Details
| Original language | English |
|---|---|
| Pages (from-to) | e4733 |
| Journal | Bio-protocol |
| Volume | 13 |
| Issue number | 15 |
| Publication status | Published - 5 Aug 2023 |
Abstract
The study of genes and their products is an essential prerequisite for fundamental research. Characterization can be achieved by analyzing mutants or overexpression lines or by studying the localization and substrate specificities of the resulting proteins. However, functional analysis of specific proteins in complex eukaryotic organisms can be challenging. To overcome this, the use of heterologous systems to express genes and analyze the resulting proteins can save time and effort. Yeast is a preferred heterologous model organism: it is easy to transform, and tools for genomics, engineering, and metabolomics are already available. Here, we describe a well-established and simple method to analyze the activity of plant monosaccharide transporters in the baker's yeast, Saccharomyces cerevisiae, using a simple growth complementation assay. We used the famous hexose-transport-deficient yeast strain EBY.VW4000 to express candidate plant monosaccharide transporters and analyzed their transport activity. This assay does not require any radioactive labeling of substrates and can be easily extended for quantitative analysis using growth curves or by analyzing the transport rates of fluorescent substrates like the glucose analog 2-NBDG. Finally, to further simplify the cloning of potential candidate transporters, we provide level 0 modular cloning (MoClo) modules for efficient and simple Golden Gate cloning. This approach provides a convenient tool for the functional analysis of plant monosaccharide transporters in yeast.
Keywords
- Drop-out assay, EBY.VW4000, Heterologous expression, Plant monosaccharide transporters, Yeast
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Immunology and Microbiology(all)
- Neuroscience(all)
- Agricultural and Biological Sciences(all)
- Plant Science
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In: Bio-protocol, Vol. 13, No. 15, 05.08.2023, p. e4733.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Functional Analysis of Plant Monosaccharide Transporters Using a Simple Growth Complementation Assay in Yeast
AU - Streubel, Jana
AU - Fuhrmeister, Robert
N1 - This work was funded by university core funding only. We thank Jens Boch for general support. The protocol was used in Skoppek et al. (2022).
PY - 2023/8/5
Y1 - 2023/8/5
N2 - The study of genes and their products is an essential prerequisite for fundamental research. Characterization can be achieved by analyzing mutants or overexpression lines or by studying the localization and substrate specificities of the resulting proteins. However, functional analysis of specific proteins in complex eukaryotic organisms can be challenging. To overcome this, the use of heterologous systems to express genes and analyze the resulting proteins can save time and effort. Yeast is a preferred heterologous model organism: it is easy to transform, and tools for genomics, engineering, and metabolomics are already available. Here, we describe a well-established and simple method to analyze the activity of plant monosaccharide transporters in the baker's yeast, Saccharomyces cerevisiae, using a simple growth complementation assay. We used the famous hexose-transport-deficient yeast strain EBY.VW4000 to express candidate plant monosaccharide transporters and analyzed their transport activity. This assay does not require any radioactive labeling of substrates and can be easily extended for quantitative analysis using growth curves or by analyzing the transport rates of fluorescent substrates like the glucose analog 2-NBDG. Finally, to further simplify the cloning of potential candidate transporters, we provide level 0 modular cloning (MoClo) modules for efficient and simple Golden Gate cloning. This approach provides a convenient tool for the functional analysis of plant monosaccharide transporters in yeast.
AB - The study of genes and their products is an essential prerequisite for fundamental research. Characterization can be achieved by analyzing mutants or overexpression lines or by studying the localization and substrate specificities of the resulting proteins. However, functional analysis of specific proteins in complex eukaryotic organisms can be challenging. To overcome this, the use of heterologous systems to express genes and analyze the resulting proteins can save time and effort. Yeast is a preferred heterologous model organism: it is easy to transform, and tools for genomics, engineering, and metabolomics are already available. Here, we describe a well-established and simple method to analyze the activity of plant monosaccharide transporters in the baker's yeast, Saccharomyces cerevisiae, using a simple growth complementation assay. We used the famous hexose-transport-deficient yeast strain EBY.VW4000 to express candidate plant monosaccharide transporters and analyzed their transport activity. This assay does not require any radioactive labeling of substrates and can be easily extended for quantitative analysis using growth curves or by analyzing the transport rates of fluorescent substrates like the glucose analog 2-NBDG. Finally, to further simplify the cloning of potential candidate transporters, we provide level 0 modular cloning (MoClo) modules for efficient and simple Golden Gate cloning. This approach provides a convenient tool for the functional analysis of plant monosaccharide transporters in yeast.
KW - Drop-out assay
KW - EBY.VW4000
KW - Heterologous expression
KW - Plant monosaccharide transporters
KW - Yeast
UR - http://www.scopus.com/inward/record.url?scp=85169590801&partnerID=8YFLogxK
U2 - 10.21769/BioProtoc.4733
DO - 10.21769/BioProtoc.4733
M3 - Article
C2 - 37575400
VL - 13
SP - e4733
JO - Bio-protocol
JF - Bio-protocol
IS - 15
ER -