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

Skaar K, Korza HJ, Tarry M, Sekyrova P, Högbom M - PLoS ONE (2015)

Bottom Line: Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0134041.g001: 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.

Mentions: Tetraspanins are predicted to have an overall structure of a tight, rod-like shape [30,31], see Fig 1. The large extracellular domain (LED) varies in sequence and size, from 6 to 17 kDa, between homologs. The variable region of LED (Fig 1) contains the conserved CCG motif with stabilizing disulfide bonds in the extracellular domain [32–35]. The small extracellular loop of roughly 1 kDa displays much less variation. Both the N- and C-termini of tetraspanins are located in the cytoplasm and are usually as short as 5–15 amino acids. Some proteins in the family are however predicted to have longer termini tails of about 5–10 kDa. Tetraspanins are also known to be posttranslationally modified by N-linked glycosylation and/or palmitoylation, see Fig 1 [36,37]. The location of the tetraspanins in the cell membrane with key interacting domains on the extracellular side makes them amenable to manipulation by macromolecular drugs, such as monoclonal antibodies, and potentially useful as tumor biomarkers and therapeutic targets [4].


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

Skaar K, Korza HJ, Tarry M, Sekyrova P, Högbom M - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4517926&req=5

pone.0134041.g001: 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.
Mentions: Tetraspanins are predicted to have an overall structure of a tight, rod-like shape [30,31], see Fig 1. The large extracellular domain (LED) varies in sequence and size, from 6 to 17 kDa, between homologs. The variable region of LED (Fig 1) contains the conserved CCG motif with stabilizing disulfide bonds in the extracellular domain [32–35]. The small extracellular loop of roughly 1 kDa displays much less variation. Both the N- and C-termini of tetraspanins are located in the cytoplasm and are usually as short as 5–15 amino acids. Some proteins in the family are however predicted to have longer termini tails of about 5–10 kDa. Tetraspanins are also known to be posttranslationally modified by N-linked glycosylation and/or palmitoylation, see Fig 1 [36,37]. The location of the tetraspanins in the cell membrane with key interacting domains on the extracellular side makes them amenable to manipulation by macromolecular drugs, such as monoclonal antibodies, and potentially useful as tumor biomarkers and therapeutic targets [4].

Bottom Line: Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.

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.

No MeSH data available.


Related in: MedlinePlus