© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs249045. doi:10.1242/jcs.249045

RESEARCH ARTICLE Palmitoylated CKAP4 regulates mitochondrial functions through an interaction with VDAC2 at ER–mitochondria contact sites Takeshi Harada1, Ryota Sada1, Yoshito Osugi1, Shinji Matsumoto1, Tomoki Matsuda2, Mitsuko Hayashi-Nishino3, Takeharu Nagai2, Akihiro Harada4 and Akira Kikuchi1,*

ABSTRACT in cellular functions, including lipid transport, apoptosis control, 2+ Cytoskeleton-associated protein 4 (CKAP4) is a palmitoylated type II energy metabolism and Ca signaling (Marchi et al., 2018). transmembrane protein localized to the endoplasmic reticulum (ER). The ER is a continuous membrane network that can be divided into Here, we found that knockout (KO) of CKAP4 in HeLaS3 cells induces sheet-like structures connected to the nuclear envelope and a network the alteration of mitochondrial structures and increases the number of of tubules extending throughout the periphery of the cells (Shibata ER–mitochondria contact sites. To understand the involvement of et al., 2006; Westrate et al., 2015). Both the absolute and relative CKAP4 in mitochondrial functions, the binding proteins of CKAP4 were abundance of ER sheets and tubules vary with cell type and their explored, enabling identification of the mitochondrial porin voltage- balance is tightly regulated. The ER is dynamic, with sheets ‘ dependent anion-selective channel protein 2 (VDAC2), which is localized rearranging and tubules moving and fusing to form three-way ’ to the outer mitochondrial membrane. Palmitoylation at Cys100 of CKAP4 junctions (Du et al., 2004; Lee and Chen, 1988). In addition, the ER was required for the binding between CKAP4 and VDAC2. In CKAP4 KO plays a critical role in many cellular processes, including the regulation 2+ cells, the binding of inositol trisphosphate receptor (IP3R) and VDAC2 of Ca homeostasis, as well as protein synthesis, protein modification was enhanced, the intramitochondrial Ca2+ concentration increased and and lipid synthesis (Baumann and Walz, 2001; Marchi et al., 2018). 2+ the mitochondrial membrane potential decreased. In addition, CKAP4 The main effectors of the ER Ca release machinery are inositol KO decreased the oxidative consumption rate, in vitro cancer cell 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which facilitate the release 2+ proliferation under low-glucose conditions and in vivo xenograft tumor of Ca from ER stores in response to IP3 (Patel et al., 1999). Three – formation. The phenotypes were not rescued by expression of a different gene products (types I III) assemble as large tetrameric 2+ palmitoylation-deficient CKAP4 mutant. These results suggest that structures. Mitochondria can take up Ca into their matrix directly CKAP4 plays a role in maintaining mitochondrial functions through the from IP3Rs through voltage-dependent anion channels (VDACs) binding to VDAC2 at ER–mitochondria contact sites and that (Shoshan-Barmatz et al., 2006; Tsujimoto et al., 2006). The three – palmitoylation is required for this novel function of CKAP4. isoforms (VDAC1 VDAC3) show comparable channel properties, despite having different effects on cell death, and form a complex with – This article has an associated First Person interview with the first author IP3Rs at ER mitochondria contact sites (Szabadkai et al., 2006). 2+ of the paper. Under physiological conditions, Ca in mitochondria stimulates oxidative metabolism through the modulation of Ca2+-sensitive KEY WORDS: CKAP4, VDAC2, Mitochondria, Palmitoylation, dehydrogenases and metabolite carriers (McCormack et al., 1990). Mitochondria-associated ER membrane, ER However, mitochondrial Ca2+ overload damages mitochondrial morphology through the increase in ER–mitochondria contact sites INTRODUCTION by various stresses, including fragmentation by the recruitment of the Intracellular organelles coordinate complex signaling, metabolism GTPase dynamin-related protein 1 (DRP1; also known as DNM1L) and gene expression mechanisms in the cell through functional and/or cristae remodeling by optic atrophy 1 (OPA1), and also impairs or physical interactions with one another (Wu et al., 2018). Of the functions, with one consequence being decreased ATP production various combinations of interactions among organelles, that between (Jahani-Asl et al., 2010; Raffaello et al., 2016; Rizzuto et al., 2012). In the endoplasmic reticulum (ER) and mitochondria plays pivotal roles addition, loss of cristae structure leads to the release of cytochrome c and cell death (Eisner et al., 2018; Pernas and Scorrano, 2016). Cytoskeleton-associated protein 4 (CKAP4; also known as CLIMP- 63 and ERGIC-63) is a non-glycosylated type II transmembrane protein 1Department of Molecular Biology and Biochemistry, Graduate School of Medicine, located in the ER (Schweizer et al., 1993, 1995b). Several possible Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan. 2Department of Biomolecular Science and Engineering, The Institute of Scientific and Industrial functions of CKAP4 in the ER have been reported, including Research (SANKEN), Osaka University, Ibaraki, 8-1 Mihogaoka, Osaka 567-0047, segregation of ER sheets close to the nucleus (Klopfenstein et al., Japan. 3Department of Biomolecular Science and Regulation and Artificial 2001), maintenance of luminal width through intermolecular binding of Intelligence Research Center, The Institute of Scientific and Industrial Research (SANKEN), Osaka University, Ibaraki, 8-1 Mihogaoka, Osaka 567-0047, Japan. the luminal region of CKAP4 localized on opposing cisternal 4Department of Cell Biology, Graduate School of Medicine, Osaka University, membranes (Shibata et al., 2010), binding of the cytoplasmic region 2-2 Yamadaoka, Suita 565-0871, Japan. of CKAP4 to microtubules to create a link between the ER and *Author for correspondence ([email protected]) microtubules (Klopfenstein et al., 1998; Vedrenne and Hauri, 2006), acting as a Dicer-binding protein and regulating the microRNA pathway T.N., 0000-0003-2650-9895; A.H., 0000-0002-2484-9784; A.K., 0000-0003- and mRNA translation by anchoring Dicer to the ER (Pépin et al., 3378-9522 2012), and finally, through binding to gentamicin in the ER lumen, Handling Editor: David Stephens participating in gentamicin-induced apoptosis in proximal tubule cells

Received 18 May 2020; Accepted 29 September 2020 (Karasawa et al., 2010). Thus, although CKAP4 is likely to be involved Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249045. doi:10.1242/jcs.249045 in the regulation of the morphology and functions of the ER, the roles of caused by knockdown of YME1L or annexin A6 (Chlystun et al., CKAP4 in the functions of other organelles remain obscure. 2013; Stiburek et al., 2012). A small population of CKAP4 is localized to the plasma Electron microscopy examination revealed the frequency of membrane and functions as a receptor for extracellular ligands alterations in mitochondrial cristae structures in CKAP4 KO cells (Kikuchi et al., 2017). The extracellular region of plasma membrane- (Fig. 1B). CKAP4 KO mitochondrion appeared to be round, with loss located CKAP4, which is the same as the ER luminal region, of the well-defined cristae structures (An et al., 2012; Dalla Rosa et al., functions as a receptor for surfactant protein A (Gupta et al., 2006), 2014; Stiburek et al., 2012). The individual mitochondrion area was tissue plasminogen activator (Razzaq et al., 2003), anti-proliferative calculated to be 0.85 µm2 and 0.37 µm2 in control and CKAP4 KO factor (Conrads et al., 2006) and dickkopf1 (DKK1) (Kimura et al., cells. The aspect ratio of mitochondria was 1.90 and 1.34 in control and 2016), and also binds to integrin to regulate its recycling (Osugi et al., CKAP4 KO cells, respectively (Fig. 1B). The split-GFP system, which 2019). Thus, CKAP4 has multiple functions, depending on its can detect ER–mitochondria contact sites (Kakimoto et al., 2018), subcellular localization and interacting proteins. CKAP4 is modified showed that the numbers of GFP punctate increase in CKAP4 KO with palmitate at Cys100 (Schweizer et al., 1995a; Zhang et al., 2008) HeLaS3 cells (Fig. 1C). The finding was confirmed by electron and this palmitoylation is required for the localization of CKAP4 to microscopy, which revealed that the ratio of contact sites to the lipid raft of the plasma membrane, for DKK1-dependent AKT mitochondrial perimeters and the numbers of contact sites per activation and for cancer cell proliferation (Sada et al., 2019). mitochondria were increased in CKAP4 KO cells (Fig. 1D), whereas However, the role of palmitoylation of CKAP4 in the ER is unclear. the length and width of contact sites were not changed in control and In this study, we observed mitochondrial morphological changes CKAP4 KO cells (Fig. 1D). The mitochondrial morphology was also and dysfunction in CKAP4 knockout (KO) cells, in addition to ER fragmented in CKAP4 KO U2OS cells and the numbers of the contact morphological changes. We determined that CKAP4 interacts with sites were increased in the cells (Fig. S1F,G). Therefore, CKAP4 may be VDAC2 and modulates the functional coupling between IP3R and necessary to maintain healthy mitochondria through ER–mitochondria VDAC2. In addition, CKAP4 was involved in the formation of contact sites, because it is known that the interaction of the two ER–mitochondria contact sites, Ca2+ influx into mitochondria, organelles is important for mitochondrial structure and function mitochondrial respiration and cancer cell proliferation, and these (Hayashi et al., 2009). functions required palmitoylation of CKAP4. Our findings provide new roles for CKAP4 at ER–mitochondria contact sites. CKAP4 interacts with VDAC2 To address the involvement of CKAP4 in mitochondrial functions, RESULTS CKAP4–HA was stably expressed in S2-CP8 pancreatic cancer cells CKAP4 KO damages mitochondrial morphology and CKAP4-binding proteins were precipitated from whole-cell HeLaS3 cells with CKAP4 KO were generated with the CRISPR/Cas9 lysates (Fig. 2A). Among candidate proteins, voltage-dependent system (CKAP4 KO HeLaS3 cells) (Fig. S1A). CKAP4 was primarily anion-selective channel protein 2 (VDAC2) was further examined localized to ER sheets in control HeLaS3 cells (control cells) and the because VDAC2 is involved in various mitochondrial functions, ER sheets in CKAP4 KO cells were distributed throughout the including Ca2+ transport, apoptosis and oxidative phosphorylation cytoplasm (Fig. S1B). Electron microscopy revealed that the luminal (Mannella, 1992; Tsujimoto et al., 2006). The mRNA level of width was reduced from 60 nm to 40 nm (Fig. S1C). These results are VDAC2 was more abundant than that of VDAC1 and VDAC3 in consistent with previous observations (Shibata et al., 2010). To HeLaS3 cells (Fig. S2A). CKAP4 formed a complex with VDAC2 observe the mitochondrial morphology, mitochondrial matrix-targeted at the endogenous level in HeLaS3 cells and preferentially GFP (Mito-GFP) was transiently expressed in control and CKAP4 KO interacted with VDAC2 compared to VDAC1 and VDAC3 when HeLaS3 cells (Fig. 1A). The expression level of transiently transfected VDAC family proteins were transiently expressed in X293T cells Mito-GFP was examined in each cell by microscopic imaging, and (Fig. 2B,C). Although the first 11 amino acid residues of VDAC2 cells expressing about ±10% of the most frequent expression are not aligned with the sequence of other VDACs, a VDAC2 levels were selected for analyses. These cells were found to have an mutant with deletion of 11 N-terminal amino acids (Δ1–11) bound expression level of Mito-GFP of 51,014±5205 (arbitrary units to CKAP4 to a similar extent as VDAC2 wild-type (WT) of fluorescence intensity±standard deviation) in control cells and (Fig. S2B). 51,485±5953 in CKAP4 KO cells (n=100, P=0.48; Mann–Whitney The N-terminal cytoplasmic region (aa 1–106) of CKAP4 has been U-test). These results indicate that the variance of transiently expressed reported to regulate ER rearrangement (aa 2–21), link the ER to Mito-GFP is similar in control and CKAP4 KO cells. Under these microtubules (aa 24–101) and be modified with palmitate at Cys100 conditions, CKAP4 KO cells exhibited a significantly fragmented (Klopfenstein et al., 1998; Schweizer et al., 1994; Zhang et al., 2008). and attenuated mitochondrial network compared with the tubular Thus, N-terminal deletion mutants and a palmitoylation-deficient mitochondrial reticulum of control cells (Fig. 1A). Mitochondrial mutant (C100S) of CKAP4 were transiently expressed in X293T cells fragmentation was observed in 10% of control and 70% of CKAP4 KO (Fig. 2D). CKAP4 (Δ2–21)–HA and CKAP4 (Δ24–99)–HA formed cells (data not shown). In addition, the number and the individual size a complex with FLAG–VDAC2 to a similar extent and to a lesser of mitochondria were increased and decreased, respectively, in extent, respectively, to CKAP4WT–HA. On the other hand, CKAP4 CKAP4 KO cells compared with control cells under the conditions (Δ101–106)–HA and CKAP4C100S–HA did not (Fig. 2D). In X293T where the total mitochondrial area was similar (Fig. 1A). The cells, transiently expressed CKAP4WT–HA was palmitoylated but mitochondrial mass, which was analyzed through MitoTracker Green, CKAP4C100S–HA was not (Fig. 2E). Under the same conditions, and the expression levels of proteins, such as VDAC2, glucose- CKAP4 (Δ2–21)–HA and CKAP4 (Δ24–99)–HA was palmitoylated regulated protein 75 (GRP75; also known as HSPA9), adenine to a similar extent and to a lesser extent, respectively, to that of nucleotide translocase 2 (ANT2; also known as SLC25A5), COXIV, CKAP4WT–HA, but CKAP4 (Δ101–106)–HA was not (Fig. 2E). the type 1 IP3R (IP3R1; also known as ITPR1) and IP3R3 (ITPR3), These results suggest that the amino acid residues 100–106, which were not changed in control and CKAP4 KO cells (Fig. S1D,E). These include a palmitoylation site, are necessary for the binding of results are similar to the mitochondrial morphological alterations ER-located CKAP4 to mitochondrial VDAC2. Journal of Cell Science

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Fig. 1. CKAP4 KO affects mitochondrial structures. (A) The mitochondrial morphology in control and CKAP4 KO HeLaS3 cells transiently expressing Mito-GFP was observed through the green fluorescence. Left panel, Z-stack confocal images of Mito-GFP; right panel, binarized images of the left panel obtained by thresholding. The regions in white solid squares are shown as enlargements below. The Mito Morphology Macro plugin in ImageJ was used to quantify the mitochondrial number, individual mitochondrial size, and total mitochondrial area (mean±s.d.; n=30). **P<0.01; n.s., not significant (Mann–Whitney U-test). (B) The mitochondrial morphology of control and CKAP4 KO HeLaS3 cells was analyzed using electron microscopy, and the percentages of abnormal mitochondria with loss of well-defined cristae structures, individual mitochondrial size and mitochondria aspect ratio were quantified in both cell types (mean±s.d.; n=100). **P<0.01 (Mann–Whitney U-test). (C) Split-GFP was transiently expressed in control and CKAP4 KO HeLaS3 cells. The numbers of GFP dot signals, which indicate ER–mitochondria contact sites, were counted in both cell types (mean±s.d.; n=30). Cell contours are shown as white dotted lines. **P<0.01 (Mann–Whitney U-test). (D) ER–mitochondria contact sites in control and CKAP4 KO HeLaS3 cells were observed using electron microscopy. The percentage of the contact sites per mitochondrial perimeter, the contact site numbers per mitochondrion, and the length and width of the contact sites were quantified in both cell types (mean±s.d.; n=100). ER–mitochondria contacts sites are highlighted with a yellow dotted line. *P<0.05; **P<0.01; n.s., not significant (Mann–Whitney U-test). Scale bars: 10 µm (A,C); 500 nm (B,D).

There are 23 members of the ZDHHC protein family containing (Fig. 2G). Taken together, these results suggest that ER-localized palmitoyl acyltransferases (PATs) with a DHHC (Asp-His-His-Cys) CKAP4 binds to VDAC2 in a palmitoylation-dependent manner. motif (Fukata et al., 2004; Roth et al., 2002). ZDHHC2 localizes to the ER/Golgi (Ohno et al., 2006) and is required for the palmitoylation of CKAP4 regulates the formation of ER–mitochondria contact CKAP4 (Zhang et al., 2008). Consistent with the previous report, sites and the interaction of VDAC2 and IP3R palmitoylation of CKAP4 was reduced in ZDHHC2-depleted HeLaS3 ER–mitochondria contact sites are also known as mitochondria- cells (Fig. 2F; Fig. S2C). Furthermore, it was confirmed that the binding associated ER membranes (MAMs) (Giacomello and Pellegrini, of CKAP4 to VDAC2 was reduced in ZDHHC2-depleted cells 2016; Vance, 1990). Consistent with previous observations Journal of Cell Science

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Fig. 2. VDAC2 is a novel CKAP4-binding protein. (A) CKAP4-binding proteins were precipitated from lysates of S2-CP8 cells stably expressing CKAP4–HA with anti-HA antibody and the precipitated proteins were detected by silver staining. The bands indicated by the arrows were analyzed by mass spectrometry and the identified proteins are shown. (B) Lysates of HeLaS3 cells were immunoprecipitated (IP) with anti-CKAP4 antibody. The immunoprecipitates were probed with anti-CKAP4 and anti-VDAC2 antibodies. Input represents 5% of the whole cell lysate. (C) Lysates of X293T cells transiently expressing CKAP4WT-HA and FLAG-VDAC (1, 2 or 3) were immunoprecipitated with anti-HA antibody. The immunoprecipitates were probed with anti-FLAG and anti-HA antibodies. Input represents 5% of the whole cell lysate. (D) Top panels, a schematic representation of CKAP4 mutants. Bottom panels, lysates of X293T cells transiently expressing various CKAP4–HA mutants and FLAG–VDAC2 immunoprecipitated with anti-HA antibody. The immunoprecipitates were probed with anti-FLAG and anti-HA antibodies. Input represents 5% of the whole cell lysate. (E) X293T cells stably expressing various CKAP4–HA mutants were subjected to an APEGS assay. The lysates were probed with anti-HA antibody. Reactions without HAM treatment were negative controls. Asterisks indicate the band positionsof palmitoylated CKAP4. (F) ZDHHC2 in HeLaS3 cells was depleted by siRNA. Control HeLaS3 (siControl) and ZDHHC2 KD HeLaS3 (siZDHHC2) cells were subjected to an APEGS assay. Lysates were probed with anti-CKAP4 and anti-caveolin antibodies. Asterisks indicate the band positions of palmitoylated CKAP4 and palmitoylated caveolin. Caveolin was used as a loading control and positive control of palmitoylation. (G) Lysates of control HeLaS3 (siControl) and ZDHHC2 KD HeLaS3 (siZDHHC2) cells were immunoprecipitated with anti-CKAP4 antibody. The immunoprecipitates were probed with anti-CKAP4 and anti-VDAC2 antibodies. Input represents 5% of the whole cell lysate.

(Csordás et al., 2006; Poston et al., 2013), subcellular fractionation CKAP4C100S cells) (Fig. 3A). Light microscopy analysis showed of HeLaS3 cells confirmed that calnexin is present in MAM, that a part of CKAP4 is colocalized with ER-mitochondria contact microsome and crude mitochondria fractions (Fig. 3A). On the sites, which are indicated by split-GFP (Fig. S3A). other hand, IP3R was primarily observed in the MAM fraction, Immunoelectron microscopy analysis revealed that both and VDAC2 was detected in the MAM and pure mitochondrial endogenous CKAP4 and CKAP4C100S were distributed fractions, but not in the microsomal fraction (Fig. 3A). CKAP4 throughout the ER membrane (Fig. 3B). Thus, CKAP4 is was present in the MAM, microsome and crude mitochondrial present in ER membranes, including MAMs, and unlikely to be fractions, but not in the pure mitochondria fraction, of control enriched in MAMs through palmitoylation. HeLaS3 cells, with stably expressed CKAP4C100S showing a The split-GFP system revealed that overexpression of CKAP4,

similar subcellular localization in CKAP4 KO cells (CKAP4 KO/ but not that of VDAC2, decreases the numbers of ER-mitochondria Journal of Cell Science

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Fig. 3. CKAP4 is involved in the formation of ER–mitochondria contact sites. (A) Control HeLaS3 and CKAP4 KO HeLaS3 cells stably expressing CKAP4C100S (CKAP4 KO/CKAP4C100S) were fractionated into the cytosolic, microsomal, crude mitochondrial, MAM and pure mitochondrial fractions. Each fraction was probed with antibodies against CKAP4, calnexin (for microsomal and MAM fractions), IP3R3 (for microsomal and MAM fractions), VDAC2 (for mitochondrial and MAM fractions), Hsp90 (for cytosolic and microsomal fractions) and cytochrome c (mitochondrial fraction). (B) Endogenous CKAP4 in control HeLaS3 cells and CKAP4C100S in CKAP4 KO/CKAP4C100S cells were immunolabeled with anti-CKAP4 antibody, followed by silver-enhanced gold nanoparticle labeling, and the samples were observed by electron microscopy. (C) The mitochondrial morphology of control, CKAP4 KO, CKAP4 KO cells stably expressing CKAP4WT (CKAP4 KO/CKAP4WT) and CKAP4 KO/CKAP4C100S cells was analyzed using electron microscopy, and the percentages of abnormal mitochondria with loss of well-defined cristae structures and ER–mitochondria contact site numbers per mitochondria were quantified in respective cell types (mean±s.d.; n=100). CKAP4WT and CKAP4C100S were stably expressed in CKAP4 KO HeLaS3 cells. ER–mitochondria contacts sites are highlighted with a yellow dotted line. **P<0.01; n.s., not significant (ANOVA and Bonferroni post hoc test). (D) Split-GFP was transiently expressed in control, CKAP4 KO, CKAP4 KO/CKAP4WT and CKAP4 KO/CKAP4C100S cells. The numbers of GFP dot signals were counted in respective cell types (mean±s.d.; n=30). Cell contours are shown as white dotted lines. **P<0.01; n.s., not significant (ANOVA and Bonferroni post hoc test). Scale bars: 500 nm (B,C); 10 µm (D). contact sites (Fig. S3B). The increase in abnormal mitochondria and CKAP4 KO cells (Fig. 4A). Expression of CKAP4WT in CKAP4 ER-mitochondria contact sites induced by CKAP4 KO, which were KO cells decreased the number of PLA signals, unlike CKAP4C100S observed by electron and light microscopy, were rescued by the (Fig. 4A). In a co-immunoprecipitation assay, FLAG–VDAC2 expression of CKAP4WT but not by that of CKAP4C100S weakly precipitated endogenous GRP75 in control cells, and (Fig. 3C,D). At MAMs, VDAC1 is physically linked to IP3R1 CKAP4 KO enhanced the formation of the complex through glucose-regulated protein 75 (GRP75) (Szabadkai et al., (Fig. 4B). Since the expression levels of proteins constituting 2006). IP3R1 mRNA was more abundantly expressed in HeLaS3 ER–mitochondria contact sites were not changed in control and cells compared with the IP3R2 (also known as ITPR2) and IP3R3 CKAP4 KO cells, as shown in Fig. S1E, it is unlikely that the mRNAs (Fig. S3C). FLAG–VDAC2 was localized to the increase in protein–protein interaction at ER-mitochondria contact mitochondrial in HeLaS3 cells (Fig. S3D). A proximity ligation sites by CKAP4 KO is due to increased protein levels. The increase assay (PLA) showed some signals, indicating that there was in ER–mitochondria contact sites in CKAP4 KO cells expressing interaction between FLAG–VDAC2 and endogenous IP3R3 in split-GFP was also rescued by knockdown (KD) of VDAC2, IP3R1 control HeLaS3 cells, and the number of signals was increased in or IP3R3 (Fig. 4C; Fig. S3E). Taken together, these results suggest Journal of Cell Science

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Fig. 4. CKAP4 regulates the protein interactions in ER–mitochondria contact sites. (A) Control, CKAP4 KO and CKAP4 KO stably expressing CKAP4WT or CKAP4C100S (CKAP4 KO/CKAP4WT and CKAP4 KO/ CKAP4C100S, respectively) cells transiently expressing FLAG–VDAC2 were incubated with rabbit anti-FLAG and mouse anti-IP3R3 antibodies, and these primary antibodies were then combined with the secondary PLA probe. The interaction events are shown as red dots. The right graph shows the number of PLA dots per cell (mean±s.d.; n=30). **P<0.01; n.s., not significant (ANOVA and Bonferroni post hoc test). (B) Control and CKAP4 KO cells transiently expressing FLAG– VDAC2 were lysed, and the lysates were immunoprecipitated with anti- FLAG antibody. The immunoprecipitates were probed with anti-GRP75 and anti-FLAG antibodies. (C) Split-GFP was transiently expressed in control or CKAP4 KO cells with siRNA knockdown of IP3Rs (1, 2 or 3) or VDAC2. The numbers of GFP dot signals were counted in respective cell types (mean±s.d.; n=30). Cell contours were labeled with white dotted line. **P<0.01 (ANOVA and Bonferroni post hoc test). Scale bars: 10 µm.

that CKAP4 at ER–mitochondria contact sites suppresses the change compared with basal intensity, control, 2.70±0.20; CKAP4 KO, interaction between VDAC2, GRP75 and IP3R, and that CKAP4 is 2.63±0.14). In addition, the expression levels of mitochondrial Ca2+ involved in Ca2+ transport between the ER and mitochondria. uniporter (MCU) and its regulators [the regulator of MCU (MICU1; also known as CBARA1) and the essential MCU regulator (EMRE; CKAP4 regulates mitochondrial Ca2+ concentrations and also known as SMDT1)] (Patron et al., 2014) were almost similar in 2+ membrane potential control and CKAP4 KO cells (Fig. 5E). Thus, the increased [Ca ]m in To examine the role of CKAP4 in the regulation of Ca2+ flow from the CKAP4KOcellscouldbecausedbythe direct influx of ER-released ER into the mitochondria, intramitochondrial Ca2+ concentrations Ca2+ due to the increased number of ER–mitochondrial sites. 2+ 2+ 2+ ([Ca ]m) in HeLaS3 cells were imaged by expressing a mitochondrial Ca is required for metabolic regulation, but Ca overload also Ca2+ sensor CEPIA3mt with mCherry, which was used for causes the collapse of the mitochondrial membrane potential (Rizzuto 2+ normalization of the expression levels (Fig. 5A). [Ca ]m were et al., 2012). Indeed, when the mitochondrial membrane potential in increased in CKAP4 KO cells under the conditions where CKAP4 KO cells was measured using JC-1 (Smiley et al., 1991), the mitochondrial mass, which was measured using MitoTracker Deep intensity of JC-1 polymer (red) was reduced, whereas that of JC-1 Red, was not changed (Fig. S4A). The phenotype was rescued by monomer (green) was increased (Fig. 6A), indicating a decrease CKAP4WT expression and also rescued by VDAC2 KO, IP3R1 KD or in the mitochondrial membrane potential. Experiments with IP3R3 KD (Fig. 5A,B; Fig. S4B). These results indicate that the basal MitoTracker Orange, whose intensity is dependent upon the Ca2+ levels are increased in the mitochondria of CKAP4 KO cells. To mitochondrial membrane potential, and MitoTracker Green, whose examine whether CKAP4 KO affects stimulation-dependent Ca2+ intensity is independent of the mitochondrial membrane potential release from the ER, HeLaS3 cells were stimulated with ATPγSto (Cottet-Rousselle et al., 2011), showed that the MitoTracker Orange 2+ 2+ 2+ measure in [Ca ]m and Ca concentrations in the cytosol ([Ca ]c) intensity was decreased in CKAP4 KO cells under the condition that 2+ chronologically. In control HeLaS3 cells, ATPγS increased [Ca ]m the MitoTracker Green intensity was the same, further confirming that 2+ with a plateau at ∼30 s (Fig. 5C), while it gradually increased [Ca ]c, CKAP4 KO reduces the mitochondrial membrane potential in reaching a plateau at ∼120 s (Fig. 5D). In CKAP4 KO HeLaS3 cells, HeLaS3 cells (Fig. 6B). A similar phenotype was also observed in 2+ basal [Ca ]m were higher than control cells, but the fold-increase of other cell lines, including U2OS cells, MDCK dog kidney epithelial 2+ [Ca ]m observed upon ATPγS stimulation was similar to that of control cells and Eph4 mouse mammary epithelial cells using MitoTracker 2+ HeLaS3 cells [Fig. 5C, fold change compared with basal intensity Orange (Fig. S4C–E).Aswellasanincreasein[Ca ]m,the (mean±s.d.), control, 2.38±0.10; CKAP4 KO, 2.34±0.11]. The phenotype observed in CKAP4 KO cells was also rescued by 2+ WT stimulation-dependent change in [Ca ]c did not show significant CKAP4 expression, VDAC2 KO, IP3R1 KD or IP3R3 KD differences in control and CKAP4 KO HeLaS3 cells (Fig. 5D, fold (Fig. 6C,D). Thus, CKAP4 may inhibit the binding of IP3R and Journal of Cell Science

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2+ 2+ Fig. 5. CKAP4 controls mitochondrial Ca levels. (A) CEPIA3mt and mCherry were transiently expressed to measure [Ca ]m in control, CKAP4 KO, VDAC2 KO, CKAP4 KO stably expressing CKAP4WT or CKAP4C100S (CKAP4 KO/CKAP4WT and CKAP4 KO/CKAP4C100S, respectively), and CKAP4 KO/VDAC2 KO cells. The intensities of CEPIA3mt signal normalized to those of mCherry signal were measured (mean±s.d.; n=30). The regions in white solid square are shown as enlargements below. The relative fluorescence intensities of the indicated cells are expressed as fold changes compared with control cells (set at 1). 2+ **P<0.01 (ANOVA and Bonferroni post hoc test). (B) [Ca ]m was measured in control or CKAP4 KO cells with siRNA knockdown of IP3Rs (1, 2 or 3) (n=30). **P<0.01 (ANOVA and Bonferroni post hoc test). (C) Control and CKAP4 KO cells transiently expressing CEPIA3mt and mCherry were treated with 100 µM ATPγS 2+ at the indicated time. The time course of changes in [Ca ]m are shown (mean±s.d.; n=15). (D) Control and CKAP4 KO cells transiently expressing cyto-RCaMP1h and EGFP were treated with 100 µM ATPγS at the indicated time. The intensity of cyto-RCaMP1h signal normalized by that of EGFP signal was measured. 2+ The time course of changes in [Ca ]c were shown (mean±s.d.; n=15). (E) Lysates of control and CKAP4 KO HeLaS3 cells were probed with the indicated antibodies. Scale bars: 10 µm.

2+ VDAC2, and thereby appropriately control [Ca ]m and maintain the (cleavage of L-OPA1 into S-OPA1 impairs mitochondrial fusion; mitochondrial membrane potential. CKAP4C100S did not rescue the Ishihara et al., 2006) was unchanged in control and CKAP4 KO cells phenotypes induced by CKAP4 KO (Fig. 6C), suggesting that (Fig. 6E), suggesting that mitochondrial fragmentation induced by palmitoylation is necessary for the functions of CKAP4 at MAMs. It CKAP4 KO does not depend on OPA1. has been reported that the decrease in the mitochondrial membrane potential induces the cleavage of OPA1 and controls mitochondrial CKAP4 is required for mitochondrial respiratory functions fission and fusion (Ishihara et al., 2006). When treated with a m- and cell proliferation under low-glucose conditions chlorophenylhydrazone (CCCP), a chemical inhibitor of oxidative VDAC is a master regulator of mitochondrial bioenergetics (Fang and phosphorylation, the ratio of long (L)-OPA1 to short (S)-OPA1 Maldonado, 2018). When the bioenergetics profiles of control and Journal of Cell Science

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Fig. 6. CKAP4 is required for maintenance of mitochondrial membrane potential. (A,B) Mitochondrial membrane potentials of control and CKAP4 KO cells were measured using JC-1 (A) or MitoTracker Orange and Green (B). The right graphs show the relative fluorescence intensities of the indicated cells and the results are expressed as fold changes compared with control cells (set at 1; mean±s.d.; n=120–150). **P<0.01; n.s., not significant (Mann–Whitney U-test). (C,D) Mitochondrial membrane potentials of the indicated cells were measured using MitoTracker Orange. The bottom graphs show the relative fluorescence intensities of the indicated cells and the results are expressed as fold changes compared with control cells (set at 1; mean±s.d.; n=120–150). **P<0.01 (ANOVA and Bonferroni post hoc test). (E) Control and CKAP4 KO cells were treated with 10 µM CCCP for the indicated time. The lysates were probed with anti-OPA1 antibody. Scale bars: 100 µm.

CKAP4 KO HeLaS3 cells were analyzed, the baseline oxygen but not the quantity of mitochondrial proteins, resulting in decreased consumption rate (OCR), which reflects the mitochondrial function respiratory functions in mitochondria. of healthy oxidative phosphorylation, and the maximal respiration It is generally believed that cancer cells undergo metabolic were decreased in CKAP4 KO cells (Fig. 7A), even though the reprogramming and mainly generate their ATP from aerobic extracellular acidification rate (ECAR), which reflects the cellular glycolysis rather than oxidative phosphorylation (Ward and aerobic glycolysis activity, was unchanged by CKAP4 KO (Fig. 7B). Thompson, 2012). We found that the proliferation of HeLaS3 cells The decrease in the OCR in CKAP4 KO cells was rescued by the was dependent on glucose concentration (Fig. S5A). When HeLaS3 expression of CKAP4WT but not by that of CKAP4C100S (Fig. 7A). cells were cultured in the presence of 1 mg/ml glucose (high-glucose The expression levels of oxidative phosphorylation (OXPHOS)- conditions), CKAP4 KO did not affect cell proliferation (Fig. 8A). related proteins were not altered in CKAP4 KO cells compared with When glucose concentrations were decreased to 0.25 mg/ml (low- control cells (Fig. 7C). Therefore, CKAP4 KO may affect the quality glucose conditions), CKAP4 KO suppressed cell proliferation and Journal of Cell Science

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Fig. 7. CKAP4 is involved in mitochondrial respiratory function. (A) The oxygen consumption rates (OCRs) of the indicated cells were measured using an XF96 metabolic analyzer, and mitochondrial inhibitors were added at the indicated time points. (B) Extracellular acidification rates (ECARs) of control and CKAP4 KO HeLa3 cells were measured, and mitochondrial effectors were added at the indicated time points. (C) Lysates of control, CKAP4 KO, and CKAP4 KO stably expressing CKAP4WT or CKAP4C100S (CKAP4 KO/CKAP4WT and CKAP4 KO/ CKAP4C100S, respectively) cells were probed with the indicated antibodies against mitochondrial electron transport chain proteins.

increased the number of propidium iodide (PI)-positive cells (dead DISCUSSION cells), and these phenotypes were rescued by the expression of CKAP4 is primarily localized to the ER of various cells and is also CKAP4WT but not by that of CKAP4C100S (Fig.8B,C).However,dead present in the plasma membrane of some types of cells, including cells did not exhibit apoptotic features, such as cleaved caspase-3 cancer cells (Kikuchi et al., 2017; Vedrenne and Hauri, 2006). In expression, in the presence of 1 mg/ml or 0.25 mg/ml glucose this study, we found that CKAP4 localized to the ER binds to (Fig. S5B), although cytochrome c wasdetectedinthecytosolof VDAC2 in mitochondria and identified new functions of CKAP4 at CKAP4 KO cells (Fig. S5C). In addition, CKAP4 KO did not affect ER–mitochondria contact sites. the interaction of VDAC2 and the pro-apoptotic proteins Bak and Bax (Fig. S5D,E). Taken together, cell death induced by CKAP4 KO under Roles of CKAP4 in ER–mitochondria contact sites low-glucose conditions is unlikely to be associated with apoptosis. There are three isoforms of VDAC in humans, VDAC1, VDAC2 Under the low-glucose conditions, the numbers of ER–mitochondria and VDAC3 (Shoshan-Barmatz and Gincel, 2003). The amino contact sites were increased, but the formation of a complex between acid sequence of VDACs is highly conserved, with 80–90% GRP75 and VDAC2 and subcellular localization of CKAP4 were not overall similarity, indicating the high degree of 3D structural changed in HeLaS3 cells (Fig. S5F–H), suggesting that the increase in similarity between the three isoforms (Bayrhuber et al., 2008; ER–mitochondria contact sites by the low-glucose conditions is not Schredelseker et al., 2014). Our study revealed that CKAP4 caused by suppression of CKAP4 function. preferentially binds to VDAC2, rather than VDAC1 or VDAC3. In addition to HeLaS3 cells, U2OS cells also showed similar VDAC2 has an N-terminal amino acid sequence that is distinct proliferation patterns, depending on glucose concentrations; in these from that of other VDACs. However, the N-terminal region of cells, CKAP4 KO did not affect cell proliferation in the presence of VDAC2 was not required for its binding to CKAP4. Thus, it is 1 mg/ml glucose, but decreased it in the presence of 0.25 mg/ml currently unclear which specific region of VDAC2 determines the glucose (Fig. S6A,B). In contrast, normal (i.e. not cancer) cell lines binding to CKAP4. such as MDCK cells and Eph4 cells showed decreased cell CKAP4 KO enhanced the interaction of IP3R and VDAC2 and that proliferation upon CKAP4 KO, even under 1 mg/ml glucose of GRP75 and VDAC2. GRP75 acts as a scaffolding, rather than conditions (Fig. S6C,D). chaperoning, protein of the IP3R and VDAC complex at ER– When HeLaS3 cells were subcutaneously injected into mitochondria contact sites to increase the efficiency of mitochondrial immunodeficient mice, the volumes and weights of the xenograft Ca2+ uptake (Rizzuto et al., 2012; Szabadkai et al., 2006). When tumors derived from CKAP4 KO HeLaS3 cells were lower than those mitochondria are exposed to Ca2+ overload, opening of the of control cells (Fig. 8D–F). These phenotypes were rescued by the mitochondrial permeability transition pore (mPTP) is triggered, expression of CKAP4WT but not by that of CKAP4C100S. In conclusion, leading to mitochondrial dysfunction (Bock and Tait, 2020; Martinou palmitoylated CKAP4 could be required for in vitro cancer cell et al., 2000; NavaneethaKrishnan et al., 2020). Similar to IP3R, proliferation under low-glucose conditions and in vivo xenograft tumor calnexin and VDAC2, CKAP4 is present in the MAM fraction. Our formation. present results in CKAP4 KO cells demonstrate an increase in Journal of Cell Science

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Fig. 8. CKAP4 is necessary for cancer cell proliferation under low-glucose conditions. (A) Control, CKAP4 KO, and CKAP4 KO stably expressing CKAP4WT or CKAP4C100S (CKAP4 KO/CKAP4WT and CKAP4 KO/CKAP4C100S, respectively) cells were subjected to a proliferation assay under 1 mg/ml glucose conditions. Fluorescence intensities were measured at the indicated time points and the results are shown as arbitrary units (mean±s.d.; n=4) compared with day 0. AU, arbitrary unit. (B) The indicated cells were subjected to a proliferation assay under low-glucose conditions (mean±s.d.; n=4). **P<0.01; n.s., not significant (ANOVA and Bonferroni post hoc test). (C) The indicated cells were cultured under 0.25 mg/ml glucose conditions for 48 h. Cells were incubated with propidium iodide (PI) and Hoechst 33342. PI-positive cells are expressed as the percentage of Hoechst 33342-stained cells per field (n=60–100). **P<0.01 (ANOVA and Bonferroni post hoc test). (D–F) The indicated cells (5.0×106 cells) were implanted into the back of nude mice. Representative appearances of xenograft tumors are shown in D. The volumes (E) and weights (F) of the xenograft tumors were measured (mean±s.d.; n=6–8). Tumor weights are plotted as a box-and-whisker plot with the median represented with a line, the 25th to 75th percentile represented with a box, and the 5th to 95th percentile indicated by the whiskers. *P<0.05; **P<0.01 (ANOVA and Bonferroni post hoc test; E) and (Wilcoxon rank sum test; F). Scale bars: 100 µm (C); 10 mm (D).

2+ [Ca ]m, without affecting the expression of MCU and its regulators, CKAP4 may appropriately suppress the formation of ER– increased numbers of ER–mitochondria contact sites, and a reduced mitochondria contact sites and the mitochondrial functions of the mitochondrial membrane potential. These phenotypes are rescued GRP75–IP3R–VDAC2 complex by binding to VDAC2, resulting in through the depletion of VDAC2, IP3R1 or IP3R3, indicating inhibition of mitochondrial Ca2+ transport and maintenance of the functional interactions between CKAP4, VDAC2 and IP3Rs. Thus, membrane potential. Journal of Cell Science

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GRP75 is localized in extramitochondrial and intramitochondrial CKAP4C100S neither binds to VDAC2 nor rescues the decrease in 2+ sites. Although we speculate that the interaction of [Ca ]m and membrane potential observed in CKAP4 KO. The extramitochondrial GRP75 and VDAC2 is increased in CKAP4 binding of CKAP4 to VDAC2 was decreased in ZDHHC2-depleted KO cells, intramitochondrial GRP75 may form a complex with cells. Because palmitate covalently bound to CKAP4 is probably VDAC2. It has recently been shown that ANT3 (SLC25A6), an inner inserted into the ER membrane, it might not directly bind to mitochondrial membrane protein, interacts with GRP75 and VDAC VDAC2. CKAP4 (Δ101–106) is neither palmitoylated nor binds to in mitochondria (Wu et al., 2020). GRP75 depletion enhances the VDAC2, even though the palmitoylation site (Cys100) is intact. interaction of ANT3 and cyclophilin D and mPTP opening, Thus, the amino acid sequence (aa 101–106) adjacent to the cell suggesting that intramitochondrial GRP75 inhibits the formation of membrane is required for CKAP4 palmitoylation, and its structure, a complex between ANT3 and cyclophilin D and decreases determined by palmitoylation at Cys100, would be important for its mitochondrial permeability. However, our results revealed that direct or indirect interaction with VDAC2. At least seven amino cytochrome c is released into the cytoplasm of CKAP4 KO cells, acids, including the palmitoylation site of CKAP4, are necessary which reflects mPTP opening. Thus, the increase in the amount of for the formation of the CKAP4 and VDAC2 complex. Taken GRP75 co-immunoprecipitated with VDAC2 in CKAP4 KO cells together, the present study proposes that palmitoylated CKAP4 in could be derived from extramitochondrial GRP75. the ER regulates mitochondrial functions via its interaction with VDAC2. Role of CKAP4 in mitochondrial morphology Fragmented mitochondria were frequently observed in CKAP4 KO CKAP4 is required for cancer cell proliferation and survival cells. Although the cleavage of L-OPA1 to S-OPA1, which impairs under low-glucose conditions mitochondrial fusion (Ishihara et al., 2006), was not enhanced in Consistent with the results on mitochondrial damage in CKAP4 KO CKAP4 KO cells, the numbers of ER–mitochondria contact sites cells, oxidative phosphorylation, but not glycolysis, was reduced in were increased. It has been reported that ER–mitochondria contact CKAP4 KO cells. Cancer cells, such as HeLaS3 cells and U2OS sites coordinate mitochondrial DNA replication with mitochondrial cells, exhibit increased glycolysis and reduced oxidative division, and that CKAP4 overexpression increases sheet-like ER phosphorylation (Ward and Thompson, 2012). This is due to the structures and reduces mitochondrial DNA synthesis (Lewis et al., enhanced glycolysis pathway resulting from an interplay between 2016). Thus, it is intriguing to speculate that the increase in ER– oncogenes and the tumor microenvironment (Zheng, 2012). mitochondria contact sites observed in CKAP4 KO might affect CKAP4 KO did not affect the proliferation of HeLaS3 cells or mitochondrial DNA synthesis and mitochondrial fission, which U2OS cells when the glucose concentration was 1 mg/ml. However, occurs independently of OPA1 cleavage. Taken together with the when the glucose concentration was reduced to 0.25 mg/ml, the previous observation that OPA1 cleavage occurs during apoptosis proliferation of CKAP4 KO cells was suppressed. Similar results (Arnoult et al., 2005) and our present result that apoptosis does not were observed in U2OS cells, but not in MDCK or Eph4 occur in CKAP4 KO cells, there may be a new mechanism of cells. Therefore, cancer cell proliferation is supported by CKAP4-dependent control of mitochondrial morphology. glycolysis when glucose is sufficient, and mitochondrial oxidative phosphorylation, which requires CKAP4, contributes Role of palmitoylation in ER–mitochondria contact sites to cancer cell proliferation under low-glucose conditions. These CKAP4 is modified with palmitate at Cys100 in a reaction catalyzed by results are consistent with a recent report showing that glycolytic ZDHHC2 and ZDHHC5 (Planey et al., 2009; Sada et al., 2019). suppression induced mitochondria dependency in cancer cells Palmitoylation of membrane proteins has been reported to be involved (Shiratori et al., 2019). In contrast, because normal cells primarily in the regulation of protein targeting, trafficking, protein–protein undergo mitochondrial respiration, their proliferation is affected interactions and protein conformation (Charollais and Van Der Goot, by CKAP4, even in the presence of high concentrations of 2009). CKAP4 is localized to the ER and plasma membrane, and glucose. In addition to cancer cell proliferation, cancer cell palmitoylation is not required for the trafficking of CKAP4 from the survival is also regulated by CKAP4 under low-glucose ER to the plasma membrane (Sada et al., 2019). In plasma membrane- conditions. CKAP4 KO increases the number of PI-positive located CKAP4, palmitoylation is required for the localization of (dead) cells under low-glucose conditions. Cytochrome c,an CKAP4 to lipid rafts, and DKK1 induces depalmitoylation of apoptosis inducer, is released into the cytoplasm of CKAP4 KO CKAP4, resulting in its moving to non-lipid rafts (Sada et al., 2019). cells, but CKAP4 KO neither increases cleaved caspase-3 nor Lipid rafts are cholesterol- and sphingolipid-rich plasma membrane affects the interaction of BAX or Bak with VDAC2. Thus, cell microdomains that are thicker than other parts of the plasma death is induced in a non-apoptotic manner by CKAP4 under low- membrane (Jacobson et al., 2007). Changes in transmembrane glucose conditions and may result from mitochondrial domain tilting caused by palmitoylation may control the effective dysfunction. length of hydrophobic CKAP4 segments, causing the protein to Compared with what was seen in the in vitro experiments, which partition into lipid rafts. depended on the glucose concentration, the effect of CKAP4 KO on In addition to local Ca2+ transfer from the ER to mitochondria, cancer cell proliferation was clearly observed in vivo. This difference the lipid transport function of ER–mitochondria contact sites, such between the in vitro and in vivo experiments may be related to the as MAMs, has been extensively studied (Hayashi et al., 2009). tumor microenvironment. Inhibition of ER-located CKAP4 in cancer MAMs are enriched in cholesterol and neutral lipids (Hayashi and cells using, for example, an antisense oligonucleotide (ASO) against Su, 2003). However, in contrast to the plasma membrane lipid rafts, CKAP4 may suppress cancer cell proliferation in vivo. Although palmitoylation of CKAP4 is not required for localization to ER– normal cells express ER-located CKAP4 in various organs, ASO mitochondria contact sites or its distribution in the ER membrane. tends to accumulate in tumor lesions rather than normal cells (Harada Thus, the difference in the CKAP4 localization in the microdomains et al., 2019; Kimura et al., 2020). Thus, CKAP4 ASO may be a novel of the plasma membrane and ER may be due to differences in their nucleic acid treatment for patients with tumors expressing high levels lipid composition. of CKAP4. Journal of Cell Science

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MATERIALS AND METHODS with uranyl acetate and lead citrate and observed under a JEM-2100 Materials transmission electron microscope (JEOL, Tokyo, Japan) at an Anti-CKAP4 monoclonal antibodies (3F11-2B10 and 5A6-17A11) and anti- accelerating voltage of 80 kV. CKAP4 polyclonal antibody were generated as described (Kimura et al., Normal or abnormal cristae morphology of mitochondria was defined as 2016, 2019). Other primary antibodies used in this study are listed in previously described (Deng et al., 2015). Briefly, normal mitochondria with Table S1. pLV-Mito-GFP (#44385), pCMV-CEPIA3mt (#58219) and pCAG numerous well-organized cristae or abnormal mitochondria showing round cyto-RCaMP1h (#105014) were from AddGene (Cambridge, MA, USA). mitochondria containing unstructured cristae were scored and quantified as The primers for real-time PCR and the siRNA target sequences used in this the percentage of total mitochondria examined (n=160–170). study are described in Tables S2 and S3, respectively. Other materials were To quantify the amount of ER–mitochondria contact sites, the percentage obtained from commercial sources. of the mitochondrial surface closely apposed to the ER was calculated (<25 nm distance between membranes, n=25–30) (Demetriadou et al., Cells 2017; Hirabayashi et al., 2017). HeLaS3 cervical cancer cells were provided by Kunihiro Matsumoto (Nagoya University, Aichi, Japan) in May 2002. MDCK type I (MDCK) dog renal Split-GFP system tubule cells and Eph4 mouse mammary epithelial cells were provided by The split-GFP system, which can detect ER–mitochondria contact sites, was Sachiko Tsukita (Osaka University, Suita, Japan) in January 2013 and described previously (Kakimoto et al., 2018). HeLa cells stably expressing October 2011. S2-CP8 pancreatic cancer cells were purchased from Cell TOM70 (1–70)–FLAG–GFP11 in mitochondria and doxycycline- Resource Center for Biomedical Research, Institute of Development, Aging dependently expressing ERj1 (1–200)–V5–GFP1-10 in the ER were and Cancer, Tohoku University, in April 2014. Lenti-X 293T (X293T) cells treated with 30 ng/ml doxycycline for 24 h to induce the expression of were purchased from Takara Bio Inc. in October 2011. U2OS osteosarcoma ERj1 (1–200)–V5–GFP1-10. Split-GFP signals were viewed and analyzed cells were purchased from American Type Culture Collection (ATCC, using an LSM810 laser scanning microscope. Manassas, VA, USA) in February 2010. Initial cell lines were frozen in liquid nitrogen and early passages of cells (<1 month in culture) were used in all Mass spectrometry experiments (no authentication was done by the authors). Cells were checked Liquid chromatography-tandem mass spectrometry analysis for for mycoplasma using the e-Myco plus Mycoplasma PCR Detection Kit. identification of CKAP4-binding proteins was performed as described HeLaS3, MDCK, S2-CP8, X293T and U2OS cells were maintained in previously (Osugi et al., 2019) using an UltiMate 3000 nano LC system Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (Thermo Fisher Scientific, Waltham, MA) coupled to a Q-Exactive hybrid fetal bovine serum (FBS). For cell proliferation assays, glucose-depleted quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) with a DMEM and dialyzed FBS by filtration (10,000 molecular weight cut-off) nanoelectrospray ionization source. were used. CKAP4 KO and VDAC2 KO cells were generated by the CRISPR/Cas9 Immunoprecipitation assay system as previously described (Harada et al., 2017). The target sequences The immunoprecipitation assay was performed as described previously for human CKAP4 (5′-GGGTGGGCACCCTTCTCCGA-3′) and human (Matsumoto et al., 2019; Osugi et al., 2019). Cells (60-mm diameter dish) VDAC2 (5′-TATGATGGAGGAATACACAT-3′) were designed with the were lysed in 500 µl of Nonidet P-40 (NP-40) buffer (20 mM Tris-HCl pH help of CRISPR Genome Engineering Resources (https://zlab.bio/guide- 8.0, 10% glycerol, 137 mM NaCl, and 1% NP-40) with protease inhibitors design-resources). To generate cells stably expressing proteins for rescue (10 µg/ml leupeptin, 20 µg/ml aprotinin and 1 mM phenylmethanesulfonyl experiments, parental cells were infected with lentivirus and selected with fluoride). After centrifugation, the lysates were incubated with primary G418 and blasticidin. antibodies and 40 µl of a 50% slurry of protein G–Sepharose beads (GE Healthcare Bio-Sciences, Buckinghamshire, UK) for 1 h at 4°C. After Immunocytochemistry washing three times with NP-40 buffer, the precipitates were probed with Immunocytochemistry was performed as described previously (Matsumoto the indicated antibodies. et al., 2019; Osugi et al., 2019). Cells were fixed with phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA) and then permeabilized in PBS containing 0.2% (w/v) Triton X-100 and 2 mg/ml bovine serum Acyl-PEGyl exchange gel shift assay albumin (BSA) for 10 min. The cells were blocked in blocking buffer (PBS The acyl-PEGyl exchange gel shift (APEGS) assay was performed as containing 2 mg/ml BSA). Samples were incubated with the primary previously described (Sada et al., 2019). X293T CKAP4 KO cells (60-mm WT C100S antibodies diluted in PBS for 1 h or overnight, washed three times with diameter dish) expressing CKAP4 –HA, CKAP4 –HA, CKAP4 PBS, and then stained with a secondary antibody (conjugated to Alexa Fluor (Δ24-99)–HA or CKAP4 (Δ2-21)–HA were then lysed in 1 ml of PBS 488 or 546; Invitrogen) diluted in PBS for 1 h. After washing, the samples containing 5 mM EDTA, 4% SDS and protease inhibitors. After sonication were covered with PBS containing 50% glycerol. Samples were viewed and and centrifugation at 20,000 g for 15 min at room temperature (RT), the analyzed using an LSM810 laser scanning microscope. soluble proteins (0.4 to 0.5 mg/ml, 1 ml) were reduced with 25 mM Tris (2-carboxyethyl) phosphine (TCEP) for 1 h at 55°C, and free cysteine residues were alkylated with 50 mM N-ethylmaleimide for 3 h at RT. After Quantification of mitochondrial morphology chloroform and methanol precipitation (CM ppt), the precipitates were The number per cell, mean size and total area of mitochondria were suspended in 250 µl of PBS containing 5 mM EDTA, 4% SDS, and 10 µg/ measured in ImageJ by using the Mito Morphology Macro plugin (Dagda ml pepstatin A and then centrifuged at 20,000 g for 10 min at RT to Z et al., 2009). -stack confocal microscopy images of Mito-GFP were completely remove the undissolved protein pellet. The supernatant (125 µl) acquired using an LSM810 laser scanning microscope and the images were was mixed with 375 µl of either 1.33 M hydroxylamine (pH 7.0), 0.2% imported into ImageJ, where the program was used to set a common Triton X-100, and 5 mM EDTA or 1.33 M Tris-HCl (pH 7.0), 0.2% Triton threshold and calculate the mitochondrial parameters. X-100, and 5 mM EDTA, and the mixtures were incubated for 1 h at 37°C. After CM ppt, the precipitates were resuspended in 100 µl of PBS Conventional electron microscopy containing 5 mM EDTA, 4% SDS and 10 µg/ml pepstatin A. Soluble Conventional electron microscopy was performed as previously proteins (0.5 to 0.75 mg/ml, 100 µl) were PEGylated with 20 mM mPEG-5k described (Hayashi-Nishino et al., 2009). Briefly, cells were fixed in in the presence of 10 mM TCEP for 1 h so that newly exposed cysteinyl thiol 2.5% glutaraldehyde in 0.1 M PBS at pH 7.4 for 1 h. The specimens were groups could be labeled with mPEG-5k, which causes a mobility shift of postfixed in buffer containing 1% OsO4 and 1.5% potassium palmitoylated proteins in SDS-PAGE gels. After CM ppt, the precipitates ferrocyanide, dehydrated in a series of graded ethanol solutions, and were resuspended in Laemmli’s SDS-sample buffer and boiled at 100°C for embedded in epoxy resin. Ultra-thin sections were collected and stained 5 min. Western blotting was then used to detect palmitoylated bands and Journal of Cell Science

12 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249045. doi:10.1242/jcs.249045 non-palmitoylated bands. Protein concentrations were measured with the Ca2+ imaging and measurement of the mitochondrial membrane bicinchoninic acid protein assay at individual steps. potential To measure intramitochondrial Ca2+ concentrations, cells were plated on Organelle fractionation glass-bottom dishes (Iwaki) and transiently transfected with CEPIA3mt Microsomes, mitochondria and MAMs from HeLaS3 cells were isolated (Suzuki et al., 2014) and mCherry using FuGENE HD (Promega) and following previously described protocols (Wieckowski et al., 2009). incubated for 1 day. The medium was replaced with DMEM supplemented HeLaS3 cells were harvested from 10 dishes (100-mm-diameter dishes) at with 2.5 mM HEPES (pH 7.4). The fluorescence intensities of CEPIA3 mt 90–100% confluence and homogenized in isolation buffer (30 mM Tris- and mCherry were measured using a laser scanning microscope (LSM810 or HCl pH 7.4, 225 mM mannitol, 75 mM sucrose and 0.1 mM EGTA) using a FV1000) and the intensity of the CEPIA3mt signal was normalized to that of 2+ Teflon pestle until 80–90% of the cells were disrupted. Then, the mCherry signal. To measure cytoplasmic Ca concentrations, cells were homogenate was centrifuged twice at 600 g for 10 min each to remove plated on glass-bottom dishes (Iwaki) and transiently transfected with nuclei and debris. The supernatant was collected and centrifuged for 15 min RCaMP1h (Hirabayashi et al., 2017) and EGFP using FuGENE HD at 7000 g to obtain crude mitochondria. For ER isolation, this step was (Promega) and incubated for 1 day. The medium was replaced with DMEM repeated with the supernatant until a pellet was no longer visible, and the supplemented with 2.5 mM HEPES (pH 7.4). The fluorescence intensities supernatant was centrifuged at 20,000 g for 30 min. Next, the mitochondria- of RCaMP1 h and EGFP were measured using a laser scanning microscope free supernatant was centrifuged again at 100,000 g for 1 h. The pellet was (LSM810 or FV1000). To monitor the mitochondrial membrane potential, then resuspended for the ER fraction and the supernatant was kept for the the cells were incubated with 2 µM JC-1 (Dojindo), 200 nM MitoTracker cytosolic fraction. For the pure mitochondria and MAM fraction, the crude Orange CMXRos (Invitrogen), 200 nM MitoTracker Green FM (Invitrogen) mitochondria pellet was resuspended in a 5× volume of resuspension buffer or 200 nM MitoTracker Deep Red FM (Invitrogen) for 30 min and then (5 mM HEPES-NaOH pH 7.4, 250 mM mannitol and 0.5 mM EGTA), and imaged using an LSM810 laser scanning microscope. the fraction was added to the top of 30% Percoll medium [25 mM HEPES- NaOH pH 7.4, 225 mM mannitol, 1 mM EGTA and 30% Percoll (v/v)] in a Mitochondrial respirometry and cell glycolysis assays centrifuge tube. Centrifugation was performed at 95,000 g for 30 min, and Respirometry of intact HeLaS3 cells was performed using an XF96 then the upper (MAMs) and lower (pure mitochondria) layers were Extracellular Flux Analyzer (Seahorse Biosciences) (VanLinden et al., collected with a Pasteur pipette. Both fractions were diluted with a 10× 2015). Cells were seeded at a density of 4.0×104 cells/well in 96-well XF volume resuspension buffer and centrifuged at 6300 g for 15 min. Then, the microplates and cultured with DMEM containing 10% FBS for 24 h. At 1 h pure mitochondria fraction was obtained from the pellet after one more wash before measurements, the medium was replaced with XF base medium under the same conditions. The supernatant containing the MAMs was supplemented with 2 mM L-glutamate and 2 mM pyruvate. After 1-h centrifuged at 100,000 g for 1 h, and the resulting pellet was resuspended in incubation in a CO2-free incubator at 37°C for temperature and pH resuspension buffer. Each fraction was evaluated by western blotting using equilibration, the baseline oxygen consumption rate (OCR) was measured. mouse anti-CKAP4 (3F11-2B10), rabbit anti-calnexin (Sigma), mouse This was followed by sequential injections with 2 μg/ml oligomycin to anti-IP3R (BD Bioscience), rabbit anti-VDAC2 (Invitrogen) and mouse measure the ATP-linked OCR, 1 μM FCCP (an oxidative phosphorylation anti-Hsp90 (BD Bioscience) antibodies. uncoupler) to determine maximal respiration, and 0.1 μM rotenone and 0.1 μM antimycin A to determine non-mitochondrial respiration. For cell Immunoelectron microscopy glycolysis measurement, HeLaS3 cells seeded in XF96 microplates as Immunoelectron microscopy was performed following the previously described above were cultured in XFbase medium supplemented with 2 mM described protocols (Sobajima et al., 2018). Control and CKAP4 KO L-glutamine in a CO2-free incubator for 1 h. For extracellular acidification HeLaS3 cells were fixed with 3% PFA and 0.1% glutaraldehyde in PBS, pH rate (ECAR) measurement, the cells were treated sequentially with 10 mM μ 7.4, for 30 min at RT, washed with PBS, and permeabilized in 5% normal (1.8 mg/ml) glucose to measure glucose metabolism, oligomycin (2 g/ml) goat serum and 0.25% saponin in PBS. After quenching with 1 mg/ml to measure glycolytic capacity, and 2-deoxy-D-glucose (2-DG) (100 mM) to measure non-glycolytic acidification. NaBH4 in the same solution for 30 min at room temperature, the cells were washed with PBS and treated with anti-CKAP4 monoclonal antibody (5A6- 17A11) for 2 h at 37°C and then with Alexa Fluor 488-labeled Cytochrome c release assay FluoroNanogold anti-mouse IgG (1:30; Nanoprobes, Yaphank, NY, USA) Cytochrome c release assays were performed as described previously in 5% normal donkey serum and 0.25% saponin in PBS for 1 h at RT. The (Waterhouse et al., 2004). Cells were homogenized in plasma membrane cells were washed with PBS and then with distilled water. The fixed cells permeabilization buffer (PBS containing 200 µg/ml digitonin and 80 mM were incubated with HQ silver enhancement solution (Nanoprobes) for KCl) and incubated on ice for 5 min. Then, the homogenate was centrifuged 7 min at RT, extensively washed with distilled water, and incubated with at 8000 g for 5 min, and the supernatant (the cytosol-enriched digitonin- selenium toner (1:20; Kodak) for 7 min at RT to prevent erosion of the silver soluble fraction) was probed with anti-cytochrome c antibody (Santa Cruz during the OsO4 fixation. Samples were postfixed in 2.5% glutaraldehyde in Biotechnology). PBS for 15 min and in 1% OsO4 for 1 h on ice, followed by staining in 4% uranyl acetate for 2 h at RT. The samples were dehydrated and embedded as Xenograft tumor assay previously described. They were examined and photographed with a JEOL- HeLaS3 cell (5×106 cells) pellet was suspended in 100 μl of PBS and 1010 electron microscope. sububcutaneous injected into the back of anesthetized 8-week-old male BALB/cAJcl-nu/nu mice (nude mice; CLEA Japan, Tokyo, Japan). The Proximity ligation assay mice were euthanized 28 days after transplantation. The areas containing PLA was performed according to the manufacturer’s protocol (Sigma) as transplanted cells were measured and weighed. Tumor volumes were described previously (Ibuka et al., 2015; Osugi et al., 2019). Cells calculated using the following formula: (major axis)×(minor axis)×(minor transiently expressing FLAG–VDAC2 grown on glass coverslips were axis)×0.5. All animal experiments were performed according to guidelines fixed for 15 min at RT in PBS containing 4% PFA. The cells were approved by the Animal Research Committee of Osaka University, Japan permeabilized in PBS containing 0.2% (w/v) Triton X-100 and 2 mg/ml (No. 21-048-1). BSA for 10 min. The glass coverslips were blocked in blocking buffer for 30 min and incubated with anti-FLAG and anti-IP3R antibodies diluted in Statistical analysis blocking buffer for 1 h at RT. After washing, the coverslips were incubated Each experiment was performed at least three times, and the results are with Duolink PLA anti-rabbit minus and PLA anti-mouse plus proximity presented as the mean±s.d. A Mann–Whitney U-test was used to determine probes (Sigma). PLA dots were counted using an LSM810 laser scanning the statistical significance between the means of two groups. Analysis of ’ microscope. variance (ANOVA) with Tukey, Bonferroni or Dunnett s post hoc tests was Journal of Cell Science

13 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249045. doi:10.1242/jcs.249045 used to compare three or more group means. Statistical analysis was Cottet-Rousselle, C., Ronot, X., Leverve, X. and Mayo, J.-F. (2011). Cytometric performed using Excel 2010 (Microsoft, Redmond, WA, USA). The assessment of mitochondria using fluorescent probes. Cytometry A 79, 405-425. doi:10.1002/cyto.a.21061 Wilcoxon rank sum test was used for analysis of xenograft tumor weight ́ ́ P Csordas, G., Renken, C., Varnai, P., Walter, L., Weaver, D., Buttle, K. F., Balla, T., (Fig. 8F). values <0.05 were considered statistically significant. Mannella, C. A. and Hajnóczky, G. (2006). Structural and functional features and significance of the physical linkage between ER and mitochondria. J. Cell Biol. Acknowledgements 174, 915-921. doi:10.1083/jcb.200604016 We are grateful to Drs N. Ishihara, Y. Shintani and H. Kato for helpful discussion Dagda, R. K., Cherra, S. J., III, Kulich, S. M., Tandon, A., Park, D. and Chu, C. T. about mitochondrial functions, and Drs Y. Tamura and S. Tashiro for donating cells. (2009). Loss of PINK1 function promotes mitophagy through effects on oxidative We also thank Grants-in-Aid for Scientific Research on Innovative Area ‘Organelle stress and mitochondrial fission. J. Biol. Chem. 284, 13843-13855. doi:10.1074/ zone’ for conventional electron microscopy and immunoelectron microscopy. We jbc.M808515200 are grateful to the technical staff, especially I. Nishimura, of the Comprehensive Dalla Rosa, I., Durigon, R., Pearce, S. F., Rorbach, J., Hirst, E. M. A., Vidoni, S., Reyes, A., Brea-Calvo, G., Minczuk, M., Woellhaf, M. W. et al. (2014). MPV17L2 Analysis Center of the Institute of Scientific and Industrial Research at Osaka is required for ribosome assembly in mitochondria. Nucleic Acids Res. 42, University. We would like to thank the Center of Medical Research and Education, 8500-8515. doi:10.1093/nar/gku513 Graduate School of Medicine, Osaka University for in-gel digestion and LC-MS/MS Demetriadou, A., Morales-Sanfrutos, J., Nearchou, M., Baba, O., Kyriacou, K., analysis. Tate, E. W., Drousiotou, A. and Petrou, P. P. (2017). Mouse Stbd1 is N- myristoylated and affects ER-mitochondria association and mitochondrial Competing interests morphology. J. Cell Sci. 130, 903-915. doi:10.1242/jcs.195263 The authors declare no competing or financial interests. Deng, J., Yang, M., Chen, Y., Chen, X., Liu, J., Sun, S., Cheng, H., Li, Y., Bigio, E. H., Mesulam, M. et al. (2015). FUS interacts with HSP60 to promote Author contributions mitochondrial damage. PLoS Genet. 11, e1005357. doi:10.1371/journal.pgen. Methodology: T.M., T.N.; Validation: T.H.; Investigation: T.H., R.S., Y.O., M.H.-N., 1005357 A.H.; Resources: Y.O.; Writing - original draft: T.H., S.M., A.K.; Writing - review & Du, Y., Ferro-Novick, S. and Novick, P. (2004). Dynamics and inheritance of the endoplasmic reticulum. J. Cell Sci. 117, 2871-2878. doi:10.1242/jcs.01286 editing: R.S., S.M., A.K.; Visualization: T.H., T.M., M.H.-N., A.H.; Supervision: A.K.; Eisner, V., Picard, M. and Hajnóczky, G. (2018). Mitochondrial dynamics in Project administration: A.K.; Funding acquisition: A.K. adaptive and maladaptive cellular stress responses. Nat. Cell. Biol. 20, 755-765. doi:10.1038/s41556-018-0133-0 Funding Fang, D. and Maldonado, E. N. (2018). VDAC regulation: a mitochondrial target to This work was supported by the Ministry of Education, Culture, Sports, Science and stop cell proliferation. Adv. Cancer Res. 138, 41-69. doi:10.1016/bs.acr.2018.02. Technology (Japan) [Grants-in-Aids for Scientific Research to A.K. (2016-2020) 002 (no. 16H06374) and Grants-in-Aid for Scientific Research on Innovative Area Fukata, M., Fukata, Y., Adesnik, H., Nicoll, R. A. and Bredt, D. S. (2004). ‘Organelle zone’ to A.K. (2018-2019) (no. 18H04861) and to A.H. and M.H.-N. Identification of PSD-95 palmitoylating enzymes. Neuron 44, 987-996. doi:10. (2017-2022) (no. 17H06422)], and by grants from the Yasuda Memorial Foundation 1016/j.neuron.2004.12.005 and the Ichiro Kanehara Foundation of the Promotion of Medical Science & Medical Giacomello, M. and Pellegrini, L. (2016). The coming of age of the mitochondria- Care to A.K., by Integrated Frontier Research for Medical Science Division, Institute ER contact: a matter of thickness. Cell Death Differ. 23, 1417-1427. doi:10.1038/ for Open and Transdisciplinary Research Initiatives, Osaka University to A.K., and cdd.2016.52 by a grant from Core Research for Evolutionary Science and Technology, Japan Gupta, N., Manevich, Y., Kazi, A. S., Tao, J.-Q., Fisher, A. B. and Bates, S. R. Science and Technology Agency to T.N. (JPMJCR15N3). (2006). Identification and characterization of p63 (CKAP4/ERGIC-63/CLIMP-63), a surfactant protein A binding protein, on type II pneumocytes. Am. J. Physiol. Lung Cell Mol. Physiol. 291, L436-L446. doi:10.1152/ajplung.00415.2005 Supplementary information Harada, T., Yamamoto, H., Kishida, S., Kishida, M., Awada, C., Takao, T. and Supplementary information available online at Kikuchi, A. (2017). Wnt5b-associated exosomes promote cancer cell migration https://jcs.biologists.org/lookup/doi/10.1242/jcs.249045.supplemental and proliferation. Cancer Sci. 108, 42-52. doi:10.1111/cas.13109 Harada, T., Matsumoto, S., Hirota, S., Kimura, H., Fujii, S., Kasahara, Y., Gon, H., Peer review history Yoshida, T., Itoh, T., Haraguchi, N. et al. (2019). Chemically modified antisense The peer review history is available online at oligonucleotide against ARL4C inhibits primary and metastatic liver tumor growth. https://jcs.biologists.org/lookup/doi/10.1242/jcs.249045.reviewer-comments.pdf Mol. Cancer Ther. 18, 602-612. doi:10.1158/1535-7163.MCT-18-0824 Hayashi, T. and Su, T.-P. (2003). σ-1 receptors (σ(1) binding sites) form raft-like References microdomains and target lipid droplets on the endoplasmic reticulum: roles in endoplasmic reticulum lipid compartmentalization and export. J. Pharmacol. Exp. An, J., Shi, J., He, Q., Lui, K., Liu, Y., Huang, Y. and Sheikh, M. S. (2012). CHCM1/ Ther. 306, 718-725. doi:10.1124/jpet.103.051284 CHCHD6, novel mitochondrial protein linked to regulation of mitofilin and Hayashi, T., Rizzuto, R., Hajnoczky, G. and Su, T.-P. (2009). MAM: more than just mitochondrial cristae morphology. J. Biol. Chem. 287, 7411-7426. doi:10.1074/ a housekeeper. Trends Cell Biol. 19, 81-88. doi:10.1016/j.tcb.2008.12.002 jbc.M111.277103 Hayashi-Nishino, M., Fujita, N., Noda, T., Yamaguchi, A., Yoshimori, T. and Arnoult, D., Grodet, A., Lee, Y.-J., Estaquier, J. and Blackstone, C. (2005). Yamamoto, A. (2009). A subdomain of the endoplasmic reticulum forms a cradle Release of OPA1 during apoptosis participates in the rapid and complete release for autophagosome formation. Nat. Cell Biol. 11, 1433-1437. doi:10.1038/ of cytochrome c and subsequent mitochondrial fragmentation. J. Biol. Chem. 280, ncb1991 35742-35750. doi:10.1074/jbc.M505970200 Hirabayashi, Y., Kwon, S.-K., Paek, H., Pernice, W. M., Paul, M. A., Lee, J., Baumann, O. and Walz, B. (2001). Endoplasmic reticulum of animal cells and its Erfani, P., Raczkowski, A., Petrey, D. S., Pon, L. A. et al. (2017). ER- organization into structural and functional domains. Int. Rev. Cytol. 205, 149-214. mitochondria tethering by PDZD8 regulates Ca(2+) dynamics in mammalian doi:10.1016/S0074-7696(01)05004-5 neurons. Science 358, 623-630. doi:10.1126/science.aan6009 Bayrhuber, M., Meins, T., Habeck, M., Becker, S., Giller, K., Villinger, S., Ibuka, S., Matsumoto, S., Fujii, S. and Kikuchi, A. (2015). The P2Y(2) receptor Vonrhein, C., Griesinger, C., Zweckstetter, M. and Zeth, K. (2008). Structure of promotes Wnt3a- and EGF-induced epithelial tubular formation by IEC6 cells by the human voltage-dependent anion channel. Proc. Natl. Acad. Sci. USA 105, binding to integrins. J. Cell Sci. 128, 2156-2168. doi:10.1242/jcs.169060 15370-15375. doi:10.1073/pnas.0808115105 Ishihara, N., Fujita, Y., Oka, T. and Mihara, K. (2006). Regulation of mitochondrial Bock, F. J. and Tait, S. W. G. (2020). Mitochondria as multifaceted regulators of cell morphology through proteolytic cleavage of OPA1. EMBO J. 25, 2966-2977. death. Nat. Rev. Mol. Cell Biol. 21, 85-100. doi:10.1038/s41580-019-0173-8 doi:10.1038/sj.emboj.7601184 Charollais, J. and Van Der Goot, F. G. (2009). Palmitoylation of membrane proteins Jacobson, K., Mouritsen, O. G. and Anderson, R. G. W. (2007). Lipid rafts: at a crossroad between cell biology and physics. Nat. Cell Biol. 9, 7-14. doi:10.1038/ (Review). Mol. Membr. Biol. 26, 55-66. doi:10.1080/09687680802620369 ncb0107-7 Chlystun, M., Campanella, M., Law, A.-L., Duchen, M. R., Fatimathas, L., Levine, Jahani-Asl, A., Germain, M. and Slack, R. S. (2010). Mitochondria: joining forces T. P., Gerke, V. and Moss, S. E. (2013). Regulation of mitochondrial to thwart cell death. Biochim. Biophys. Acta 1802, 162-166. doi:10.1016/j.bbadis. morphogenesis by annexin A6. PLoS ONE 8, e53774. doi:10.1371/journal. 2009.09.006 pone.0053774 Kakimoto, Y., Tashiro, S., Kojima, R., Morozumi, Y., Endo, T. and Tamura, Y. Conrads, T. P., Tocci, G. M., Hood, B. L., Zhang, C.-O., Guo, L., Koch, K. R., (2018). Visualizing multiple inter-organelle contact sites using the organelle- Michejda, C. J., Veenstra, T. D. and Keay, S. K. (2006). CKAP4/p63 is a receptor targeted split-GFP system. Sci. Rep. 8, 6175. doi:10.1038/s41598-018-24466-0 for the frizzled-8 protein-related antiproliferative factor from interstitial cystitis Karasawa, T., Wang, Q., David, L. L. and Steyger, P. S. (2010). CLIMP-63 is a patients. J. Biol. Chem. 281, 37836-37843. doi:10.1074/jbc.M604581200 gentamicin-binding protein that is involved in drug-induced cytotoxicity. Cell Death

Dis. 1, e102. doi:10.1038/cddis.2010.80 Journal of Cell Science

14 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249045. doi:10.1242/jcs.249045

Kikuchi, A., Fumoto, K. and Kimura, H. (2017). The Dickkopf1-cytoskeleton- vascular smooth muscle cells by the type-II transmembrane protein p63 (CKAP4). associated protein 4 axis creates a novel signalling pathway and may represent a J. Biol. Chem. 278, 42679-42685. doi:10.1074/jbc.M305695200 molecular target for cancer therapy. Br. J. Pharmacol. 174, 4651-4665. doi:10. Rizzuto, R., De Stefani, D., Raffaello, A. and Mammucari, C. (2012). Mitochondria 1111/bph.13863 as sensors and regulators of calcium signalling. Nat. Rev. Mol. Cell Biol. 13, Kimura, H., Fumoto, K., Shojima, K., Nojima, S., Osugi, Y., Tomihara, H., 566-578. doi:10.1038/nrm3412 Eguchi, H., Shintani, Y., Endo, H., Inoue, M. et al. (2016). CKAP4 is a Dickkopf1 Roth, A. F., Feng, Y., Chen, L. and Davis, N. G. (2002). The yeast DHHC cysteine- receptor and is involved in tumor progression. J. Clin. Invest. 126, 2689-2705. rich domain protein Akr1p is a palmitoyl transferase. J. Cell Biol. 159, 23-28. doi:10.1172/JCI84658 doi:10.1083/jcb.200206120 Kimura, H., Yamamoto, H., Harada, T., Fumoto, K., Osugi, Y., Sada, R., Maehara, Sada, R., Kimura, H., Fukata, Y., Fukata, M., Yamamoto, H. and Kikuchi, A. N., Hikita, H., Mori, S., Eguchi, H. et al. (2019). CKAP4, a DKK1 receptor, is a (2019). Dynamic palmitoylation controls the microdomain localization of the DKK1 biomarker in exosomes derived from pancreatic cancer and a molecular target for receptors CKAP4 and LRP6. Sci. Signal. 12, eaat9519. doi:10.1126/scisignal. therapy. Clin. Cancer Res. 25, 1936-1947. doi:10.1158/1078-0432.CCR-18-2124 aat9519 Kimura, K., Matsumoto, S., Harada, T., Morii, E., Nagatomo, I., Shintani, Y. and Schredelseker, J., Paz, A., López, C. J., Altenbach, C., Leung, C. S., Drexler, Kikuchi, A. (2020). ARL4C is associated with initiation and progression of lung M. K., Chen, J.-N., Hubbell, W. L. and Abramson, J. (2014). High resolution adenocarcinoma and represents a therapeutic target. Cancer Sci. 111, 951-961. structure and double electron-electron resonance of the zebrafish voltage- doi:10.1111/cas.14303 dependent anion channel 2 reveal an oligomeric population. J. Biol. Chem. 289, Klopfenstein, D. R. C., Kappeler, F. and Hauri, H.-P. (1998). A novel direct 12566-12577. doi:10.1074/jbc.M113.497438 interaction of endoplasmic reticulum with microtubules. EMBO J. 17, 6168-6177. Schweizer, A., Ericsson, M., Bachi, T., Griffiths, G. and Hauri, H. P. (1993). doi:10.1093/emboj/17.21.6168 Characterization of a novel 63 kDa membrane protein. Implications for the Klopfenstein, D. R., Klumperman, J., Lustig, A., Kammerer, R. A., Oorschot, V. organization of the ER-to-Golgi pathway. J. Cell Sci. 104, 671-683. and Hauri, H.-P. (2001). Subdomain-specific localization of CLIMP-63 (p63) in Schweizer, A., Rohrer, J., Hauri, H. P. and Kornfeld, S. (1994). Retention of p63 in the endoplasmic reticulum is mediated by its luminal α-helical segment. J. Cell an ER-Golgi intermediate compartment depends on the presence of all three of its Biol. 153, 1287-1300. doi:10.1083/jcb.153.6.1287 domains and on its ability to form oligomers. J. Cell Biol. 126, 25-39. doi:10.1083/ Lee, C. and Chen, L. B. (1988). Dynamic behavior of endoplasmic reticulum in living jcb.126.1.25 cells. Cell 54, 37-46. doi:10.1016/0092-8674(88)90177-8 Schweizer, A., Rohrer, J. and Kornfeld, S. (1995a). Determination of the structural Lewis, S. C., Uchiyama, L. F. and Nunnari, J. (2016). ER-mitochondria contacts requirements for palmitoylation of p63. J. Biol. Chem. 270, 9638-9644. doi:10. couple mtDNA synthesis with mitochondrial division in human cells. Science 353, 1074/jbc.270.16.9638 aaf5549. doi:10.1126/science.aaf5549 Schweizer, A., Rohrer, J., Slot, J. W., Geuze, H. J. and Kornfeld, S. (1995b). Mannella, C. A. (1992). The ‘ins’ and ‘outs’ of mitochondrial membrane channels. Reassessment of the subcellular localization of p63. J. Cell Sci. 108, 2477-2485. Trends Biochem. Sci. 17, 315-320. doi:10.1016/0968-0004(92)90444-E Shibata, Y., Voeltz, G. K. and Rapoport, T. A. (2006). Rough sheets and smooth Marchi, S., Patergnani, S., Missiroli, S., Morciano, G., Rimessi, A., Wieckowski, tubules. Cell 126, 435-439. doi:10.1016/j.cell.2006.07.019 M. R., Giorgi, C. and Pinton, P. (2018). Mitochondrial and endoplasmic reticulum Shibata, Y., Shemesh, T., Prinz, W. A., Palazzo, A. F., Kozlov, M. M. and calcium homeostasis and cell death. Cell Calcium 69, 62-72. doi:10.1016/j.ceca. Rapoport, T. A. (2010). Mechanisms determining the morphology of the 2017.05.003 peripheral ER. Cell 143, 774-788. doi:10.1016/j.cell.2010.11.007 Martinou, J.-C., Desagher, S. and Antonsson, B. (2000). Cytochrome c release Shiratori, R., Furuichi, K., Yamaguchi, M., Miyazaki, N., Aoki, H., Chibana, H., Ito, K. from mitochondria: all or nothing. Nat. Cell Biol. 2, E41-E43. doi:10.1038/ and Aoki, S. (2019). Glycolytic suppression dramatically changes the intracellular 35004069 metabolic profile of multiple cancer cell lines in a mitochondrial metabolism-dependent Matsumoto, S., Yamamichi, T., Shinzawa, K., Kasahara, Y., Nojima, S., manner. Sci. Rep. 9, 18699. doi:10.1038/s41598-019-55296-3 Kodama, T., Obika, S., Takehara, T., Morii, E., Okuyama, H. et al. (2019). Shoshan-Barmatz, V. and Gincel, D. (2003). The voltage-dependent anion GREB1 induced by Wnt signaling promotes development of hepatoblastoma by channel: characterization, modulation, and role in mitochondrial function in cell life suppressing TGFβ signaling. Nat. Commun. 10, 3882. doi:10.1038/s41467-019- and death. Cell Biochem. Biophys. 39, 279-292. doi:10.1385/CBB:39:3:279 11533-x Shoshan-Barmatz, V., Israelson, A., Brdiczka, D. and Sheu, S. S. (2006). The McCormack, J. G., Halestrap, A. P. and Denton, R. M. (1990). Role of calcium ions voltage-dependent anion channel (VDAC): function in intracellular signalling, cell in regulation of mammalian intramitochondrial metabolism. Physiol. Rev. 70, life and cell death. Curr. Pharm. Des. 12, 2249-2270. doi:10.2174/ 391-425. doi:10.1152/physrev.1990.70.2.391 138161206777585111 NavaneethaKrishnan, S., Rosales, J. L. and Lee, K.-Y. (2020). mPTP opening Smiley, S. T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T. W., caused by Cdk5 loss is due to increased mitochondrial Ca(2+) uptake. Oncogene Steele, G. D., Jr. and Chen, L. B. (1991). Intracellular heterogeneity in 39, 2797-2806. doi:10.1038/s41388-020-1188-5 mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic Ohno, Y., Kihara, A., Sano, T. and Igarashi, Y. (2006). Intracellular localization and cation JC-1. Proc. Natl. Acad. Sci. USA 88, 3671-3675. doi:10.1073/pnas.88.9.3671 tissue-specific distribution of human and yeast DHHC cysteine-rich domain- Sobajima, T., Yoshimura, S.-I., Maeda, T., Miyata, H., Miyoshi, E. and Harada, A. containing proteins. Biochim. Biophys. Acta. 1761, 474-483. doi:10.1016/j.bbalip. (2018). The Rab11-binding protein RELCH/KIAA1468 controls intracellular 2006.03.010 cholesterol distribution. J. Cell Biol. 217, 1777-1796. doi:10.1083/jcb.201709123 Osugi, Y., Fumoto, K. and Kikuchi, A. (2019). CKAP4 regulates cell migration via Stiburek, L., Cesnekova, J., Kostkova, O., Fornuskova, D., Vinsova, K., the interaction with and recycling of integrin. Mol. Cell. Biol. 39, e00073-19. doi:10. Wenchich, L., Houstek, J. and Zeman, J. (2012). YME1L controls the 1128/MCB.00073-19 accumulation of respiratory chain subunits and is required for apoptotic Patel, S., Joseph, S. K. and Thomas, A. P. (1999). Molecular properties of inositol 1,4,5- resistance, cristae morphogenesis, and cell proliferation. Mol. Biol. Cell 23, trisphosphate receptors. Cell Calcium 25, 247-264. doi:10.1054/ceca.1999.0021 1010-1023. doi:10.1091/mbc.e11-08-0674 Patron, M., Checchetto, V., Raffaello, A., Teardo, E., Vecellio Reane, D., Suzuki, J., Kanemaru, K., Ishii, K., Ohkura, M., Okubo, Y. and Iino, M. (2014). Mantoan, M., Granatiero, V., Szabo,̀ I., De Stefani, D. and Rizzuto, R. (2014). Imaging intraorganellar Ca2+ at subcellular resolution using CEPIA. Nat. MICU1 and MICU2 finely tune the mitochondrial Ca2+ uniporter by exerting Commun. 5, 4153. doi:10.1038/ncomms5153 opposite effects on MCU activity. Mol. Cell 53, 726-737. doi:10.1016/j.molcel. Szabadkai, G., Bianchi, K., Várnai, P., De Stefani, D., Wieckowski, M. R., 2014.01.013 Cavagna, D., Nagy, A. I., Balla, T. and Rizzuto, R. (2006). Chaperone-mediated Pépin, G., Perron, M. P. and Provost, P. (2012). Regulation of human Dicer by the coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. J. Cell Biol. resident ER membrane protein CLIMP-63. Nucleic Acids Res. 40, 11603-11617. 175, 901-911. doi:10.1083/jcb.200608073 doi:10.1093/nar/gks903 Tsujimoto, Y., Nakagawa, T. and Shimizu, S. (2006). Mitochondrial membrane Pernas, L. and Scorrano, L. (2016). Mito-morphosis: mitochondrial fusion, fission, permeability transition and cell death. Biochim. Biophys. Acta 1757, 1297-1300. and cristae remodeling as key mediators of cellular function. Annu. Rev. Physiol. doi:10.1016/j.bbabio.2006.03.017 78, 505-531. doi:10.1146/annurev-physiol-021115-105011 Vance, J. E. (1990). Phospholipid synthesis in a membrane fraction associated with Planey, S. L., Keay, S. K., Zhang, C.-O. and Zacharias, D. A. (2009). mitochondria. J. Biol. Chem. 265, 7248-7256. Palmitoylation of cytoskeleton associated protein 4 by DHHC2 regulates VanLinden, M. R., Dölle, C., Pettersen, I. K. N., Kulikova, V. A., Niere, M., Agrimi, antiproliferative factor-mediated signaling. Mol. Biol. Cell 20, 1454-1463. doi:10. G., Dyrstad, S. E., Palmieri, F., Nikiforov, A. A., Tronstad, K. J. et al. (2015). 1091/mbc.e08-08-0849 Subcellular distribution of NAD+ between cytosol and mitochondria determines Poston, C. N., Krishnan, S. C. and Bazemore-Walker, C. R. (2013). In-depth the metabolic profile of human cells. J. Biol. Chem. 290, 27644-27659. doi:10. proteomic analysis of mammalian mitochondria-associated membranes (MAM). 1074/jbc.M115.654129 J. Proteomics 79, 219-230. doi:10.1016/j.jprot.2012.12.018 Vedrenne, C. and Hauri, H.-P. (2006). Morphogenesis of the endoplasmic Raffaello, A., Mammucari, C., Gherardi, G. and Rizzuto, R. (2016). Calcium at the reticulum: beyond active membrane expansion. Traffic 7, 639-646. doi:10.1111/ center of cell signaling: interplay between endoplasmic reticulum, mitochondria, and j.1600-0854.2006.00419.x lysosomes. Trends Biochem. Sci. 41, 1035-1049. doi:10.1016/j.tibs.2016.09.001 Ward, P. S. and Thompson, C. B. (2012). Metabolic reprogramming: a cancer Razzaq, T. M., Bass, R., Vines, D. J., Werner, F., Whawell, S. A. and Ellis, V. hallmark even warburg did not anticipate. Cancer Cell 21, 297-308. doi:10.1016/j.

(2003). Functional regulation of tissue plasminogen activator on the surface of ccr.2012.02.014 Journal of Cell Science

15 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs249045. doi:10.1242/jcs.249045

Waterhouse, N. J., Steel, R., Kluck, R. and Trapani, J. A. (2004). Assaying Wu, P.-K., Hong, S.-K., Chen, W., Becker, A. E., Gundry, R. L., Lin, C.-W., Shao, cytochrome c translocation during apoptosis. Methods Mol. Biol. 284, 307-313. H., Gestwicki, J. E. and Park, J.-I. (2020). Mortalin (HSPA9) facilitates BRAF- doi:10.1385/1-59259-816-1:307 mutant tumor cell survival by suppressing ANT3-mediated mitochondrial Westrate, L. M., Lee, J. E., Prinz, W. A. and Voeltz, G. K. (2015). Form follows membrane permeability. Sci. Signal. 13, eaay1478. doi:10.1126/scisignal. function: the importance of endoplasmic reticulum shape. Annu. Rev. Biochem. aay1478 84, 791-811. doi:10.1146/annurev-biochem-072711-163501 Zhang, J., Planey, S. L., Ceballos, C., Stevens, S. M., Jr, Keay, S. K. and Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J. and Pinton, P. Zacharias, D. A. (2008). Identification of CKAP4/p63 as a major substrate of the (2009). Isolation of mitochondria-associated membranes and mitochondria palmitoyl acyltransferase DHHC2, a putative tumor suppressor, using a novel from animal tissues and cells. Nat. Protoc. 4, 1582-1590. doi:10.1038/nprot. proteomics method. Mol. Cell Proteomics 7, 1378-1388. doi:10.1074/mcp. 2009.151 M800069-MCP200 Wu, H., Carvalho, P. and Voeltz, G. K. (2018). Here, there, and everywhere: The Zheng, J. (2012). Energy metabolism of cancer: Glycolysis versus importance of ER membrane contact sites. Science 361, eaan5835. doi:10.1126/ oxidative phosphorylation (Review). Oncol. Lett. 4, 1151-1157. doi:10.3892/ol. science.aan5835 2012.928 Journal of Cell Science

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