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The human transport protein SLC35B1 was recently postulated to mediate ATP/ADP exchange in the ER; however, the underlying molecular mechanisms mediating ATP uptake are not completely understood. Here, we extensively characterized the transport kinetics of human SLC35B1 expressed in yeast that was purified and reconstituted into liposomes. Using [alpha 32P]ATP uptake assays, we tested the nucleotide concentration dependence of ATP/ ADP exchange activity on both sides of the membrane. We found that the apparent affinities of SLC35B1 for ATP/ADP on the internal face were approximately 13 times higher than those on the external side. Because SLC35B1-containing liposomes were preferentially inside-out oriented, these results suggest a low-affinity external site and a high-affinity internal site in the ER. Three different experimental approaches indicated that ATP/ADP exchange by SLC35B1 was not strict, and that other di-and tri-nucleotides could act as suitable counter substrates for ATP, although mononucleotides and nucleotide sugars were not transported. Finally, bioinformatic analysis and site-directed mutagenesis identified that conserved residues K117 and K120 from transmembrane helix 4 and K277 from transmembrane helix 9 play critical roles in transport. The fact that SLC35B1 can promote ATP transport in exchange for ADP or UDP suggest a more direct coupling between ATP import requirements and the need for eliminating ADP and UDP, which are generated as side products of reactions taking place in the ER-lumen.", "keywords" : [ { "otype" : "Keyword", "mtid" : 1644, "link" : "/api/keyword/1644", "label" : "STRUCTURAL BASIS", "published" : true, "oldId" : 1644, "snippet" : true }, { "otype" : "Keyword", "mtid" : 6084, "link" : "/api/keyword/6084", "label" : "ENDOPLASMIC-RETICULUM", "published" : true, "oldId" : 6084, "snippet" : true }, { "otype" : "Keyword", "mtid" : 6487, "link" : "/api/keyword/6487", "label" : "SACCHAROMYCES-CEREVISIAE", "published" : true, "oldId" : 6487, "snippet" : true }, { "otype" : "Keyword", "mtid" : 1119715, "link" : "/api/keyword/1119715", "label" : "SECRETORY PATHWAY", "published" : true, "oldId" : 1119715, "snippet" : true }, { "otype" : "Keyword", "mtid" : 1573471, "link" : "/api/keyword/1573471", "label" : "extracellular ATP", "published" : true, "snippet" : true }, { "otype" : "Keyword", "mtid" : 1642616, "link" : "/api/keyword/1642616", "label" : "sugar transporters", "published" : true, "snippet" : true }, { "otype" : "Keyword", "mtid" : 2654059, "link" : "/api/keyword/2654059", "label" : "DIFFERENT SUBCELLULAR COMPARTMENTS", "published" : true, "snippet" : true }, { "otype" : "Keyword", "mtid" : 2654060, "link" : "/api/keyword/2654060", "label" : "NUCLEOSIDE DIPHOSPHATASE", "published" : true, "snippet" : true }, { "otype" : "Keyword", "mtid" : 2654061, "link" : "/api/keyword/2654061", "label" : "GALACTOSE TRANSPORTER", "published" : true, "snippet" : true } ], "digital" : null, "printed" : true, "sourceYear" : 2022, "foreignEdition" : true, "foreignLanguage" : true, "fullPublication" : true, "conferencePublication" : false, "nationalOrigin" : false, "missingAuthor" : false, "oaType" : "NONE", "oaCheckDate" : "2023-08-26", "oaFree" : false, "citationCount" : 0, "citationCountUnpublished" : 0, "citationCountWoOther" : 0, "independentCitCountWoOther" : 0, "doiCitationCount" : 0, "wosCitationCount" : 0, "scopusCitationCount" : 0, "independentCitationCount" : 0, "unhandledCitationCount" : 0, "citingPubCount" : 0, "independentCitingPubCount" : 0, "unhandledCitingPubCount" : 0, "citedPubCount" : 1, "citedCount" : 1, "ratings" : [ { "otype" : "SjrRating", "mtid" : 11283040, "link" : "/api/sjrrating/11283040", "label" : "sjr:Q1 (2022) Scopus - Biochemistry JOURNAL OF BIOLOGICAL CHEMISTRY 0021-9258 1083-351X", "listPos" : 48, "rankValue" : 0.24, "type" : "journal", "ratingType" : { "otype" : "RatingType", "mtid" : 10002, "link" : "/api/ratingtype/10002", "label" : "sjr", "code" : "sjr", "published" : true, "snippet" : true }, "subject" : { "otype" : "ClassificationExternal", "mtid" : 1303, "link" : "/api/classificationexternal/1303", "label" : "Scopus - Biochemistry", "published" : true, "oldId" : 1303, "snippet" : true }, "ranking" : "Q1", "calculation" : "DIRECT", "published" : true, "snippet" : true } ], "ratingsForSort" : "Q1", "hasCitationDuplums" : false, "userChangeableUntil" : "2022-09-21T21:43:04.852+0000", "directInstitutesForSort" : "", "ownerAuthorCount" : 4, "ownerInstituteCount" : 13, "directInstituteCount" : 0, "authorCount" : 3, "contributorCount" : 0, "hasQualityFactor" : true, "link" : "/api/publication/32901768", "label" : "Schwarzbaum Pablo J. et al. 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