interactor across all the HA-IPs. Line color stands for the statistical significance with which each protein was found. (B) FLAG-IP of overexpressed FLAG-RAB5C in HeLa cells. FLAG and MFN1 immunoblots of input and eluate (IP) fractions are shown. N = 6. (C) FLAG-IP of overexpressed SLC27A2-FLAG in HeLa cells. FLAG and MFN2 immunoblots of input and eluate (IP) fractions are shown. N = 6.

Figure 4.

RAB5C ablation alters mitochondrial homeostasis. (A) RAB5C immunoblot of WT cells and RAB5C KO cells lysates. (B) MFN1 and MFN2 immunoblots of WT cells and RAB5C KO cells lysates. (C) TOMM20, VDAC1, TIMM44, OPA1, DNM1L, FIS1, TFAM, NDUFB8 (complex 1), UQCRC2 (complex 3), MT-CO1 (complex 4) and ATP5F1A (complex 5) immunoblots of WT cells and RAB5C KO cells lysates. (D) quantification of immunoblots of mitochondrial proteins in WT and RAB5C KO cells. N = 5–6. (E) immunoblots of TOMM20, VDAC1 and RAB5C in WT and RAB5C KO HeLa cells, or RAB5C KO cells transfected with FLAG-RAB5C (KO+OE), and total protein staining. (F) quantification of TOMM20 immunoblots of WT and RAB5C KO cells. Data were expressed relative to the WT group, and the dashed line indicates WT group levels. N = 4. (G) quantification of VDAC1 immunoblots of WT and RAB5C KO cells. Data were expressed relative to the WT group, and the dashed line indicates WT group levels. N = 4. (H) quantification of mitochondrial morphology in WT and RAB5C KO cells. Percentages of each category of cells (elongated, short, oval) per experiment are shown. Three independent experiments were performed and > 120 cells were counted per cell line per experiment. N = 3. (I) mitochondrial mass quantified as relative fluorescence units using FACS after MitoTracker green staining. N = 6. (J) Representative images of mitochondrial DNA (mtDNA) staining in WT and RAB5C KO cells. Scale bar: 5 μm. (K) quantification of mitochondrial DNA nucleoids of WT and RAB5C KO cells. 22 WT and 17 RAB5C KO cells were analyzed. Data are represented as mean ± SEM. Values were analyzed for normality with Saphiro-Wilk test and compared using paired T-test or one-way ANOVA when following a normal distribution. ((D), (F), (G) and (I)) or with Wilcoxon test when the distribution was not normal ((K)). Unless indicated, * refers to comparisons between WT and RAB5C KO cells. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001.

Figure 5.

RAB5C depletion induces mitochondrial degradation. (A) PINK1 immunoblot of WT and RAB5C KO cells. (B) quantification of PINK1 protein levels in WT and RAB5C KO cells. N = 6. (C) immunoblots of TOMM20, MFN1 and VDAC1 in WT and RAB5C KO cells with and without 4 h of baf A1 treatment. (D-F) quantification of TOMM20, VDAC1 and MFN1 levels in WT and RAB5C KO cells. N = 3. (G) basal autophagic (AP) flux of TOMM20, VDAC1 and MFN1 after 4 h of baf A1 treatment. (H) Representative images of WT and RAB5C KO cells in basal conditions and after baf A1 treatment stained for mtDNA. We counted 22 WT cells in basal conditions and 20 after baf A1 treatment. Scale bar: 5 μm. (I) quantification of mitochondrial DNA nucleoids under basal conditions and after baf A1 treatment. 17 RAB5C KO cells in basal conditions and 22 after baf A1 treatment were analyzed. (J) halo, RAB5C and ACTB immunoblots in pSu9-halo-mGFP control and RAB5C KO HeLa cells treated with halo-ligand for 0 h or 4 h. (K) quantification of basal mitochondrial degradation in pSu9-halo-mGFP control and RAB5C KO HeLa cells. Dashed line at 1 indicates baseline level of mitochondrial removal at 0 h. N = 4. Data are represented as mean ± SEM. Values were analyzed for normality with Saphiro-Wilk test and compared using a t-test (B, G, K) or two-way ANOVA with Tukey test (D, E, F) when following a normal distribution or with Wilcoxon test when the distribution was non-normal (I). Comparisons between RAB5C KO and WT are indicated by *. Comparisons between basal condition and baf A1 treatment are indicated by #. * or #: p ≤ 0.05, ** or ##: p ≤ 0.01, *** or ###: p ≤ 0.001, **** or ####: p ≤ 0.0001.

Figure 6.

RAB5C interacts with MFN1 through Trp75, Arg82, Tyr83 and Tyr90. (A, and B) predicted aligned error (PAE) generated with AlphaFold2 multimer showing the confidence measure of the interaction between RAB5C and MFN1 (A) or RAB5C and MFN2 (B). The coordinates (x,y) correspond to the residues, and the color is mapped to the expected position error at residue x if the predicted and true structures were aligned on residue y (in Å), as shown in the color scale at the right side of the plots. Chain a is either MFN1 or MFN2, and chain B is RAB5C. (C) Model of the potential RAB5C-MFN2 interaction according to AlphaFold2 multimer. Residues colored in red and named are RAB5C residues relevant in the interaction. (D) Model of RAB5C-MFN1 interaction. Residues colored in red and named are RAB5C residues relevant in the interaction. (E) HA-IP of overexpressed FLAG-RAB5C WT or mutant version in MFN1-HA HeLa cells. FLAG, and MFN1 immunoblots of eluate (IP) and input fractions are shown. N = 2.

Figure 7.

SLC27A2 ablation results in impaired autophagy. (A) LC3B-II immunoblot of control cells and cells with transient ablation of MFN2 or SLC27A2 after 1 h of EBSS and Baf A1 treatment. (B) LC3B-II starvation autophagic flux of siCtrl, siMFN2 and siSLC27A2 treated cells. siCtrl N = 8, siMFN2 N = 3, siSLC27A2 N = 7. (C) Representative images of LC3B staining in 48 h siCtrl, siMFN2 and siSLC27A2 treated cells in basal conditions and starved for 1 h with EBSS. Scale bar: 5 μm. (D) quantification of the number of LC3B puncta in basal and starvation conditions. More than 50 cells were analyzed per experimental condition. (E) quantification of LC3 total puncta in mCherry-GFP-LC3 HeLa cells after silencing MFN2 or SLC27A2 in basal and 1 h EBSS treatment. (F) quantification of mCherry⁺ and GFP⁻ puncta in mCherry-GFP-LC3 HeLa cells after silencing MFN2 or SLC27A2 in basal and 1 h EBSS treatment. (G-I) quantification of ATG5, WIPI2 and ATG2A puncta in HeLa cells after silencing MFN2 or SLC27A2 in basal and 1 h EBSS treatment. In figure H, data of 2 different experiments are shown, where ● indicates “experiment 1” values and indicates “experiment 2” values. Data are represented as mean ± SEM. Outlier test was performed, and outliers were removed. Values were analyzed for normality with Saphiro-Wilk test and compared using one-way ANOVA when following a normal distribution (B), or with mann-whitney test when the distribution was non-normal (D, E, F, G, H, I). Comparisons between siCtrl and siMFN2 or siSLC27A2 are indicated by *. Comparisons between basal and starvation conditions are indicated by #. * or #: p ≤ 0.05, ** or ##: p ≤ 0.01, *** or ###: p ≤ 0.001, **** or ####: p ≤ 0.0001.

Figure 8.

SLC27A2 interacts with autophagy proteins and functionally interacts with MFN2 upon starvation stimulus. (A) FLAG-IP of overexpressed SLC27A2-FLAG in HeLa cells. ULK1, BECN1, ATG14, ATG16L1, LC3B, GABARAP and FLAG immunoblots of eluate (IP) and input fractions are shown. N = 1. (B) FLAG-IP of overexpressed SLC27A2-FLAG in HeLa cells with or without 1 h of EBSS treatment. ATG2A, ATG2B and FLAG immunoblots of eluate (IP) and input fractions are shown. N = 4 for ATG2A; N = 2 for ATG2B. (C) MFN2, SLC27A2 and LC3B immunoblots of total cellular lysate and subcellular fraction enriched in autophagosomes (AP fraction). Cells were treated for 4 h using baf A1. N = 3. (D) quantification of LC3B co-distribution with SLC27A2-FLAG in MFN2-silenced cells in basal conditions and after 1 h of EBSS treatment. siCtrl basal N = 35 cells, siMFN2 basal N = 34 cells, siCtrl starvation N = 36 cells, siMFN2 N = 24 cells. (E) quantification of LC3B co-distribution with MFN2 in SLC27A2-silenced cells in basal conditions and after 1 h of EBSS treatment. siCtrl basal N = 44 cells, siSLC27A2 basal N = 59 cells, siCtrl starvation N = 37 cells, siSLC27A2 N = 45 cells. (F) LC3B, FLAG, and MFN2 immunoblots of eluate (FLAG-IP) and input fractions of FLAG-IP of overexpressed SLC27A2-FLAG in WT or MFN2 KD HeLa cells with or without 1 h of EBSS treatment. (G) quantification of LC3B-II:FLAG ratio in IP fractions. N = 3. Data are represented as mean ± SEM. Outlier test was performed, and outliers were removed. Values were analyzed for normality with Saphiro-Wilk test and compared using one-way ANOVA with Tukey test (E, G) when following a normal distribution or mann-whitney test when the distribution was non-normal (D). Comparisons between siCtrl and siMFN2 or siSLC27A2 are indicated by *. Comparisons between basal and starvation conditions are indicated by #. * or #: p ≤ 0.05, ** or ##: p ≤ 0.01, *** or ###: p ≤ 0.001, **** or ####: p ≤ 0.0001.

Figure 9.

Proposed new functions of MFN1 and MFN2.