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

PLoS One. 2015 Jul 28;10(7):e0134041. doi: 10.1371/journal.pone.0134041. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Glycosylation
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Humans
  • Microscopy, Confocal
  • Protein Transport
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Subcellular Fractions
  • Tetraspanins / genetics
  • Tetraspanins / metabolism*

Substances

  • Recombinant Fusion Proteins
  • Tetraspanins
  • Green Fluorescent Proteins

Grant support

This work was supported by the Swedish Cancer Society (CAN 2012/571), www.cancerfonden.se and the Swedish Foundation for Strategic Research (SSF FFL4), http://www.stratresearch.se. The confocal microscopy facility was supported by the Knut and Alice Wallenberg foundation, www.wallenberg.com/kaw/.