#METABOLOMICS WORKBENCH lje3080_20210303_204644 DATATRACK_ID:2516 STUDY_ID:ST001717 ANALYSIS_ID:AN002798 PROJECT_ID:PR001101 VERSION 1 CREATED_ON March 4, 2021, 6:40 am #PROJECT PR:PROJECT_TITLE Phospholipid transfer function of PTPIP51 at mitochondria-associated ER PR:PROJECT_TITLE membranes PR:PROJECT_TYPE MS-based lipid profiling PR:PROJECT_SUMMARY LC/MS-based lipid profiling of mitochondria obtained HeLa cell line PR:INSTITUTE Korea Basic Science Institute PR:DEPARTMENT Western Seoul Center PR:LABORATORY Integrated Metabolomics Research Group PR:LAST_NAME Lee PR:FIRST_NAME Jueun PR:ADDRESS 150, Bugahyeon-ro, Seoul, Seoul, 03759, Korea, South PR:EMAIL lje3080@kbsi.re.kr PR:PHONE +82-2-6908-6256 #STUDY ST:STUDY_TITLE Phospholipid transfer function of PTPIP51 at mitochondria-associated ER ST:STUDY_TITLE membranes ST:STUDY_SUMMARY In eukaryotic cells, mitochondria are closely tethered to the endoplasmic ST:STUDY_SUMMARY reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca2+ ST:STUDY_SUMMARY ion and phospholipid transfer occurs at MAMs to support diverse cellular ST:STUDY_SUMMARY functions. Unlike those in yeast, the protein complexes involved in phospholipid ST:STUDY_SUMMARY transfer at MAMs in humans have not been identified. Here, we determined the ST:STUDY_SUMMARY crystal structure of the tetratricopeptide repeat domain of PTPIP51 ST:STUDY_SUMMARY (PTPIP51_TPR), a mitochondrial protein that interacts with the ER-anchored VAPB ST:STUDY_SUMMARY protein at MAMs. The structure of PTPIP51_TPR showed an archetypal TPR fold, and ST:STUDY_SUMMARY an electron density corresponding to an unidentified lipid-like molecule ST:STUDY_SUMMARY probably derived from the protein expression host was found in the structure. We ST:STUDY_SUMMARY revealed functions of PTPIP51 in phospholipid binding/transfer, particularly of ST:STUDY_SUMMARY phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduced the ST:STUDY_SUMMARY mitochondrial cardiolipin level. Additionally, we confirmed that the ST:STUDY_SUMMARY PTPIP51–VAPB interaction is mediated by the FFAT-like motif of PTPIP51 and the ST:STUDY_SUMMARY MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer ST:STUDY_SUMMARY protein with a MAM-tethering function similar to the ERMES complex in yeast. ST:INSTITUTE Korea Basic Science Institute ST:DEPARTMENT Western Seoul Center ST:LABORATORY Integrated Metabolomics Research Group ST:LAST_NAME Lee ST:FIRST_NAME Jueun ST:ADDRESS 150, Bugahyeon-ro, Seodaemun-gu, Seoul, Republic of Korea (Zip code: 03759) ST:EMAIL lje3080@kbsi.re.kr ST:PHONE +82-2-6908-6256 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:GENDER Not applicable #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Mito_001_po Group:Mock RAW_FILE_NAME=Mito_001_po.mzml SUBJECT_SAMPLE_FACTORS - Mito_002_po Group:Mock(+) RAW_FILE_NAME=Mito_002_po.mzml SUBJECT_SAMPLE_FACTORS - Mito_003_po Group:FL(+) RAW_FILE_NAME=Mito_003_po.mzml SUBJECT_SAMPLE_FACTORS - Mito_004_po Group:FFAT(+) RAW_FILE_NAME=Mito_004_po.mzml SUBJECT_SAMPLE_FACTORS - Mito_005_po Group:Mock RAW_FILE_NAME=Mito_005_po.mzml SUBJECT_SAMPLE_FACTORS - Mito_006_po Group:Mock(+) RAW_FILE_NAME=Mito_006_po.mzml SUBJECT_SAMPLE_FACTORS - Mito_011_po Group:Mock(+) RAW_FILE_NAME=Mito_011_po.mzml SUBJECT_SAMPLE_FACTORS - Mito_007_po Group:FL(+) RAW_FILE_NAME=Mito_007_po.mzml SUBJECT_SAMPLE_FACTORS - Mito_008_po Group:FFAT(+) RAW_FILE_NAME=Mito_008_po.mzml SUBJECT_SAMPLE_FACTORS - Mito_012_po Group:FFAT(+) RAW_FILE_NAME=Mito_012_po.mzml SUBJECT_SAMPLE_FACTORS - Mito_009_po Group:Mock RAW_FILE_NAME=Mito_009_po.mzml SUBJECT_SAMPLE_FACTORS - Mito_010_po Group:FL(+) RAW_FILE_NAME=Mito_010_po.mzml SUBJECT_SAMPLE_FACTORS - QC_mito_01_po Group:QC RAW_FILE_NAME=QC_mito_01_po.mzml SUBJECT_SAMPLE_FACTORS - QC_mito_02_po Group:QC RAW_FILE_NAME=QC_mito_02_po.mzml SUBJECT_SAMPLE_FACTORS - QC_mito_03_po Group:QC RAW_FILE_NAME=QC_mito_03_po.mzml SUBJECT_SAMPLE_FACTORS - tQC_mito_03_po Group:QC RAW_FILE_NAME=tQC_mito_03_po.mzml SUBJECT_SAMPLE_FACTORS - Mito_001_ne Group:Mock RAW_FILE_NAME=Mito_001_ne.mzml SUBJECT_SAMPLE_FACTORS - Mito_002_ne Group:Mock(+) RAW_FILE_NAME=Mito_002_ne.mzml SUBJECT_SAMPLE_FACTORS - Mito_003_ne Group:FL(+) RAW_FILE_NAME=Mito_003_ne.mzml SUBJECT_SAMPLE_FACTORS - Mito_004_ne Group:FFAT(+) RAW_FILE_NAME=Mito_004_ne.mzml SUBJECT_SAMPLE_FACTORS - Mito_005_ne Group:Mock RAW_FILE_NAME=Mito_005_ne.mzml SUBJECT_SAMPLE_FACTORS - Mito_006_ne Group:Mock(+) RAW_FILE_NAME=Mito_006_ne.mzml SUBJECT_SAMPLE_FACTORS - Mito_011_ne Group:Mock(+) RAW_FILE_NAME=Mito_011_ne.mzml SUBJECT_SAMPLE_FACTORS - Mito_007_ne Group:FL(+) RAW_FILE_NAME=Mito_007_ne.mzml SUBJECT_SAMPLE_FACTORS - Mito_008_ne Group:FFAT(+) RAW_FILE_NAME=Mito_008_ne.mzml SUBJECT_SAMPLE_FACTORS - Mito_012_ne Group:FFAT(+) RAW_FILE_NAME=Mito_012_ne.mzml SUBJECT_SAMPLE_FACTORS - Mito_009_ne Group:Mock RAW_FILE_NAME=Mito_009_ne.mzml SUBJECT_SAMPLE_FACTORS - Mito_010_ne Group:FL(+) RAW_FILE_NAME=Mito_010_ne.mzml SUBJECT_SAMPLE_FACTORS - QC_mito_01_ne Group:QC RAW_FILE_NAME=QC_mito_01_ne.mzml SUBJECT_SAMPLE_FACTORS - QC_mito_02_ne Group:QC RAW_FILE_NAME=QC_mito_02_ne.mzml SUBJECT_SAMPLE_FACTORS - QC_mito_03_ne Group:QC RAW_FILE_NAME=QC_mito_03_ne.mzml SUBJECT_SAMPLE_FACTORS - tQC_mito_03_ne Group:QC RAW_FILE_NAME=tQC_mito_03_ne.mzml #COLLECTION CO:COLLECTION_SUMMARY Mitochondria were isolated from HeLa cells using a mitochondria isolation kit CO:COLLECTION_SUMMARY for tissues (cat. no. 89874, Thermo Fisher Scientific, USA) according to the CO:COLLECTION_SUMMARY manufacturer’s instructions. Mitochondrial pellets were washed and stored in CO:COLLECTION_SUMMARY 1xTBS buffer supplemented with 0.1% CHAPS on ice until further use. Isolated CO:COLLECTION_SUMMARY mitochondrial fractions were used for lipidomic analyses. CO:SAMPLE_TYPE HeLa cells #TREATMENT TR:TREATMENT_SUMMARY Conditional knockdown and reconstitution of PTPIP51 in HeLa cells were performed TR:TREATMENT_SUMMARY as previously reported (Bong et al, 2020). Huma PTPIP51-targeting small hairpin TR:TREATMENT_SUMMARY RNA (shRNA) sequences (5’-ATGACTTGATGCCACTATTTA-3’) were inserted into the TR:TREATMENT_SUMMARY Tet-pLKO-blasticidin vector. Lentiviruses were produced according to a method TR:TREATMENT_SUMMARY described previously (Kim et al, 2018). HeLa cells infected with lentiviruses TR:TREATMENT_SUMMARY were selected with blasticidin (10 μg/ml, Invitrogen, USA) for at least 7 days TR:TREATMENT_SUMMARY and named HeLa Tet-on-shPTPIP51 cells. The genes encoding human full-length TR:TREATMENT_SUMMARY PTPIP51 and PTPIP51_ΔFFAT were cloned into the pCAG-Flag-IRES-puro vector. HeLa TR:TREATMENT_SUMMARY Tet-on-shPTPIP51 cells were transfected with pCAG-Flag-IRES-puro empty vector TR:TREATMENT_SUMMARY (Mock), PTPIP51, and PTPIP51_ΔFFAT using Lipofectamine 3000 (Life Technologies, TR:TREATMENT_SUMMARY USA). Transfected cells were selected with puromycin (2 μg/ml, Amresco, USA) TR:TREATMENT_SUMMARY for at least 4 days. For endogenous PTPIP51 knockdown, doxycycline (1 μg/ml, TR:TREATMENT_SUMMARY Sigma-Aldrich, USA) was added every two days. Because the shRNAs were designed TR:TREATMENT_SUMMARY to target the 3’ UTR of PTPIP51, exogenously added constructs were not TR:TREATMENT_SUMMARY targeted. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For mitochondrial analysis, isolated mitochondria from 2x10^7 cells were mixed SP:SAMPLEPREP_SUMMARY with 500 µl of 100 mM hydrochloric acid solution in methanol/water (8:2, v/v), SP:SAMPLEPREP_SUMMARY 700 µl of chloroform and 500 µl of 100 mM hydrochloric acid solution in water. SP:SAMPLEPREP_SUMMARY The mitochondrial sample was homogenized with 2.8 mm zirconium oxide beads for 5 SP:SAMPLEPREP_SUMMARY min. After centrifugation at 30,130 xg and 4°C for 15 min, 600 µl of the lower SP:SAMPLEPREP_SUMMARY phase was collected. Both the cell and mitochondrial extracts were dried under a SP:SAMPLEPREP_SUMMARY gentle nitrogen stream at room temperature and reconstituted in 300 µL of SP:SAMPLEPREP_SUMMARY isopropanol/acetonitrile/water (2:1:1, v/v/v). Eighty microliters of each sample SP:SAMPLEPREP_SUMMARY was mixed with 20 µL of SPLASH LIPIDOMIX internal standard mix (Avanti Polar SP:SAMPLEPREP_SUMMARY Lipids, USA). Finally, 5 µl of each sample was injected into the UPLC-QTOF MS SP:SAMPLEPREP_SUMMARY system. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Chromatographic separation of lipids in cells and mitochondria was performed CH:CHROMATOGRAPHY_SUMMARY with an Acquity UPLC system (Waters, USA) using an Acquity UPLC CSH C18 column CH:CHROMATOGRAPHY_SUMMARY (2.1100 mm, 1.7 µm; Waters) at 55°C and a flow rate of 0.4 ml/min. The CH:CHROMATOGRAPHY_SUMMARY mobile phase for positive ion mode comprised 10 mM ammonium formate in CH:CHROMATOGRAPHY_SUMMARY water/acetonitrile (40:60, v/v) containing 0.1% formic acid (solvent A) and CH:CHROMATOGRAPHY_SUMMARY isopropanol/acetonitrile (90:10, v/v) containing 0.1% formic acid (solvent B). CH:CHROMATOGRAPHY_SUMMARY The mobile phase for negative ion mode comprised 10 mM ammonium acetate in CH:CHROMATOGRAPHY_SUMMARY water/acetonitrile (60:40, v/v) (solvent A) and isopropanol/acetonitrile (90:10, CH:CHROMATOGRAPHY_SUMMARY v/v) (solvent B). The UPLC gradient was programmed as follows: 40% to 43% B from CH:CHROMATOGRAPHY_SUMMARY 0 min to 2 min, 43% to 50% B from 2 min to 2.1 min, 50% to 54% B from 2.1 min to CH:CHROMATOGRAPHY_SUMMARY 12 min, 54% to 70% B from 12 min to 12.1 min, 70% to 99% B from 12.1 min to 18 CH:CHROMATOGRAPHY_SUMMARY min, 99% to 40% B from 18 min to 18.1 min, and 40% B for 2 min to equilibrate CH:CHROMATOGRAPHY_SUMMARY for the next run. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity I-Class CH:COLUMN_NAME Waters Acquity CSH C18 (100 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5600 TripleTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS m/z range of 80-1500 The following parameter settings were used: ion spray MS:MS_COMMENTS voltage, 5500 V (positive mode) and 4500 V (negative mode); source temperature, MS:MS_COMMENTS 500°C; nebulizer gas pressure, 50 psi; drying gas pressure, 60 psi; and curtain MS:MS_COMMENTS gas pressure, 30 psi. An atmospheric pressure chemical ionization calibration MS:MS_COMMENTS solvent was used to maintain mass accuracy with an automated calibrant delivery MS:MS_COMMENTS system (Sciex). Information-dependent acquisition (IDA) was used to acquire MS:MS_COMMENTS MS/MS spectra for ions. All samples were pooled in equal amounts to generate MS:MS_COMMENTS quality control (QC) samples, which were injected after every 4 samples to MS:MS_COMMENTS calculate the coefficient of variation (CV) and assess analytical MS:MS_COMMENTS reproducibility. MS:MS_RESULTS_FILE ST001717_AN002798_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END