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Palmitoylated CKAP4 Regulates Mitochondrial Functions Through an Interaction with VDAC2 at ER–Mitochondria Contact Sites

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Palmitoylated CKAP4 Regulates Mitochondrial Functions Through an Interaction with VDAC2 at ER–Mitochondria Contact Sites © 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
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