Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae

Abstract

Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in S. cerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in S. cerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid) polymer technique for use in further biochemical or biophysical studies.

Fig 1. Tetraspanin topology scheme.

Transmembrane helices are numbered 1–4, conserved helices in the large extracellular domain indicated with letters A,B,E according to nomenclature by Seigneuret et al. [32]. Conserved residues are shown in circles, where x stands for any amino acid. Possible post-translational modifications are indicated as palmitoylation sites shown as waves close to the intracellular side of the protein and available N-linked glycosylation sites shown as forks on the extracellular domains.

Fig 2. Recombinant expression of tetraspanins in S. cerevisiae.

(A) SDS/PAGE in-gel fluorescence of crude membranes isolated from S. cerevisiae expressing GFP fusion of TSPAN7, CO-029, TSPAN12, TSPAN18 and CD63. (B) Quantification of whole cell fluorescence of TSPAN-GFP fusion proteins during induction supplemented with either 2.5% DMSO or 0.04% histidine, or both.

Fig 3. Cellular localization and posttranslational modification of recombinant human tetraspanins.

(A) Representative confocal images of S. cerevisiae cells expressing human tetraspanin proteins with a C-terminal GFP tag. ER membranes were visualized by ER-tracker Blue-White DPX (cyan), tetraspanin-GFP fusion proteins were visualized by a native GFP fluorescence (green), membranes were visualized by FM 4-64FX (red) and cell wall was visualized by Concanavalin A Alexa Fluor 647 Conjugate (magenta). Each channel is shown separately for clarity and then merged. Scale bar: 2 μm (B) SDS-PAGE in-gel fluorescence of crude S. cerevisiae membranes containing GFP-tagged tetraspanins after EndoH treatment (+) and controls prepared in the same way but without EndoH (-). A shift of the fluorescent band to a lower molecular mass after EndoH treatment is observed only in case of TSPAN7.

Fig 4. Solubilization efficiency of TSPAN-GFP fusion proteins.

The solubilization degree of five tetraspanin-containing yeast membranes with the help of detergents and SMA polymers. The solubilization ratio was determined by comparing the fluorescence counts of solubilized material after ultracentrifugation to the mix before separation of solubilized and non-solubilized material.

Fig 5. Size exclusion chromatogram of purified CO-029.

Size exclusion of the purified CO-029 protein after proteolytic cleavage and removal of the GFP-tag. The elution was monitored with the absorbance at 280 nm.

Fig 6. Sequence alignment of selected tetraspanins.

Predicted localization of transmembrane helices with TOPCONS server [78] are indicated with a gray background. Residues conserved across the family–indicated in yellow and predicted glycosylation sites [79] are indicated with a red background. Sequence alignments were generated with the ClustalW server [80] by feeding 219 tetraspanin sequences from diverge spices to improve alignment statistics.