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Inter-species complementation of the translocon beta subunit requires only its transmembrane domain.

Leroux A, Rokeach LA - PLoS ONE (2008)

Bottom Line: This protein translocation channel is composed of three major subunits, called Sec61alpha, beta and gamma in mammals.Nevertheless, the beta subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined.Here, we show that the knockout of sbh1(+) results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23 degrees C.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Université de Montréal, Montréal, Québec, Canada.

ABSTRACT
In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61alpha, beta and gamma in mammals. Unlike the other subunits, the beta subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61beta encoding genes results in different phenotypes in different species. Nevertheless, the beta subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61beta in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1(+) results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23 degrees C. Sec61beta homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1(+) in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61beta is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61beta from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61beta exerts a cellular function that is conserved across species.

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The translocon β subunit is conserved from yeast to human.Amino-acid sequence comparison between translocon beta subunits of S. pombe (SP_Sec61β), S. cerevisiae (SC_Sec61β1 and SC_Sec61β2) and human (HS_Sec61β). Identical amino acids are shaded in black, similar amino acids are shaded in grey. The predicted conserved transmembrane domain is underlined.
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pone-0003880-g001: The translocon β subunit is conserved from yeast to human.Amino-acid sequence comparison between translocon beta subunits of S. pombe (SP_Sec61β), S. cerevisiae (SC_Sec61β1 and SC_Sec61β2) and human (HS_Sec61β). Identical amino acids are shaded in black, similar amino acids are shaded in grey. The predicted conserved transmembrane domain is underlined.

Mentions: Based on Wu-Blast2 analyses of the fission yeast proteome [38], S. pombe encodes a single Sec61β homolog at ORF SPBC2G2.03c (sbh1+), hereafter designed as SP_Sec61β for the sake of clarity. The deduced primary sequence of the protein is presented in Figure 1. As expected for a translocon subunit, the predicted protein displays a high level of sequence conservation with both paralogs of S. cerevisiae (SC) and that of human (HS). SP_Sec61β is 35% identical (66% similar) to SC_Sec61β1 (Sbh1p), 43% identical (65% similar) to SC_Sec61β2 (Sbh2p), and 29% identical (58% similar) to HS_Sec61β (human Sec61β). Multiple sequence alignment using Clustal W2 [39] revealed that SP_Sec61β is evolutionary closer to HS_Sec61β than its S. cerevisiae counterparts (not shown), thus validating our choice of S. pombe as a model organism to study the translocon β subunit of higher eukaryotes. Analyses of SP_Sec61β using various ExPASy proteomic tools [40] allowed the identification of a single domain, constituted of a continuous stretch of 26 amino acids near the C-terminus. This hydrophobic tail is predicted to form an alpha helix crossing the lipid bilayer once, with the N-terminus of the protein exposed to the cytoplasm. Thus, as in S. cerevisiae and human, SP_Sec61β is predicted to be a tail-anchored protein. Interestingly, the TMDs of Sec61β homologs are much more conserved than their N-terminal domains, 53–61% identical from yeast to human as compared to only 18–41% for the cytosolic part. In comparison, the tail anchor of the translocon γ subunit is only 40–50% identical from yeast to human, a level of sequence homology comparable to the entire protein, which is 44–48% identical. This conservation suggests a critical function for the TMD region of Sec61β homologs, not solely explained by its role as a membrane anchor, especially as almost any stretch of hydrophobic amino acids can perform this function [41].


Inter-species complementation of the translocon beta subunit requires only its transmembrane domain.

Leroux A, Rokeach LA - PLoS ONE (2008)

The translocon β subunit is conserved from yeast to human.Amino-acid sequence comparison between translocon beta subunits of S. pombe (SP_Sec61β), S. cerevisiae (SC_Sec61β1 and SC_Sec61β2) and human (HS_Sec61β). Identical amino acids are shaded in black, similar amino acids are shaded in grey. The predicted conserved transmembrane domain is underlined.
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Related In: Results  -  Collection

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pone-0003880-g001: The translocon β subunit is conserved from yeast to human.Amino-acid sequence comparison between translocon beta subunits of S. pombe (SP_Sec61β), S. cerevisiae (SC_Sec61β1 and SC_Sec61β2) and human (HS_Sec61β). Identical amino acids are shaded in black, similar amino acids are shaded in grey. The predicted conserved transmembrane domain is underlined.
Mentions: Based on Wu-Blast2 analyses of the fission yeast proteome [38], S. pombe encodes a single Sec61β homolog at ORF SPBC2G2.03c (sbh1+), hereafter designed as SP_Sec61β for the sake of clarity. The deduced primary sequence of the protein is presented in Figure 1. As expected for a translocon subunit, the predicted protein displays a high level of sequence conservation with both paralogs of S. cerevisiae (SC) and that of human (HS). SP_Sec61β is 35% identical (66% similar) to SC_Sec61β1 (Sbh1p), 43% identical (65% similar) to SC_Sec61β2 (Sbh2p), and 29% identical (58% similar) to HS_Sec61β (human Sec61β). Multiple sequence alignment using Clustal W2 [39] revealed that SP_Sec61β is evolutionary closer to HS_Sec61β than its S. cerevisiae counterparts (not shown), thus validating our choice of S. pombe as a model organism to study the translocon β subunit of higher eukaryotes. Analyses of SP_Sec61β using various ExPASy proteomic tools [40] allowed the identification of a single domain, constituted of a continuous stretch of 26 amino acids near the C-terminus. This hydrophobic tail is predicted to form an alpha helix crossing the lipid bilayer once, with the N-terminus of the protein exposed to the cytoplasm. Thus, as in S. cerevisiae and human, SP_Sec61β is predicted to be a tail-anchored protein. Interestingly, the TMDs of Sec61β homologs are much more conserved than their N-terminal domains, 53–61% identical from yeast to human as compared to only 18–41% for the cytosolic part. In comparison, the tail anchor of the translocon γ subunit is only 40–50% identical from yeast to human, a level of sequence homology comparable to the entire protein, which is 44–48% identical. This conservation suggests a critical function for the TMD region of Sec61β homologs, not solely explained by its role as a membrane anchor, especially as almost any stretch of hydrophobic amino acids can perform this function [41].

Bottom Line: This protein translocation channel is composed of three major subunits, called Sec61alpha, beta and gamma in mammals.Nevertheless, the beta subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined.Here, we show that the knockout of sbh1(+) results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23 degrees C.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Université de Montréal, Montréal, Québec, Canada.

ABSTRACT
In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61alpha, beta and gamma in mammals. Unlike the other subunits, the beta subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61beta encoding genes results in different phenotypes in different species. Nevertheless, the beta subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61beta in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1(+) results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23 degrees C. Sec61beta homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1(+) in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61beta is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61beta from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61beta exerts a cellular function that is conserved across species.

Show MeSH
Related in: MedlinePlus