Structure–function insights into direct lipid transfer between membranes by Mmm1–Mdm12 of ERMES

“ScMdm12-bound lipids as well as total lipids were extracted from E. coli cells, without coexpression of Mmm1, using the Folch procedure (Folch et al., 1957).”

“The force fields were AMB ER ff99SB (Hornak et al., 2006), Lipid11 (Skjevik et al., 2012), and TIP3P water model (Jorgensen et al., 1983) for Mdm12, POPC, and explicit water molecules, respectively.”

“Pressure regulation was achieved with isotropic position scaling, a Berendsen barostat (Berendsen et al., 1984) of ∼1 atm, and a pressure relaxation time of 1 ps. Bonds involving hydrogen were constrained using the SHA KE algorithm (Ryckaert et al., 1977).”

“The structure was refined by refmac5 (Murshudov et al., 2011) and CNS (Brünger et al., 1998), and manual model building was performed by Coot (Emsley et al., 2010).”

“using particle mesh Ewald method (Darden et al., 1993) to treat long-”

“The phase problem was solved by an automated software suite (PHE NIX; Adams et al., 2010) using single-wavelength anomalous dispersion data from the crystals of SeMet-labeled HMdm12.”

“Pressure regulation was achieved with isotropic position scaling, a Berendsen barostat (Berendsen et al., 1984) of ∼1 atm, and a pressure relaxation time of 1 ps. Bonds involving hydrogen were constrained using the SHA KE algorithm (Ryckaert et al., 1977).”

“The structures of native or chemically modified proteins were solved by the molecular replacement method using the constructed model from SeMet-labeled HMdm12 as a search model by PHA SER (McCoy et al., 2007) in the CCP4 suite (Collaborative Computational Project, Number 4, 1994).”

“To monitor lipid transfer between liposomes via the HMmm1s– Mdm12 complex and MBP-HMmm1s, we adopted fluorescent dequenching assays as described previously (Connerth et al., 2012; Watanabe et al., 2015; Miyata et al., 2016).”

“The structure was refined by refmac5 (Murshudov et al., 2011) and CNS (Brünger et al., 1998), and manual model building was performed by Coot (Emsley et al., 2010).”

“Homology modeling of KlMmm1s based on the KlMdm12 structure was performed using Modeller software (Šali and Blundell, 1993).”

“were built using the LEaP module in AmberTools (Case et al., 2005) and then minimized and equilibrated in three stages: heating for 200 ps from 10 K to 300 K with heavy atom positional restraints of strength 10 kcal mol−1 Å−2, equilibration for 5 ns with the restraints, and equil-”

“The structure was refined by refmac5 (Murshudov et al., 2011) and CNS (Brünger et al., 1998), and manual model building was performed by Coot (Emsley et al., 2010).”

“fields were AMB ER ff99SB (Hornak et al., 2006), Lipid11 (Skjevik et al.”

“The determined structures were validated by Rampage (Lovell et al., 2003), and secondary structure analysis was performed by DSSP (Kabsch and Sander, 1983; Joosten et al., 2011).”

“However, the recent identification of direct physical contacts between the membranes of two organelles (Kornmann et al., 2009; Elbaz-Alon et al., 2014; Hönscher et al., 2014; Lahiri et al., 2014; Murley et al., 2015) highlights the need for coordinated functions of distinct organelles through…”

“cording to methods reported previously (Bokoch et al., 2010), with”

“Mutations or deletions of ERM ES components lead to altered morphology and/or cellular lipid composition (Dimmer et al., 2002; Kornmann et al., 2009; Osman et al., 2009; Nguyen et al., 2012; Tamura et al., 2012; Voss et al., 2012; Tan et al., 2013).”

“The solvent-accessible pockets were searched by query in the POC ASSA server (http ://altair .sci .hokudai .ac .jp /g6 .service /pocassa /; Yu et al., 2010) and visualized.”

“However, the recent identification of direct physical contacts between the membranes of two organelles (Kornmann et al., 2009; Elbaz-Alon et al., 2014; Hönscher et al., 2014; Lahiri et al., 2014; Murley et al., 2015) highlights the need for coordinated functions of distinct organelles through direct exchange of metabo…”

“…the recent identification of direct physical contacts between the membranes of two organelles (Kornmann et al., 2009; Elbaz-Alon et al., 2014; Hönscher et al., 2014; Lahiri et al., 2014; Murley et al., 2015) highlights the need for coordinated functions of distinct organelles through direct…”

“ERM ES also consists of the peripheral components Gem1 (Frederick et al., 2004; Kornmann et al., 2011; Stroud et al., 2011) and Tom7 (Yamano et al.”

“…feature of the E-SYT2 dimer is that it has a hydrophobic continuous tunnel running through the cylinder-like dimer and accommodates two lipid-like molecules per monomer, although the continuous tunnel is still too short to bridge two membranes for lipid transport (Schauder et al., 2014; Fig.”

“Mutations or deletions of ERM ES components lead to altered morphology and/or cellular lipid composition (Dimmer et al., 2002; Kornmann et al., 2009; Osman et al., 2009; Nguyen et al., 2012; Tamura et al., 2012; Voss et al., 2012; Tan et al., 2013).”

“However, the recent identification of direct physical contacts between the membranes of two organelles (Kornmann et al., 2009; Elbaz-Alon et al., 2014; Hönscher et al., 2014; Lahiri et al., 2014; Murley et al., 2015) highlights the need for coordinated functions of distinct organelles through direct exchange of metabo…”

“…physical contacts between the membranes of two organelles (Kornmann et al., 2009; Elbaz-Alon et al., 2014; Hönscher et al., 2014; Lahiri et al., 2014; Murley et al., 2015) highlights the need for coordinated functions of distinct organelles through direct exchange of metabolites and information.”

“, 1994); Mdm10, a β-barrel mitochondrial outer membrane (OM) protein (Sogo and Yaffe, 1994); Mdm34/Mmm2, an OM protein without an obvious transmembrane segment (Youngman et al.”

“ERM ES consists of four core subunits: Mmm1, an Nanchor ER membrane protein (Burgess et al., 1994); Mdm10, a β-barrel mitochondrial outer membrane (OM) protein (Sogo and Yaffe, 1994); Mdm34/Mmm2, an OM protein without an obvious transmembrane segment (Youngman et al., 2004), and Mdm12, a peripheral…”

“ERM ES also consists of the peripheral components Gem1 (Frederick et al., 2004; Kornmann et al., 2011; Stroud et al., 2011) and Tom7 (Yamano et al.”

“4 do not allow evaluation of the kinetics of lipid transfer between membranes, we performed a fluorescent-based lipid transfer kinetics assay between membranes, which is similar to the PA or PS transport assay reported previously (Connerth et al., 2012; Watanabe et al., 2015; Miyata et al., 2016).”

“Further complicating the interpretation is that an increase in other ER–mitochondria contacts and/or expansion of the area of the ER–vacuole contact region may compensate for a loss of ERM ES (Elbaz-Alon et al., 2014, 2015; Lahiri et al., 2014; Lang et al., 2015; Murley et al., 2015).”

“Three ERM ES subunits (Mmm1, Mdm12, and Mdm34) contain a synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain, which may belong to the TUL IP superfamily (Kopec et al., 2010, 2011), suggesting that these proteins can bind to phospholipids.”

“Further complicating the interpretation is that an increase in other ER–mitochondria contacts and/or expansion of the area of the ER–vacuole contact region may compensate for a loss of ERM ES (Elbaz-Alon et al., 2014, 2015; Lahiri et al., 2014; Lang et al., 2015; Murley et al., 2015).”

“The force fields were AMB ER ff99SB (Hornak et al., 2006), Lipid11 (Skjevik et al., 2012), and TIP3P water model (Jorgensen et al., 1983) for Mdm12, POPC, and explicit water molecules, respectively.”

“Studies using in vitro lipid displacement assays and/or mass spectrometry analyses of bound lipids in S. cerevisiae cells have reported a moderate lipid binding preference of PC (and PG) to PA and to PS for ScMdm12 (AhYoung et al., 2015; Jeong et al., 2016).”

“, 2004), and Mdm12, a peripheral OM protein (Berger et al., 1997).”

“…of Lys and applied to a Superdex 200 10/300 GL column to obtain purified target proteins. lipid transfer by mmm1–mdm12 of erm eS • Kawano et al. 971 Reductive methylation of Lys in KlMdm12 was performed according to methods reported previously (Bokoch et al., 2010), with minor modifications.”

“The liposome flotation assay was performed as described previously (Krick et al., 2012), with minor modifications.”

“Mutations or deletions of ERM ES components lead to altered morphology and/or cellular lipid composition (Dimmer et al., 2002; Kornmann et al., 2009; Osman et al., 2009; Nguyen et al., 2012; Tamura et al., 2012; Voss et al., 2012; Tan et al., 2013).”

“, 1994); Mdm10, a β-barrel mitochondrial outer membrane (OM) protein (Sogo and Yaffe, 1994); Mdm34/Mmm2, an OM protein without an obvious transmembrane segment (Youngman et al., 2004), and Mdm12, a peripheral OM protein (Berger et al.”

“ERM ES also consists of the peripheral components Gem1 (Frederick et al., 2004; Kornmann et al., 2011; Stroud et al., 2011) and Tom7 (Yamano et al.”

“Because most phospholipid biosynthetic pathways are present in the ER and mitochondria, production of cellular phospholipids relies on efficient transport of their precursor lipids between these organelles (Scharwey et al., 2013; Tamura et al., 2014; Tatsuta and Langer, 2017).”

“Mutations or deletions of ERM ES components lead to altered morphology and/or cellular lipid composition (Dimmer et al., 2002; Kornmann et al., 2009; Osman et al., 2009; Nguyen et al., 2012; Tamura et al., 2012; Voss et al., 2012; Tan et al., 2013).”

“Mutations or deletions of ERM ES components lead to altered morphology and/or cellular lipid composition (Dimmer et al., 2002; Kornmann et al., 2009; Osman et al., 2009; Nguyen et al., 2012; Tamura et al., 2012; Voss et al., 2012; Tan et al., 2013).”

“ERM ES also consists of the peripheral components Gem1 (Frederick et al., 2004; Kornmann et al., 2011; Stroud et al., 2011) and Tom7 (Yamano et al., 2010).”

“to the method described previously (Kojima et al., 2016).”

“4 do not allow evaluation of the kinetics of lipid transfer between membranes, we performed a fluorescent-based lipid transfer kinetics assay between membranes, which is similar to the PA or PS transport assay reported previously (Connerth et al., 2012; Watanabe et al., 2015; Miyata et al., 2016).”

“4 do not allow evaluation of the kinetics of lipid transfer between membranes, we performed a fluorescent-based lipid transfer kinetics assay between membranes, which is similar to the PA or PS transport assay reported previously (Connerth et al., 2012; Watanabe et al., 2015; Miyata et al., 2016).”

“Lipidomic analysis was performed as described previously (Guan et al., 2010) on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific).”

“Mutational analyses, including that of the Mdm12 I5P mutant, showed that the head-to-head homodimer formation of Mdm12 observed for the ScMdm12 x-ray structure (Jeong et al., 2016) is not necessary for the function of Mdm12 in vivo.”

“Three ERM ES subunits (Mmm1, Mdm12, and Mdm34) contain a synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain, which may belong to the TUL IP superfamily (Kopec et al., 2010, 2011), suggesting that these proteins can bind to phospholipids.”

“ing to the methods reported previously (Ichihara et al., 2011).”

“Phospholipid extraction was performed as described (Kawano et al., 2018).”

“To examine whether ATG2A has such activity, we adapted a lipid extraction assay in which liposomes containing fluorescent lipids (nitrobenzoxadiazole-conjugated PE: NBD-PE) are incubated with proteins, followed by liposome flotation to isolate the lipid-bound proteins (Kawano et al., 2018).”