Phospholipid transfer function of PTPIP51 at mitochondria‐associated ER membranes
2021; Springer Nature; Volume: 22; Issue: 6 Linguagem: Inglês
10.15252/embr.202051323
ISSN1469-3178
AutoresHyun Ku Yeo, Tae Hyun Park, Hee Yeon Kim, Hyonchol Jang, Ju‐Eun Lee, Geum‐Sook Hwang, Seong Eon Ryu, Si Hoon Park, Hyun Kyu Song, Hyun Seung Ban, Hye‐Jin Yoon, Byung Il Lee,
Tópico(s)Protein Tyrosine Phosphatases
ResumoReport2 May 2021free access Transparent process Phospholipid transfer function of PTPIP51 at mitochondria-associated ER membranes Hyun Ku Yeo Hyun Ku Yeo orcid.org/0000-0002-8701-8902 Research Institute, National Cancer Center, Goyang-si, KoreaThese authors contributed equally to this work. Search for more papers by this author Tae Hyun Park Tae Hyun Park orcid.org/0000-0001-8059-8273 Research Institute, National Cancer Center, Goyang-si, Korea Department of Bioengineering, Hanyang University, Seoul, KoreaThese authors contributed equally to this work. Search for more papers by this author Hee Yeon Kim Hee Yeon Kim orcid.org/0000-0001-5104-7579 Research Institute, National Cancer Center, Goyang-si, Korea Search for more papers by this author Hyonchol Jang Hyonchol Jang orcid.org/0000-0003-1436-457X Research Institute, National Cancer Center, Goyang-si, Korea Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, Goyang-si, Korea Search for more papers by this author Jueun Lee Jueun Lee Integrated Metabolomics Research Group, Western Seoul Center, Korea Basic Science Institute, Seoul, Korea Search for more papers by this author Geum-Sook Hwang Geum-Sook Hwang orcid.org/0000-0002-1600-1556 Integrated Metabolomics Research Group, Western Seoul Center, Korea Basic Science Institute, Seoul, Korea Department of Chemistry and Nano Science, Ewha Womans University, Seoul, Korea Search for more papers by this author Seong Eon Ryu Seong Eon Ryu orcid.org/0000-0003-3335-326X Department of Bioengineering, Hanyang University, Seoul, Korea Search for more papers by this author Si Hoon Park Si Hoon Park orcid.org/0000-0003-3518-7808 Department of Life Sciences, Korea University, Seoul, Korea Search for more papers by this author Hyun Kyu Song Hyun Kyu Song orcid.org/0000-0001-5684-4059 Department of Life Sciences, Korea University, Seoul, Korea Search for more papers by this author Hyun Seung Ban Hyun Seung Ban orcid.org/0000-0002-2698-6037 Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea Search for more papers by this author Hye-Jin Yoon Hye-Jin Yoon orcid.org/0000-0003-4724-4208 Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul, Korea Search for more papers by this author Byung Il Lee Corresponding Author Byung Il Lee [email protected] orcid.org/0000-0003-1270-8439 Research Institute, National Cancer Center, Goyang-si, Korea Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, Goyang-si, Korea Search for more papers by this author Hyun Ku Yeo Hyun Ku Yeo orcid.org/0000-0002-8701-8902 Research Institute, National Cancer Center, Goyang-si, KoreaThese authors contributed equally to this work. Search for more papers by this author Tae Hyun Park Tae Hyun Park orcid.org/0000-0001-8059-8273 Research Institute, National Cancer Center, Goyang-si, Korea Department of Bioengineering, Hanyang University, Seoul, KoreaThese authors contributed equally to this work. Search for more papers by this author Hee Yeon Kim Hee Yeon Kim orcid.org/0000-0001-5104-7579 Research Institute, National Cancer Center, Goyang-si, Korea Search for more papers by this author Hyonchol Jang Hyonchol Jang orcid.org/0000-0003-1436-457X Research Institute, National Cancer Center, Goyang-si, Korea Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, Goyang-si, Korea Search for more papers by this author Jueun Lee Jueun Lee Integrated Metabolomics Research Group, Western Seoul Center, Korea Basic Science Institute, Seoul, Korea Search for more papers by this author Geum-Sook Hwang Geum-Sook Hwang orcid.org/0000-0002-1600-1556 Integrated Metabolomics Research Group, Western Seoul Center, Korea Basic Science Institute, Seoul, Korea Department of Chemistry and Nano Science, Ewha Womans University, Seoul, Korea Search for more papers by this author Seong Eon Ryu Seong Eon Ryu orcid.org/0000-0003-3335-326X Department of Bioengineering, Hanyang University, Seoul, Korea Search for more papers by this author Si Hoon Park Si Hoon Park orcid.org/0000-0003-3518-7808 Department of Life Sciences, Korea University, Seoul, Korea Search for more papers by this author Hyun Kyu Song Hyun Kyu Song orcid.org/0000-0001-5684-4059 Department of Life Sciences, Korea University, Seoul, Korea Search for more papers by this author Hyun Seung Ban Hyun Seung Ban orcid.org/0000-0002-2698-6037 Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea Search for more papers by this author Hye-Jin Yoon Hye-Jin Yoon orcid.org/0000-0003-4724-4208 Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul, Korea Search for more papers by this author Byung Il Lee Corresponding Author Byung Il Lee [email protected] orcid.org/0000-0003-1270-8439 Research Institute, National Cancer Center, Goyang-si, Korea Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, Goyang-si, Korea Search for more papers by this author Author Information Hyun Ku Yeo1, Tae Hyun Park1,2, Hee Yeon Kim1, Hyonchol Jang1,3, Jueun Lee4, Geum-Sook Hwang4,5, Seong Eon Ryu2, Si Hoon Park6, Hyun Kyu Song6, Hyun Seung Ban7, Hye-Jin Yoon8 and Byung Il Lee *,1,3 1Research Institute, National Cancer Center, Goyang-si, Korea 2Department of Bioengineering, Hanyang University, Seoul, Korea 3Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, Goyang-si, Korea 4Integrated Metabolomics Research Group, Western Seoul Center, Korea Basic Science Institute, Seoul, Korea 5Department of Chemistry and Nano Science, Ewha Womans University, Seoul, Korea 6Department of Life Sciences, Korea University, Seoul, Korea 7Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea 8Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul, Korea *Corresponding author. Tel: +82 31 920 2223; E-mail: [email protected] EMBO Reports (2021)22:e51323https://doi.org/10.15252/embr.202051323 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca2+ ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER-anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid-like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51–VAPB interaction is mediated by the FFAT-like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM-tethering function. SYNOPSIS The crystal structure and biochemical analyses of PTPIP51, a mitochondrial protein localized at the mitochondria-associated ER membrane (MAM), revealed its phospholipid binding and transfer activity. The crystal structure of the TPR domain of PTPIP51 at 1.45 Å resolution revealed the presence of a lipid-like serpentine electron density. PTPIP51 has phospholipid (especially phosphatidic acid) binding and transfer functions in vitro. Mitochondrial cardiolipin levels are affected by PTPIP51. Introduction One of the remarkable features of eukaryotic cells is their partitioning into individual organelles with specific functions. Since some essential biochemical pathways function across multiple organelles, interorganelle communication through vesicular or non-vesicular trafficking to translocate various cellular materials in cells is required (Gatta & Levine, 2017). In particular, translocation of many important cellular materials occurs at interorganelle contact sites and is closely related to cellular physiology and many human diseases (Bittremieux et al, 2016; Filadi et al, 2017; Gomez-Suaga et al, 2018). Among the various organelle contact sites, the mitochondria–endoplasmic reticulum (ER) interaction site is one of the best characterized (Rowland & Voeltz, 2012; Phillips & Voeltz, 2016). Approximately 5–20% of the mitochondrial membrane is closely attached to the ER membrane with a gap of 10–30 nm; these membranes are named mitochondria-associated ER membranes (MAMs) (Rizzuto et al, 1998; Rowland & Voeltz, 2012; Stoica et al, 2014; Paillusson et al, 2016; Hirabayashi et al, 2017). The formation of MAMs is mediated by several membrane-anchored MAM-tethering protein complexes. The vesicle-associated membrane protein-associated protein B (VAPB)–protein tyrosine phosphatase-interacting protein 51 (PTPIP51), oxysterol-binding protein-related protein 5/8 (ORP5/8)–PTPIP51, B-cell receptor-associated protein 31 (BAP31)–mitochondrial fission 1 protein (FIS1), inositol 1,4,5-trisphosphate receptor (IP3R)–glucose-regulated protein 75 (GRP75)–voltage-dependent anion channel (VDAC)–mitochondrial calcium uniporter (MCU), mitofusin (MFN2)–MFN1 (or MFN2), and ribosome-binding protein (RRBP1)–synaptojanin 2-binding protein (SYNJ2BP) complexes have been discovered in mammalian cells and have been studied (Gomez-Suaga et al, 2018). MAMs are intimately related to various cellular processes, including ER stress, mitochondrial dynamics, inflammation, autophagy, and apoptosis (Filadi et al, 2017). These functions mainly result from the exchange of cellular materials, such as Ca2+ ions and phospholipids (Rizzuto et al, 1998; Paillusson et al, 2016; Filadi et al, 2017). In detail, Ca2+ can be transferred from the ER, which functions as a Ca2+ reservoir in cells, to mitochondria through the IP3R–GRP75–VDAC Ca2+ uptake/release channel complex (Bittremieux et al, 2016; Phillips & Voeltz, 2016). These supplied Ca2+ ions can stimulate mitochondrial bioenergetic reactions, including NADH formation, respiratory chain activity, and the activity of some enzymes in the TCA cycle, which favors cell survival (Filadi et al, 2017). However, Ca2+ overload has opposite physiological effects, such as the induction of mitochondrial depolarization and release of cytochrome c, which induces apoptosis. Phospholipids are another major cellular material transferred at MAMs. While many phospholipids are usually synthesized in the ER membrane, some phospholipids, such as phosphatidylethanolamine (PE) and cardiolipin (CL), are synthesized in the inner mitochondrial membrane (IMM) after the transport of precursor phospholipids from other cellular organelles (Holthuis & Menon, 2014; Tamura et al, 2014; Tatsuta et al, 2014; Muallem et al, 2017). For example, the pathways by which PE and phosphatidylcholine (PC), major phospholipids in most cellular organelles, are synthesized function across the ER and mitochondria (Rowland & Voeltz, 2012; Tamura et al, 2014). Phosphatidic acid (PA) is converted to phosphatidylserine (PS) via cytidine diphosphate diacylglycerol (CDP-DAG) as an intermediate at the ER membrane. The produced PS molecules are transferred to mitochondria because the enzyme PSD1, which catalyzes the conversion of PS to PE, localizes at the IMM (Tatsuta et al, 2014). The produced PE molecules move back to the ER membrane, and PC molecules are synthesized from PE. For synthesis of these phospholipids, each phospholipid must be properly transferred with the aid of lipid transfer proteins. In yeast cells, the ER–mitochondria encounter structure (ERMES) complex, which is composed of several synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain-containing proteins, interacts with various glycerophospholipids and governs phospholipid transportation at MAMs (Kornmann et al, 2009; Tatsuta et al, 2014; AhYoung et al, 2015). However, the protein complex in mammalian cells that corresponds to the ERMES complex has not yet been identified. Instead, phospholipid trafficking in mammalian cells may be mediated by different kinds of proteins. For example, MFN2 binds and transfers PS, and STARD7 transfers PC (Horibata & Sugimoto, 2010; Horibata et al, 2017; Hernandez-Alvarez et al, 2019). Two oxysterol-binding proteins, ORP5 and ORP8, are also localized to MAMs and related to the exchange of PS (Rochin et al, 2020). Among the components of MAM-tethering protein complexes, PTPIP51 (also called RMDN3) is a mitochondrial membrane-anchored protein that interacts with ER membrane-bound VAPB (De Vos et al, 2012; Stoica et al, 2014). PTPIP51 was originally identified as a cellular protein that regulates the differentiation, apoptosis, proliferation, and mitosis of cells by forming protein complexes with PTP1B or 14-3-3 proteins (Brobeil et al, 2017). Previous studies have suggested that the PTPIP51−VAPB association contributes to MAM formation and the regulation of Ca2+ homeostasis and autophagy (De Vos et al, 2012; Gomez-Suaga et al, 2017), and disruption of this complex by TDP-43 or GSK-3β modulates cellular Ca2+ homeostasis and is related to neurodegenerative disease (Stoica et al, 2014; Stoica et al, 2016). Furthermore, PTPIP51 interacts with ORP5/8, suggesting a functional link to lipid transfer (Galmes et al, 2016). However, the exact biochemical functions of PTPIP51 beyond MAM tethering have not yet been elucidated. Here, we determined the crystal structure of the C-terminal tetratricopeptide repeat (TPR) domain of PTPIP51 (PTPIP51_TPR), which is the largest globular domain in PTPIP51, to gain molecular and functional insights into MAM-localized PTPIP51. Although the TPR domain has been suggested to be a protein–protein interaction module, structural analysis of PTPIP51_TPR provided evidence of a lipid binding function. Based on this finding, we revealed the phospholipid binding and transfer activity of PTPIP51. These results suggest that the PTPIP51−VAPB complex is a functional human counterpart of the yeast ERMES complex. Results and Discussion Structure of the PTPIP51 TPR domain To understand the molecular functions of PTPIP51, we aimed to study the structure of PTPIP51 and successfully crystallized its C-terminal domain (residues 236–470, Appendix Fig S1). The crystallized domain was predicted to be a TPR (PTPIP51_TPR) domain (Stenzinger et al, 2009). The crystal structure of PTPIP51_TPR was determined at 1.45 Å resolution by the single-wavelength anomalous dispersion (SAD) method (Fig 1A; Appendix Table S1). PTPIP51_TPR adopts an all-α-helical structure and contains twelve α-helices: α1 (239–250), α2 (252–270), α3 (272–290), α4 (294–314), α5 (317–335), α6 (338–358), α7 (362–379), α8 (381–390), α9 (398–412), α10 (418–431), α11 (434–446), and α12 (452–468). These α-helices form several antiparallel TPR units, joined mainly by hydrophobic interactions within TPR1 (α1–α2), TPR2 (α3–α4), TPR3 (α5–α6), TPR4 (α7–α9), and TPR5 (α10–α11). The PTPIP51_TPR structure features two kinds of hydrophobic interactions: intra-TPR and inter-TPR interactions. In the former interaction, the residues that form the hydrophobic patches are directed toward the inside of each TPR structure: TPR1 (Leu241, Ala245, Leu248, Leu262, and Leu263), TPR2 (Leu277, Ala281, Ala310, and Ala311), TPR3 (Ala319, Ala326, Phe348, Ala355, and Leu358), TPR4 (Pro363, Phe367, Ala402, Ala409, and Leu412), and TPR5 (Val422 and Ala444). The latter inter-TPR interactions involve four regions: TPR1–TPR2 (Phe260, Leu264, Leu280, and Met287), TPR3–TPR4 (Val352, Ile356, and Leu369), TPR4–TPR5 (Phe406 and Ile424), and TPR5–α12 (Val422, Ala437, Ala444, Leu461, Leu464, and Leu468) (Fig 1B). Ten of the twelve antiparallel α-helices form double-helical layers consisting of concave inner layers (α1, α3, α5, α7, and α10) and convex outer layers (α2, α4, α6, α9, and α11) (Fig 1A). Helix α8 generates a bulge, and helix α12 covers half of the concave region of PTPIP51_TPR. Analytical ultracentrifugation (AUC) experiments with PTPIP51_TPR or PTPIP51_ΔTM (residues 36–470) indicated that these proteins exist as monomers (PTPIP51_TPR) or tetramers (PTPIP51_ΔTM) in solution (Fig 1C). Interestingly, prediction of the coiled-coil oligomeric state by LOGICOIL (http://coiledcoils.chm.bris.ac.uk/LOGICOIL/) (Vincent et al, 2013) also predicted tetramerization of PTPIP51. These results suggest that tetramerization of PTPIP51 may be mediated by the coiled-coil domain of the protein. We also tried to measure the molecular weight of PTPIP51 without the TPR domain (PTPIP51_ΔTPR, residues 36–235 or 36–130). However, these constructs existed as high-molecular-weight aggregates in solution, and we could not estimate their oligomeric states. A DALI server (http://ekhidna2.biocenter.helsinki.fi/dali) search with PTPIP51_TPR identified structurally similar proteins (Holm & Laakso, 2016). The proteins with the greatest structural similarity are the hypothetical conserved protein TTC0263 (PDB ID 2PL2, Z score = 18.1) (Lim et al, 2007), the type 4 fimbrial biogenesis protein PILF (PDB ID 2FI7, Z score = 17.0) (Kim et al, 2006), and anaphase-promoting complex subunit CUT9 (PDB ID 2XPI, Z score = 17.0) (Zhang et al, 2010). Although the amino acid sequence identities between PTPIP51_TPR and these three structures were very low (< 15%), their structural topologies were quite similar (Fig 1D). Figure 1. Crystal structure of PTPIP51_TPR Domain architecture of PTPIP51 and overall structure of PTPIP51_TPR. The transmembrane (TM), coiled-coil (CC), and tetratricopeptide repeat (TPR) domains are presented. Each TPR pair is marked with a different color: red, TPR1 (α1 and α2); orange, TPR2 (α3 and α4); yellow, TPR3 (α5 and α6); green, TPR4 (α7 and α9); and blue, TPR5 (α10 and α11). Two α-helices that are not involved in a TPR (α8 and α12) are highlighted in light blue. The lower panel shows helical wheel projections for the PTPIP51_TPR structure showing one of the features typical of a multi-TPR protein: TPR pairs forming a superhelical twist. The diagram was drawn using NetWheels (http://lbqp.unb.br/NetWheels). Detailed views of the hydrophobic interactions of TPRs. There are two kinds of hydrophobic interactions, intra-TPR and inter-TPR. The hydrophobic residues associated with inter-TPR interactions are indicated with asterisks (*). Analytical ultracentrifugation (AUC) of PTPIP51_TPR and PTPIP51_ΔTM. The molecular weights of recombinant PTPIP51 calculated and estimated in AUC experiments are shown. All fusion tags were removed from the respective recombinant proteins for the AUC experiments. Topology diagrams of PTPIP51_TPR and other TPRs: TTC0263_TPR (PDB ID 2PL2), PILF_TPR (PDB ID 2FI7), and CUT9_TPR (PDB ID 2XPI). TPR pairs are presented in different color, which are identical to those in the topology diagrams. Electrostatic surface potentials of TTC0263_TPR, PILF_TPR, CUT9_TPR, and PTPIP51_TPR, showing two different views of the electrostatic surface potential of each TPR. The concave and convex regions of each TPR are shown. The electrostatic surface potentials were calculated using the APBS electrostatics plugin (https://www.poissonboltzmann.org/) at ±3 kT/e. Download figure Download PowerPoint Among the three structures with the greatest similarity to PTPIP51_TPR identified by the DALI server search, the TPR domain of TTC0263 contains a weakly positively charged concave surface that may provide a docking site for its binding partners (Fig 1E) (Lim et al, 2007). However, the corresponding surfaces in the TPR domains of PILF, CUT9, and PTPIP51 are negatively charged (Fig 1E) (Kim et al, 2006; Zhang et al, 2010). Identification of a serpentine electron density in the PTPIP51_TPR structure Interestingly, an elongated, deep cavity generated by TPR2 (α3–α4) and TPR3 (α5–α6) and a bulge region by α8 was found in PTPIP51 (Fig 2A). The sizes of the cavities in PTPIP51_TPR and other structurally similar TPR proteins were analyzed with CASTp 3.0 (http://sts.bioe.uic.edu/castp/) (Tian et al, 2018), and the cavities in the TTC0263_TPR, CUT9_TPR, and PTPIP51_TPR structures were found to be large. The calculated sizes of the cavities in TTC0263_TPR, CUT9_TPR, and PTPIP51_TPR were 827.7, 3009.6, and 709.4 Å3, respectively. We could not find a large cavity in the PILF_TPR structure. Compared to the cavities in other TPR structures, the cavity in PTPIP51 forms a channel in the absence of α8, and this non-TPR helix covers one end of the channel like a plug. Therefore, the resulting cavity resembles a cave with a wide entrance (Fig 2). The inside surface of the cavity is relatively hydrophobic and weakly positively charged (Fig 2B). In the cave-like cavity, electron density maps for two nonprotein molecules were found, and based on the shapes of the electron density maps, we tentatively assigned these molecules as glycerol derived from the cryoprotectant solution and p-toluenesulfonic acid (Fig 2B; Appendix Fig S2A and B). The p-toluenesulfonic acid molecule in the cavity is mainly surrounded by hydrophobic residues (Trp372, Val376, and Leu395) and two hydrophilic residues (Arg341 and Gln375), which form water-mediated interactions with this p-toluenesulfonic acid molecule. An additional p-toluenesulfonic acid molecule with few hydrophobic contacts (only Gln258) is located in another part of the protein structure. The glycerol molecule in the cavity participates in two direct hydrogen bonds, with Ser344 and Gln375, and two water-mediated interactions, with Leu368 and Arg371. Figure 2. Calculated cavities in the TPR structures and tube-shaped electron density map in the PTPIP51_TPR structure Cavities of TTC0263_TPR, PILF_TPR, CUT9_TPR, and PTPIP51_TPR. The cavities were calculated as solvent-accessible regions using the CASTp program. The surface properties of the PTPIP51_TPR structure. Hydrophilic and hydrophobic residues are shown in pink and yellow, respectively. Sliced views of PTPIP51_TPR showing the cavity and its hydrophobic property. The α8 helix is highlighted with a dotted line in the sliced view. Detailed view of the electron density map indicative of a tube-shaped molecule. Hydrophilic and hydrophobic amino acid residues adjacent to the electron density map are shown in pink and yellow, respectively. The mFo-DFc omit maps were generated using the program “phenix.composite_omit_map” in the Phenix program suite. The contour level of the omit map is 0.8 σ. Download figure Download PowerPoint Notably, a long tube-shaped electron density map was found in the entrance of the cave-like cavity (Fig 2C). The crystallization reservoir solution, which contained the Sokalan® CP42 polymer, suggests that the density map corresponds to part of this polymer. However, the electron density map was relatively weak, and we could not precisely determine its identity. Since many phospholipids can be exchanged at MAMs, and the electron density map looked like the hydrocarbon chain of a lipid, we presumed that this electron density could be assigned to part of an unknown phospholipid from E. coli, the expression host for the recombinant PTPIP51_TPR protein. We tried to identify lipids in the PTPIP51 structure by mass spectrometry with PTPIP51 crystals and identified various kinds of lipid species (Appendix Fig S3). However, we could not specify and assign this electron density map to a phospholipid because no electron density map for a phospholipid head group was seen. This tube-shaped electron density map is surrounded by many hydrophobic and hydrophilic patches (Fig 2C). Therefore, our crystal structure suggests that the electron density map found in the PTPIP51 structure corresponds possibly to some lipid species and that the PTPIP51 may functionally associate with lipids, notwithstanding that the role of the cavity in the TPR domain is indefinitive. PTPIP51 has phospholipid binding and phospholipid transfer activity In addition to Ca2+ ion exchange, an important biochemical event at MAMs is the transfer of phospholipids for the synthesis of various kinds of phospholipids. Although the ERMES complex, a MAM-tethering macromolecular complex involved in phospholipid exchange, has been relatively well studied in yeast (Phillips & Voeltz, 2016; Jeong et al, 2017; Muallem et al, 2017), MAM-tethering complexes that also function in phospholipid exchange have not been fully identified in humans. Recently, the amino acid sequence of PDZD8, an SMP domain-containing ER protein, was shown to partially align with that of yeast MMM1 (a component of the yeast ERMES complex), suggesting that PDZD8 is a human ortholog or paralog of the ERMES complex (Hirabayashi et al, 2017; Wideman et al, 2018). However, while the ERMES complex is known to regulate the transfer of phospholipids between the ER and mitochondria (Kornmann et al, 2009; AhYoung et al, 2015; Jeong et al, 2017; Cockcroft & Raghu, 2018), PDZD8 regulates Ca2+ dynamics in neurons (Hirabayashi et al, 2017). Since the presence of the unexplained serpentine electron density map in the crystal structure of PTPIP51_TPR suggests a possible phospholipid binding function, we sought to determine whether PTPIP51 carries out phospholipid-related functions. First, we performed a series of in vitro phospholipid- and lipid-binding assays with purified recombinant PTPIP51_ΔTM, PTPIP51_TPR, and PTPIP51_ΔTPR (residues 36–235) proteins using a dot blot overlay (PIP strips) assay (Fig 3A). Two of these PTPIP51 protein constructs—PTPIP51_ΔTM and PTPIP51_TPR—interacted with various phospholipids with different affinities. PTPIP51_ΔTM bound phosphatidylinositol (PtdIns) mono-, di-, and triphosphate, PA, PS, and CL. PTPIP51_TPR interacted with PtdIns(4)P, PtdIns(5)P, and PA. While PTPIP51_ΔTM bound CL and di-PtdIns, PTPIP51_TPR barely bound these phospholipids. Two phospholipids (PA and PtdIns(4)P) bound both PTPIP51 constructs. PtdIns(5)P interacted with all three constructs of PTPIP51. Interestingly, PTPIP51 did not bind phospholipids with positively charged head groups, such as PE and PC. Other lipids, such as sphingomyelin and cholesterol, whose biosynthesis does not occur in mitochondria (Holthuis & Menon, 2014; Tatsuta et al, 2014), did not interact with PTPIP51. Figure 3. Phospholipid binding and phospholipid transfer functions of PTPIP51 PIP strip lipid-binding assay with recombinant PTPIP51_ΔTM, PTPIP51_TPR, and PTPIP51_ΔTPR. PA binding of PTPIP51 (PTPIP51_ΔTM and PTPIP51_TPR) was monitored by a PA precipitation assay and a liposome flotation assay. The POPC:POPE:POPA ratios in each liposome were 50:50:0, 50:40:10, 50:30:20, and 50:20:30. Diagrams of the liposome precipitation and flotation assays are illustrated separately. The PA transfer activity of PTPIP51 (PTPIP51_ΔTM and PTPIP51_TPR) was monitored by a FRET-based lipid transfer assay. A schematic cartoon of the FRET-based in vitro lipid transfer assay is presented on the left. a.u.: arbitrary unit. The alternative bead pulldown-based lipid transfer assay with PA and MLCL. A schematic cartoon representation of the fluorescent lipid transfer assay is shown on the left. The y-axes in the graphs show the percentage of acceptor liposome fluorescence calculated using the following formula: 100×Facceptor/(Facceptor+Fdonor). Data information: The data are presented as the mean ± SD of technical triplicate (C) or quadruplicate (D) from one representative experiment (n = 2), and P-values calculated using one-way ANOVA are shown. Download figure Download PowerPoint Considering the cellular localization of PTPIP51 and the biological significance of PA transport between the ER and mitochondria, we further investigated the interaction between PTPIP51 and PA using a PA precipitation assay (Fig 3B). PTPIP51_ΔTM or PTPIP51_TPR was mixed with liposomes with different lipid contents (POPC:POPE:POPA = 50:50:0, 50:40:10, 50:30:20, and 50:20:30 (%)), and ultracentrifugation experiments were performed. PTPIP51_ΔTM showed increased binding to liposomes with increasing PA concentration. PTPIP51_TPR also interacted with liposomes containing PA, but this interaction did not depend on the PA concentration. These results suggest that the middle region of PTPIP51, which includes the coiled-coil domain and FFAT (two phenylalanines (FF) in acidic tract)-like motifs, partially contributes to its phospholipid binding and specificity (Appendix Fig S1). The interaction between PTPIP51 and PA was also confirmed by a sucrose gradient liposome flotation assay (Fig 3B). While PTPIP51_ΔTM floated in the less-dense fractions of the sucrose gradient due to its binding to PA-containing liposomes, it was distributed in the denser layers when mixed with liposomes not containing PA. These results showed that PTPIP51 selectively interacts with PA and not PC or PE, as also shown in the PIP strip assay. We also investigated the interaction between PTPIP51_ΔTM and other phospholipids that exhibited binding in the PIP strip assay. We selected PS, PtdIns(4)P, and CL for the liposome precipitation assay and found that PtdIns(4)P and CL interacted wit
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