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Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocation complex beta-subunit.

Zhao X, Jäntti J - BMC Cell Biol. (2009)

Bottom Line: At the same time, these mutations do not affect Sbh1p interaction with Rtn1p.Our results identify functionally important amino acids in the Sbh1p trans-membrane domain.In addition, our results provide additional support for the involvement of Sec61beta in processes unlinked to protein translocation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Program in Cell and Molecular Biology, Institute of Biotechnology, P,O, Box 56, 00014 University of Helsinki, Finland. xqzhao@genetics.ac.cn

ABSTRACT

Background: In eukaryotic cells co- and post-translational protein translocation is mediated by the trimeric Sec61 complex. Currently, the role of the Sec61 complex beta-subunit in protein translocation is poorly understood. We have shown previously that in Saccharomyces cerevisiae the trans-membrane domain alone is sufficient for the function of the beta-subunit Sbh1p in co-translational protein translocation. In addition, Sbh1p co-purifies not only with the protein translocation channel subunits Sec61p and Sss1p, but also with the reticulon family protein Rtn1p.

Results: We used random mutagenesis to generate novel Sbh1p mutants in order to functionally map the Sbh1p trans-membrane domain. These mutants were analyzed for their interactions with Sec61p and how they support co-translational protein translocation. The distribution of mutations identifies one side of the Sbh1p trans-membrane domain alpha-helix that is involved in interactions with Sec61p and that is important for Sbh1p function in protein translocation. At the same time, these mutations do not affect Sbh1p interaction with Rtn1p. Furthermore we show that Sbh1p is found in protein complexes containing not only Rtn1p, but also the two other reticulon-like proteins Rtn2p and Yop1p.

Conclusion: Our results identify functionally important amino acids in the Sbh1p trans-membrane domain. In addition, our results provide additional support for the involvement of Sec61beta in processes unlinked to protein translocation.

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Functional characterization of the Sbh1p tm-domain mutants. A, Multicopy suppression of temperature-sensitivity of sbh1Δ sbh2Δ cells (H3232) by different mutant forms of SBH1 tm-domain. The sbh1Δ sbh2Δ cells were transformed with BIO-tagged SBH1 tm-domain mutants expressed from the ADH1 promoter in p426ADH and the growth of four independent transformants was monitored at 38 and 24°C. B, Lysates prepared from sbh1Δ cells (H3429) expressing BIO-tagged Sbh1p TM, mutants TM-1 to TM-7 or the empty vector were subjected to pull-down with streptavidin-coated magnetic beads. Beads and input samples were analyzed by Western blotting with anti-Sec61 antibodies or with HRP conjugated streptavidin to detect different versions of BIO-Sbh1p.
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Figure 2: Functional characterization of the Sbh1p tm-domain mutants. A, Multicopy suppression of temperature-sensitivity of sbh1Δ sbh2Δ cells (H3232) by different mutant forms of SBH1 tm-domain. The sbh1Δ sbh2Δ cells were transformed with BIO-tagged SBH1 tm-domain mutants expressed from the ADH1 promoter in p426ADH and the growth of four independent transformants was monitored at 38 and 24°C. B, Lysates prepared from sbh1Δ cells (H3429) expressing BIO-tagged Sbh1p TM, mutants TM-1 to TM-7 or the empty vector were subjected to pull-down with streptavidin-coated magnetic beads. Beads and input samples were analyzed by Western blotting with anti-Sec61 antibodies or with HRP conjugated streptavidin to detect different versions of BIO-Sbh1p.

Mentions: In order to understand where the obtained mutations are located in the Sbh1p tm-domain structure, we made use of the similarity of SecG tm-domains from different species and deduced a model for Sbh1p association with Sec61α (Figure 1C and 1D). The tm-domains of Sbh1p homologues have a highly conserved proline residue at the membrane/cytosol boundary complex [3]. We used this residue to align the Sbh1p tm-domain with the Methanococcus jannaschii SecG tm-domain (Figure 1C). The M. jannaschii SecG tm-domain has one side of its tm domain facing the tm-domains 1 and 4 of SecY (Figure 1B). To test the effect of mutations in different locations of the Sbh1p tm-domain α-helix, eight mutants were selected for further analysis (Figure 1C). Based on the model, mutations in TM-1, TM-2, TM-3 and TM-4 are mostly oriented away from the Sec61 whereas mutations in TM-5, TM-6, TM-7 and TM-8 are facing Sec61 (Figure 1C). When tested for their capability to rescue the temperature-sensitivity of the sbh1Δ sbh2Δ cells, mutants TM-5, TM-6, TM-7, and TM-8 were unable to rescue temperature-sensitivity of this strain. Mutant TM-3 was partially functional and mutants TM-1, TM-2 and TM-4 displayed normal growth (Figure 2A).


Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocation complex beta-subunit.

Zhao X, Jäntti J - BMC Cell Biol. (2009)

Functional characterization of the Sbh1p tm-domain mutants. A, Multicopy suppression of temperature-sensitivity of sbh1Δ sbh2Δ cells (H3232) by different mutant forms of SBH1 tm-domain. The sbh1Δ sbh2Δ cells were transformed with BIO-tagged SBH1 tm-domain mutants expressed from the ADH1 promoter in p426ADH and the growth of four independent transformants was monitored at 38 and 24°C. B, Lysates prepared from sbh1Δ cells (H3429) expressing BIO-tagged Sbh1p TM, mutants TM-1 to TM-7 or the empty vector were subjected to pull-down with streptavidin-coated magnetic beads. Beads and input samples were analyzed by Western blotting with anti-Sec61 antibodies or with HRP conjugated streptavidin to detect different versions of BIO-Sbh1p.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2770995&req=5

Figure 2: Functional characterization of the Sbh1p tm-domain mutants. A, Multicopy suppression of temperature-sensitivity of sbh1Δ sbh2Δ cells (H3232) by different mutant forms of SBH1 tm-domain. The sbh1Δ sbh2Δ cells were transformed with BIO-tagged SBH1 tm-domain mutants expressed from the ADH1 promoter in p426ADH and the growth of four independent transformants was monitored at 38 and 24°C. B, Lysates prepared from sbh1Δ cells (H3429) expressing BIO-tagged Sbh1p TM, mutants TM-1 to TM-7 or the empty vector were subjected to pull-down with streptavidin-coated magnetic beads. Beads and input samples were analyzed by Western blotting with anti-Sec61 antibodies or with HRP conjugated streptavidin to detect different versions of BIO-Sbh1p.
Mentions: In order to understand where the obtained mutations are located in the Sbh1p tm-domain structure, we made use of the similarity of SecG tm-domains from different species and deduced a model for Sbh1p association with Sec61α (Figure 1C and 1D). The tm-domains of Sbh1p homologues have a highly conserved proline residue at the membrane/cytosol boundary complex [3]. We used this residue to align the Sbh1p tm-domain with the Methanococcus jannaschii SecG tm-domain (Figure 1C). The M. jannaschii SecG tm-domain has one side of its tm domain facing the tm-domains 1 and 4 of SecY (Figure 1B). To test the effect of mutations in different locations of the Sbh1p tm-domain α-helix, eight mutants were selected for further analysis (Figure 1C). Based on the model, mutations in TM-1, TM-2, TM-3 and TM-4 are mostly oriented away from the Sec61 whereas mutations in TM-5, TM-6, TM-7 and TM-8 are facing Sec61 (Figure 1C). When tested for their capability to rescue the temperature-sensitivity of the sbh1Δ sbh2Δ cells, mutants TM-5, TM-6, TM-7, and TM-8 were unable to rescue temperature-sensitivity of this strain. Mutant TM-3 was partially functional and mutants TM-1, TM-2 and TM-4 displayed normal growth (Figure 2A).

Bottom Line: At the same time, these mutations do not affect Sbh1p interaction with Rtn1p.Our results identify functionally important amino acids in the Sbh1p trans-membrane domain.In addition, our results provide additional support for the involvement of Sec61beta in processes unlinked to protein translocation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Program in Cell and Molecular Biology, Institute of Biotechnology, P,O, Box 56, 00014 University of Helsinki, Finland. xqzhao@genetics.ac.cn

ABSTRACT

Background: In eukaryotic cells co- and post-translational protein translocation is mediated by the trimeric Sec61 complex. Currently, the role of the Sec61 complex beta-subunit in protein translocation is poorly understood. We have shown previously that in Saccharomyces cerevisiae the trans-membrane domain alone is sufficient for the function of the beta-subunit Sbh1p in co-translational protein translocation. In addition, Sbh1p co-purifies not only with the protein translocation channel subunits Sec61p and Sss1p, but also with the reticulon family protein Rtn1p.

Results: We used random mutagenesis to generate novel Sbh1p mutants in order to functionally map the Sbh1p trans-membrane domain. These mutants were analyzed for their interactions with Sec61p and how they support co-translational protein translocation. The distribution of mutations identifies one side of the Sbh1p trans-membrane domain alpha-helix that is involved in interactions with Sec61p and that is important for Sbh1p function in protein translocation. At the same time, these mutations do not affect Sbh1p interaction with Rtn1p. Furthermore we show that Sbh1p is found in protein complexes containing not only Rtn1p, but also the two other reticulon-like proteins Rtn2p and Yop1p.

Conclusion: Our results identify functionally important amino acids in the Sbh1p trans-membrane domain. In addition, our results provide additional support for the involvement of Sec61beta in processes unlinked to protein translocation.

Show MeSH
Related in: MedlinePlus