{"title":"一种新的研究酿酒葡萄球菌Gtr异源二聚体鸟嘌呤核苷酸结合的交联试验。","authors":"Dylan D Doxsey, Kristen Veinotte, Kuang Shen","doi":"10.1080/21541248.2022.2141019","DOIUrl":null,"url":null,"abstract":"<p><p>The mechanistic target of rapamycin (mTOR) complex is responsible for coordinating nutrient availability with eukaryotic cell growth. Amino acid signals are transmitted towards mTOR via the Rag/Gtr heterodimers. Due to the obligatory heterodimeric architecture of the Rag/Gtr GTPases, investigating their biochemical properties has been challenging. Here, we describe an updated assay that allows us to probe the guanine nucleotide-binding affinity and kinetics to the Gtr heterodimers in <i>Saccharomyces cerevisiae</i>. We first identified the structural element that Gtr2p lacks to enable crosslinking. By using a sequence conservation-based mutation, we restored the crosslinking between Gtr2p and the bound nucleotides. Using this construct, we determined the nucleotide-binding affinities of the Gtr heterodimer, and found that it operates under a different form of intersubunit communication than human Rag GTPases. Our study defines the evolutionary divergence of the Gtr/Rag-mTOR axis of nutrient sensing.</p>","PeriodicalId":22139,"journal":{"name":"Small GTPases","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/e4/6b/KSGT_13_2141019.PMC9639563.pdf","citationCount":"1","resultStr":"{\"title\":\"A New Crosslinking Assay to Study Guanine Nucleotide Binding in the Gtr Heterodimer of <i>S. cerevisiae</i>.\",\"authors\":\"Dylan D Doxsey, Kristen Veinotte, Kuang Shen\",\"doi\":\"10.1080/21541248.2022.2141019\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The mechanistic target of rapamycin (mTOR) complex is responsible for coordinating nutrient availability with eukaryotic cell growth. Amino acid signals are transmitted towards mTOR via the Rag/Gtr heterodimers. Due to the obligatory heterodimeric architecture of the Rag/Gtr GTPases, investigating their biochemical properties has been challenging. Here, we describe an updated assay that allows us to probe the guanine nucleotide-binding affinity and kinetics to the Gtr heterodimers in <i>Saccharomyces cerevisiae</i>. We first identified the structural element that Gtr2p lacks to enable crosslinking. By using a sequence conservation-based mutation, we restored the crosslinking between Gtr2p and the bound nucleotides. Using this construct, we determined the nucleotide-binding affinities of the Gtr heterodimer, and found that it operates under a different form of intersubunit communication than human Rag GTPases. Our study defines the evolutionary divergence of the Gtr/Rag-mTOR axis of nutrient sensing.</p>\",\"PeriodicalId\":22139,\"journal\":{\"name\":\"Small GTPases\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2022-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/e4/6b/KSGT_13_2141019.PMC9639563.pdf\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Small GTPases\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1080/21541248.2022.2141019\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"Biochemistry, Genetics and Molecular Biology\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Small GTPases","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1080/21541248.2022.2141019","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
A New Crosslinking Assay to Study Guanine Nucleotide Binding in the Gtr Heterodimer of S. cerevisiae.
The mechanistic target of rapamycin (mTOR) complex is responsible for coordinating nutrient availability with eukaryotic cell growth. Amino acid signals are transmitted towards mTOR via the Rag/Gtr heterodimers. Due to the obligatory heterodimeric architecture of the Rag/Gtr GTPases, investigating their biochemical properties has been challenging. Here, we describe an updated assay that allows us to probe the guanine nucleotide-binding affinity and kinetics to the Gtr heterodimers in Saccharomyces cerevisiae. We first identified the structural element that Gtr2p lacks to enable crosslinking. By using a sequence conservation-based mutation, we restored the crosslinking between Gtr2p and the bound nucleotides. Using this construct, we determined the nucleotide-binding affinities of the Gtr heterodimer, and found that it operates under a different form of intersubunit communication than human Rag GTPases. Our study defines the evolutionary divergence of the Gtr/Rag-mTOR axis of nutrient sensing.