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.

Keywords: Drop-out assay; EBY.VW4000; Heterologous expression; Plant monosaccharide transporters; Yeast.

Figure 1.. Golden Gate cloning strategy with the provided modular cloning (MoClo) modules.

A. We provide the level 0 MoClo modules for the PMA1 promoter (Pro+5U module) and the ADH2 terminator (3U+Ter module). The candidate transporter gene has to be amplified with CDS1 stop or without stop (CDS1 no stop) according to the MoClo guidelines. The latter can be used to fuse a C-terminal tag (CT). The required BsaI overhangs for cloning are indicated for each module. B. In this example, the yeast-compatible level 1 acceptor vector pAGT572 with uracil selection was used. Cloning of the level 0 modules into the level 1 acceptor vector is done in a cut-ligation with BsaI. C. The final level 1 transcription unit contains all parts required for yeast transformation and expression of the candidate transporter.

Figure 2.. Preparation and procedure of the yeast drop-out assay.

A. Material needed. Prepare a vortex, pipette, and pipette tips. Prepare the plates needed with a pipetting scheme. In this case, a grid line was used, with the yeast strains ordered vertically and serial dilutions horizontally. Prepare a rack with the tubes ordered in the same scheme as the plate. B. A multichannel pipette is used to place the drops on the plate. C. Liquid drops visible on the plate after pipetting.

Figure 3.. Example pictures of a drop-out assay result for different sugar transport protein (STP) variants (STP_V1 to STP_V6) expressed in yeast.

The STP variants (STP_V1 to STP_V6) were transformed to the hexose-transport-deficient yeast strain EBY.VW4000. A serial dilution of the respective strains was dropped on selective medium either containing 2% maltose or 2% glucose as carbon source. Selective medium lacking uracil was used (-Ura). Plates were grown for two days at 28 °C and growth was documented using the Chemidoc from Bio-Rad. GFP was used as negative control and the yeast hexose transporter Hxt1 served as positive control.