#METABOLOMICS WORKBENCH diwu_20230914_023703 DATATRACK_ID:4313 STUDY_ID:ST002875 ANALYSIS_ID:AN004712 PROJECT_ID:PR001797 VERSION 1 CREATED_ON September 25, 2023, 9:28 am #PROJECT PR:PROJECT_TITLE LAT1-4F2hc Lipidomics PR:PROJECT_TYPE MS identification PR:PROJECT_SUMMARY The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino PR:PROJECT_SUMMARY acids, hormones and drugs. Its dysfunction is associated with many cancers and PR:PROJECT_SUMMARY immune/neurological disorders. Here, we apply native mass spectrometry PR:PROJECT_SUMMARY (MS)-based approaches to provide evidence of super-dimer formation PR:PROJECT_SUMMARY (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we PR:PROJECT_SUMMARY discover four endogenous phosphatidylethanolamine (PE) molecules at the PR:PROJECT_SUMMARY interface and C-terminus of both LAT1 subunits. We find that interfacial PE PR:PROJECT_SUMMARY binding is regulated by 4F2hc-R183 and is critical for regulation of PR:PROJECT_SUMMARY palmitoylation on neighbouring LAT1-C187. Combining native MS with mass PR:PROJECT_SUMMARY photometry (MP) we reveal that super-dimerization is sensitive to pH, and PR:PROJECT_SUMMARY modulated by complex N-glycans on the 4F2hc subunit. Free LAT1 is also present PR:PROJECT_SUMMARY in the lysosome after interferon-γ (IFN-γ) stimulation suggesting that the PR:PROJECT_SUMMARY assembly of LAT1-4F2hc is regulated by redox dynamics in vivo. Together our PR:PROJECT_SUMMARY results link PTM and lipid binding with regulation and trafficking of the PR:PROJECT_SUMMARY LAT1-4F2hc super dimer. PR:INSTITUTE University of Oxford PR:DEPARTMENT Kavli Institute for Nanoscience Discovery PR:LABORATORY Prof. Carol Robinson Group PR:LAST_NAME Wu PR:FIRST_NAME Di PR:ADDRESS Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford, OX1 PR:ADDRESS 3QU, UK PR:EMAIL di.wu2@chem.ox.ac.uk PR:PHONE +4401865275278 #STUDY ST:STUDY_TITLE LAT1-4F2hc Lipidomics ST:STUDY_SUMMARY The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino ST:STUDY_SUMMARY acids, hormones and drugs. Its dysfunction is associated with many cancers and ST:STUDY_SUMMARY immune/neurological disorders. Here, we apply native mass spectrometry ST:STUDY_SUMMARY (MS)-based approaches to provide evidence of super-dimer formation ST:STUDY_SUMMARY (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we ST:STUDY_SUMMARY discover four endogenous phosphatidylethanolamine (PE) molecules at the ST:STUDY_SUMMARY interface and C-terminus of both LAT1 subunits. We find that interfacial PE ST:STUDY_SUMMARY binding is regulated by 4F2hc-R183 and is critical for regulation of ST:STUDY_SUMMARY palmitoylation on neighbouring LAT1-C187. Combining native MS with mass ST:STUDY_SUMMARY photometry (MP) we reveal that super-dimerization is sensitive to pH, and ST:STUDY_SUMMARY modulated by complex N-glycans on the 4F2hc subunit. Free LAT1 is also present ST:STUDY_SUMMARY in the lysosome after interferon-γ (IFN-γ) stimulation suggesting that the ST:STUDY_SUMMARY assembly of LAT1-4F2hc is regulated by redox dynamics in vivo. Together our ST:STUDY_SUMMARY results link PTM and lipid binding with regulation and trafficking of the ST:STUDY_SUMMARY LAT1-4F2hc super dimer. ST:INSTITUTE University of Oxford ST:DEPARTMENT Kavli Institute for Nanoscience Discovery ST:LABORATORY Prof. Carol Robinson Group ST:LAST_NAME Wu ST:FIRST_NAME Di ST:ADDRESS Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford, OX1 ST:ADDRESS 3QU, UK ST:EMAIL di.wu2@chem.ox.ac.uk ST:PHONE +4401865275278 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS HEK293F #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 - LAT1-4F2hc_WT_1 Treatment:Untreated RAW_FILE_NAME=LAT1-4F2hc_WT_1.mzML SUBJECT_SAMPLE_FACTORS - LAT1-4F2hc_WT_2 Treatment:Untreated RAW_FILE_NAME=LAT1-4F2hc_WT_2.mzML SUBJECT_SAMPLE_FACTORS - LAT1-4F2hc_WT_delipidated_1 Treatment:Delipidated RAW_FILE_NAME=LAT1-4F2hc_WT_delipidated_1.mzML SUBJECT_SAMPLE_FACTORS - LAT1-4F2hc_WT_delipidated_2 Treatment:Delipidated RAW_FILE_NAME=LAT1-4F2hc_WT_delipidated_2.mzML #COLLECTION CO:COLLECTION_SUMMARY The LAT1-4F2hc complex was buffer-exchanged into 1M ammonium acetate, pH 7.0 CO:COLLECTION_SUMMARY with 2 mM OGNG and incubated with 1 µg trypsin overnight at 37 °C. The CO:COLLECTION_SUMMARY digested peptide/lipid mixture was dried using a SpeedVac vacuum concentrator CO:COLLECTION_SUMMARY (Thermo Fisher Scientific) CO:SAMPLE_TYPE HEK cells #TREATMENT TR:TREATMENT_SUMMARY The LAT1-4F2hc complex was buffer-exchanged into 1M ammonium acetate (pH 7.0) TR:TREATMENT_SUMMARY with 20 mM OGNG using a 100 kD MWCO centrifugal filter (Amicon Ultra-0.5 ml, TR:TREATMENT_SUMMARY Millipore) and incubated for 4 h at room temperature with gentle mixing. Excess TR:TREATMENT_SUMMARY lipomicelles and detergents were removed by buffer-exchanging into 1 M ammonium TR:TREATMENT_SUMMARY acetate with 2 mM OGNG using the same filter. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The lipid mixture was reconstituted with 70% mobile phase A (acetonitrile/H2O: SP:SAMPLEPREP_SUMMARY 60/40, 10 mM ammonium formate and 0.1% formic acid) and 30% mobile phase B SP:SAMPLEPREP_SUMMARY (isopropanol/acetonitrile: 90/10, 10 mM ammonium formate and 0.1% formic acid) SP:SAMPLEPREP_SUMMARY for the following LC-MS/MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Column information: https://www.thermofisher.com/order/catalog/product/164568 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Thermo Acclaim PepMap 100 C18 (150 x 0.007 mm, 3um, 100A) CH:SOLVENT_A 60% acetonitrile/40% water; 10 mM ammonium formate; 0.1% formic acid CH:SOLVENT_B 90% isopropanol/10% acetonitrile; 10 mM ammonium formate; 0.1% formic acid CH:FLOW_GRADIENT 30%-99% CH:FLOW_RATE 300 nl/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo LTQ XL MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS - MS:MS_RESULTS_FILE ST002875_AN004712_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END