A plasmid module for PCR-based gene modification for the accurate measurement of vacuolar delivery of specific proteins in yeast Saccharomyces cerevisiae
Monitoring the delivery of single proteins and protein complexes to the vacuole by autophagy or other processes in yeast Saccharomyces cerevisiae mainly relies on western blot or fluorescence microscopy analyses using endogenous tagging of the protein of interest with GFP. However, these approaches...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2025-12-01
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| Series: | Autophagy Reports |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/27694127.2025.2511724 |
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| author | Jakob Valdbjørn Kanne Fulvio Reggiori |
| author_facet | Jakob Valdbjørn Kanne Fulvio Reggiori |
| author_sort | Jakob Valdbjørn Kanne |
| collection | DOAJ |
| description | Monitoring the delivery of single proteins and protein complexes to the vacuole by autophagy or other processes in yeast Saccharomyces cerevisiae mainly relies on western blot or fluorescence microscopy analyses using endogenous tagging of the protein of interest with GFP. However, these approaches are semi-quantitative and next to impossible with proteins of low abundancy because of the insensitive nature of the methods. Here, we describe the creation of a new PCR-based integration cassette to endogenously tag specific proteins with the truncated version of the vacuolar phosphatase Pho8. The vacuolar activation of Pho8 allows the quantitative measurement of vacuolar delivery using a colorimetric enzymatic assay. This approach has the advantages of a more quantitative interpretation of data and relies on the appearance of a signal rather than its disappearance. As a proof-of-principle, we examined the vacuolar delivery of known cargoes of bulk autophagy and endocytosis. This new system will be of great value to the whole community working within the field of autophagy and other transport pathways to the vacuole. |
| format | Article |
| id | doaj-art-a5a9938d67aa41d78eaa002c338be54a |
| institution | OA Journals |
| issn | 2769-4127 |
| language | English |
| publishDate | 2025-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Autophagy Reports |
| spelling | doaj-art-a5a9938d67aa41d78eaa002c338be54a2025-08-20T02:00:47ZengTaylor & Francis GroupAutophagy Reports2769-41272025-12-014110.1080/27694127.2025.2511724A plasmid module for PCR-based gene modification for the accurate measurement of vacuolar delivery of specific proteins in yeast Saccharomyces cerevisiaeJakob Valdbjørn Kanne0Fulvio Reggiori1Department of Biomedicine, Aarhus University, Aarhus C, DenmarkDepartment of Biomedicine, Aarhus University, Aarhus C, DenmarkMonitoring the delivery of single proteins and protein complexes to the vacuole by autophagy or other processes in yeast Saccharomyces cerevisiae mainly relies on western blot or fluorescence microscopy analyses using endogenous tagging of the protein of interest with GFP. However, these approaches are semi-quantitative and next to impossible with proteins of low abundancy because of the insensitive nature of the methods. Here, we describe the creation of a new PCR-based integration cassette to endogenously tag specific proteins with the truncated version of the vacuolar phosphatase Pho8. The vacuolar activation of Pho8 allows the quantitative measurement of vacuolar delivery using a colorimetric enzymatic assay. This approach has the advantages of a more quantitative interpretation of data and relies on the appearance of a signal rather than its disappearance. As a proof-of-principle, we examined the vacuolar delivery of known cargoes of bulk autophagy and endocytosis. This new system will be of great value to the whole community working within the field of autophagy and other transport pathways to the vacuole.https://www.tandfonline.com/doi/10.1080/27694127.2025.2511724autophagyendocytosisPho8∆60turnovervacuolevacuolar phosphatase |
| spellingShingle | Jakob Valdbjørn Kanne Fulvio Reggiori A plasmid module for PCR-based gene modification for the accurate measurement of vacuolar delivery of specific proteins in yeast Saccharomyces cerevisiae Autophagy Reports autophagy endocytosis Pho8∆60 turnover vacuole vacuolar phosphatase |
| title | A plasmid module for PCR-based gene modification for the accurate measurement of vacuolar delivery of specific proteins in yeast Saccharomyces cerevisiae |
| title_full | A plasmid module for PCR-based gene modification for the accurate measurement of vacuolar delivery of specific proteins in yeast Saccharomyces cerevisiae |
| title_fullStr | A plasmid module for PCR-based gene modification for the accurate measurement of vacuolar delivery of specific proteins in yeast Saccharomyces cerevisiae |
| title_full_unstemmed | A plasmid module for PCR-based gene modification for the accurate measurement of vacuolar delivery of specific proteins in yeast Saccharomyces cerevisiae |
| title_short | A plasmid module for PCR-based gene modification for the accurate measurement of vacuolar delivery of specific proteins in yeast Saccharomyces cerevisiae |
| title_sort | plasmid module for pcr based gene modification for the accurate measurement of vacuolar delivery of specific proteins in yeast saccharomyces cerevisiae |
| topic | autophagy endocytosis Pho8∆60 turnover vacuole vacuolar phosphatase |
| url | https://www.tandfonline.com/doi/10.1080/27694127.2025.2511724 |
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