Yeast Heterologous Expression Systems for the Study of Plant Membrane Proteins

Abstract

Researchers are often interested in proteins that are present in cells in small ratios compared to the total amount of proteins. These proteins include transcription factors, hormones and specific membrane proteins. However, sufficient amounts of well-purified protein preparations are required for functional and structural studies of these proteins, including the creation of artificial proteoliposomes and the growth of protein 2D and 3D crystals. This aim can be achieved by the expression of the target protein in a heterologous system. This review describes the applications of yeast heterologous expression systems in studies of plant membrane proteins. An initial brief description introduces the widely used heterologous expression systems of the baker's yeast Saccharomyces cerevisiae and the methylotrophic yeast Pichia pastoris. S. cerevisiae is further considered a convenient model system for functional studies of heterologously expressed proteins, while P. pastoris has the advantage of using these yeast cells as factories for producing large quantities of proteins of interest. The application of both expression systems is described for functional and structural studies of membrane proteins from plants, namely, K⁺- and Na⁺-transporters, various ATPases and anion transporters, and other transport proteins.

Keywords: Pichia pastoris; Saccharomyces cerevisiae; baker’s yeasts; heterologous expression; methylotrophic yeasts; plant membrane proteins; recombinant proteins.

Figure 1

The key steps in the expression of heterologous protein in yeast cells. (a) The gene of interest (cDNA) is isolated from a plant sample; (b) the cDNA (blue arrow) is integrated into the host vector; (c,d) E. coli is routinely used for construct amplification; (e) yeast cells are transformed by the vector containing the cDNA of gene of interest. Selection of colonies (E. coli, yeast colonies) is carried out on selective media.

Figure 2

Scheme of yeast episome plasmid. MCS—multiple cloning site (polylinker); ori—bacterial replication site; AmpR—gene of resistance to ampicillin (selective markers for the selection of bacterial transformants); 2µ ori—ori site of yeast 2µ-plasmid; URA3—gene of orotidine-5′phosphate decarboxylase (selective markers for the selection of yeast transformants).

Figure 3

The major known plasma membrane and intracellular membrane ion transporters in the yeast cell are mentioned in the text.

Figure 4

Scheme of classic yeast two-hybrid technique. DBD—DNA-binding domain; AD—activation domain; P1—“bait” (protein of interest, POI); P2—“prey”.

Figure 5

Scheme of split-ubiquitin Y2H system. P1—“bait” (protein of interest, POI), fused with C-end of ubiquitin (Cub); P2—“prey”, fused with N-end of ubiquitin (Nub); TF—transcription factor.

Figure 6

Scheme of recombinant integration of plasmid vector into P. pastoris genome. pAOX1—promoter of alcohol oxidase AOX1 gene; AmpR and ZeoR—selective genes of ampicillin resistance and zeomycin resistance, respectively. TT—yeast terminator.

Figure 7

Gene (cDNA) of P-type ATPase from the marine microalga Dunaliella maritima similar to higher plant H⁺-ATPases (DmHA2; GenBank ID: KX 832225.1) fused with GFP coding sequence is expressed (green colour) in transformed P. pastoris cells (the GS115 strain). The plasma membranes of the yeast cells are stained with a fluorescent lipophilic dye FM 4-64 (molecular probes) (red colour).