Functional Analysis of Plant Monosaccharide Transporters Using a Simple Growth Complementation Assay in Yeast.

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. Key features Comprehensive, simple protocol for analysis of plant monosaccharide transporters in yeast Includes optional MoClo parts for cloning with Golden Gate method Includes protocol for the production and transformation of competent yeast cells Does not require hazardous solutions, radiolabeled substrates, or specialized equipment.