Intra-FCY1: a novel system to identify mutations that cause protein misfolding
Protein misfolding is a common intracellular occurrence. Most mutations to coding sequences increase the propensity of the encoded protein to misfold. These misfolded molecules can have devastating effects on cells. Despite the importance of protein misfolding in human disease and protein evolution,...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-09-01
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| Series: | Frontiers in Genetics |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fgene.2023.1198203/full |
| _version_ | 1797691050061463552 |
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| author | N. Quan N. Quan Y. Eguchi K. Geiler-Samerotte K. Geiler-Samerotte |
| author_facet | N. Quan N. Quan Y. Eguchi K. Geiler-Samerotte K. Geiler-Samerotte |
| author_sort | N. Quan |
| collection | DOAJ |
| description | Protein misfolding is a common intracellular occurrence. Most mutations to coding sequences increase the propensity of the encoded protein to misfold. These misfolded molecules can have devastating effects on cells. Despite the importance of protein misfolding in human disease and protein evolution, there are fundamental questions that remain unanswered, such as, which mutations cause the most misfolding? These questions are difficult to answer partially because we lack high-throughput methods to compare the destabilizing effects of different mutations. Commonly used systems to assess the stability of mutant proteins in vivo often rely upon essential proteins as sensors, but misfolded proteins can disrupt the function of the essential protein enough to kill the cell. This makes it difficult to identify and compare mutations that cause protein misfolding using these systems. Here, we present a novel in vivo system named Intra-FCY1 that we use to identify mutations that cause misfolding of a model protein [yellow fluorescent protein (YFP)] in Saccharomyces cerevisiae. The Intra-FCY1 system utilizes two complementary fragments of the yeast cytosine deaminase Fcy1, a toxic protein, into which YFP is inserted. When YFP folds, the Fcy1 fragments associate together to reconstitute their function, conferring toxicity in media containing 5-fluorocytosine and hindering growth. But mutations that make YFP misfold abrogate Fcy1 toxicity, thus strains possessing misfolded YFP variants rise to high frequency in growth competition experiments. This makes such strains easier to study. The Intra-FCY1 system cancels localization of the protein of interest, thus can be applied to study the relative stability of mutant versions of diverse cellular proteins. Here, we confirm this method can identify novel mutations that cause misfolding, highlighting the potential for Intra-FCY1 to illuminate the relationship between protein sequence and stability. |
| first_indexed | 2024-03-12T02:08:51Z |
| format | Article |
| id | doaj.art-1863c3397fc440cca7842582bb5a0639 |
| institution | Directory Open Access Journal |
| issn | 1664-8021 |
| language | English |
| last_indexed | 2024-03-12T02:08:51Z |
| publishDate | 2023-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genetics |
| spelling | doaj.art-1863c3397fc440cca7842582bb5a06392023-09-06T17:39:33ZengFrontiers Media S.A.Frontiers in Genetics1664-80212023-09-011410.3389/fgene.2023.11982031198203Intra-FCY1: a novel system to identify mutations that cause protein misfoldingN. Quan0N. Quan1Y. Eguchi2K. Geiler-Samerotte3K. Geiler-Samerotte4Biodesign Center for Mechanisms of Evolution, Arizona State University, Tempe, AZ, United StatesSchool of Life Sciences, Arizona State University, Tempe, AZ, United StatesBiodesign Center for Mechanisms of Evolution, Arizona State University, Tempe, AZ, United StatesBiodesign Center for Mechanisms of Evolution, Arizona State University, Tempe, AZ, United StatesSchool of Life Sciences, Arizona State University, Tempe, AZ, United StatesProtein misfolding is a common intracellular occurrence. Most mutations to coding sequences increase the propensity of the encoded protein to misfold. These misfolded molecules can have devastating effects on cells. Despite the importance of protein misfolding in human disease and protein evolution, there are fundamental questions that remain unanswered, such as, which mutations cause the most misfolding? These questions are difficult to answer partially because we lack high-throughput methods to compare the destabilizing effects of different mutations. Commonly used systems to assess the stability of mutant proteins in vivo often rely upon essential proteins as sensors, but misfolded proteins can disrupt the function of the essential protein enough to kill the cell. This makes it difficult to identify and compare mutations that cause protein misfolding using these systems. Here, we present a novel in vivo system named Intra-FCY1 that we use to identify mutations that cause misfolding of a model protein [yellow fluorescent protein (YFP)] in Saccharomyces cerevisiae. The Intra-FCY1 system utilizes two complementary fragments of the yeast cytosine deaminase Fcy1, a toxic protein, into which YFP is inserted. When YFP folds, the Fcy1 fragments associate together to reconstitute their function, conferring toxicity in media containing 5-fluorocytosine and hindering growth. But mutations that make YFP misfold abrogate Fcy1 toxicity, thus strains possessing misfolded YFP variants rise to high frequency in growth competition experiments. This makes such strains easier to study. The Intra-FCY1 system cancels localization of the protein of interest, thus can be applied to study the relative stability of mutant versions of diverse cellular proteins. Here, we confirm this method can identify novel mutations that cause misfolding, highlighting the potential for Intra-FCY1 to illuminate the relationship between protein sequence and stability.https://www.frontiersin.org/articles/10.3389/fgene.2023.1198203/fullprotein foldingfitness competitionmisfoldingyeastdeep mutational scanningprotein complementation assay (PCA) |
| spellingShingle | N. Quan N. Quan Y. Eguchi K. Geiler-Samerotte K. Geiler-Samerotte Intra-FCY1: a novel system to identify mutations that cause protein misfolding Frontiers in Genetics protein folding fitness competition misfolding yeast deep mutational scanning protein complementation assay (PCA) |
| title | Intra-FCY1: a novel system to identify mutations that cause protein misfolding |
| title_full | Intra-FCY1: a novel system to identify mutations that cause protein misfolding |
| title_fullStr | Intra-FCY1: a novel system to identify mutations that cause protein misfolding |
| title_full_unstemmed | Intra-FCY1: a novel system to identify mutations that cause protein misfolding |
| title_short | Intra-FCY1: a novel system to identify mutations that cause protein misfolding |
| title_sort | intra fcy1 a novel system to identify mutations that cause protein misfolding |
| topic | protein folding fitness competition misfolding yeast deep mutational scanning protein complementation assay (PCA) |
| url | https://www.frontiersin.org/articles/10.3389/fgene.2023.1198203/full |
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