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Latency of transcription factor Stp1 depends on a modular regulatory motif that functions as cytoplasmic retention determinant and nuclear degron.

Omnus DJ, Ljungdahl PO - Mol. Biol. Cell (2014)

Bottom Line: Stp1, the effector transcription factor, is synthesized as a latent cytoplasmic precursor with an N-terminal regulatory domain that restricts its nuclear accumulation.Our results indicate that RI mediates latency by two distinct activities: it functions as a cytoplasmic retention determinant and an Asi-dependent degron.These findings provide novel insights into the SPS-sensing pathway and demonstrate for the first time that the inner nuclear membrane Asi proteins function in a degradation pathway in the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden.

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RI acts in a modular manner as a promoter exclusion determinant and an Asi-dependent degron. (A) Schematic representation of the artificial transcription factor composed of amino acids 16–50 of Stp1 fused to the bacterial DNA-binding protein lexA fused to the viral VP16 transcription AD. OPlexA-LEU2 and OPlexA-LYS2 reporter genes were used to assess binding to lexA operators. (B) Growth analysis of CAY235 (ASI1) and SHY016 (asi1Δ) carrying pDO211 (lexA-AD) or pDO260 (STP116-50-lexA-AD) on nonselective and selective media lacking leucine and lysine. Immunoblot of extracts prepared from strains. The immunoreactive forms of the lexA proteins present in cell extracts are schematically represented at their corresponding positions of migration. (C) Immunoblot analysis of extracts from strains CAY235 (ASI1) and SHY016 (asi1Δ) carrying pDO260 (STP116-50-lexA-AD) treated with CHX. Cells were pregrown in SD; at t = 0, the cultures received an aliquot of CHX (final concentration, 100 μg/ml), and samples were taken at the indicated time points. Levels of Pgk1 serve as internal control for protein loading.
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Figure 7: RI acts in a modular manner as a promoter exclusion determinant and an Asi-dependent degron. (A) Schematic representation of the artificial transcription factor composed of amino acids 16–50 of Stp1 fused to the bacterial DNA-binding protein lexA fused to the viral VP16 transcription AD. OPlexA-LEU2 and OPlexA-LYS2 reporter genes were used to assess binding to lexA operators. (B) Growth analysis of CAY235 (ASI1) and SHY016 (asi1Δ) carrying pDO211 (lexA-AD) or pDO260 (STP116-50-lexA-AD) on nonselective and selective media lacking leucine and lysine. Immunoblot of extracts prepared from strains. The immunoreactive forms of the lexA proteins present in cell extracts are schematically represented at their corresponding positions of migration. (C) Immunoblot analysis of extracts from strains CAY235 (ASI1) and SHY016 (asi1Δ) carrying pDO260 (STP116-50-lexA-AD) treated with CHX. Cells were pregrown in SD; at t = 0, the cultures received an aliquot of CHX (final concentration, 100 μg/ml), and samples were taken at the indicated time points. Levels of Pgk1 serve as internal control for protein loading.

Mentions: To ultimately test whether RI suffices to mediate cytoplasmic retention and constitutes a transferable Asi-dependent degron, we fused residues 16–50 of Stp1 containing RI to an artificial transcription factor composed of the bacterial DNA-binding protein lexA (lexA) and the strong viral VP16 transcription activation domain (AD). We used lexA operator–driven LYS2 and LEU2 genes as reporters for DNA binding of lexA-AD (Figure 7A). We then assessed cell growth in the absence of leucine and lysine as a functional readout for nuclear accumulation. When expressed in wild-type cells, the artificial transcription factor (lexA-AD) induced the reporter genes to facilitate growth in the absence of leucine and lysine (Figure 7B, dilution 1). In contrast, the fusion of lexA to Stp116-50 abolished promoter induction; no growth was observed (Figure 7B, compare dilution 2 with dilution 1). However, when expressed in cells lacking Asi1, robust growth was detected (Figure 7B, dilution 3). Of note, as judged by immunoblotting, the steady-state expression level of Stp116-50-lexA-AD was elevated in cells lacking Asi1 (Figure 7B, bottom, compare lane 3 with lane 2). Next we performed a CHX chase over a time course of 60 min to assess protein stability in wild-type and asi1Δ cells (Figure 7C). Strikingly, Stp116-50-lexA-AD was completely stable in asi1Δ cells (Figure 7C, lanes 6–10), whereas it efficiently turned over in wild-type cells (Figure 7C, lanes 1–5). Together these results show that the RI-containing domain of Stp1 comprising amino acids 16–50 contains the necessary information for Asi-dependent promoter exclusion, presumably by mediating efficient degradation.


Latency of transcription factor Stp1 depends on a modular regulatory motif that functions as cytoplasmic retention determinant and nuclear degron.

Omnus DJ, Ljungdahl PO - Mol. Biol. Cell (2014)

RI acts in a modular manner as a promoter exclusion determinant and an Asi-dependent degron. (A) Schematic representation of the artificial transcription factor composed of amino acids 16–50 of Stp1 fused to the bacterial DNA-binding protein lexA fused to the viral VP16 transcription AD. OPlexA-LEU2 and OPlexA-LYS2 reporter genes were used to assess binding to lexA operators. (B) Growth analysis of CAY235 (ASI1) and SHY016 (asi1Δ) carrying pDO211 (lexA-AD) or pDO260 (STP116-50-lexA-AD) on nonselective and selective media lacking leucine and lysine. Immunoblot of extracts prepared from strains. The immunoreactive forms of the lexA proteins present in cell extracts are schematically represented at their corresponding positions of migration. (C) Immunoblot analysis of extracts from strains CAY235 (ASI1) and SHY016 (asi1Δ) carrying pDO260 (STP116-50-lexA-AD) treated with CHX. Cells were pregrown in SD; at t = 0, the cultures received an aliquot of CHX (final concentration, 100 μg/ml), and samples were taken at the indicated time points. Levels of Pgk1 serve as internal control for protein loading.
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Related In: Results  -  Collection

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Figure 7: RI acts in a modular manner as a promoter exclusion determinant and an Asi-dependent degron. (A) Schematic representation of the artificial transcription factor composed of amino acids 16–50 of Stp1 fused to the bacterial DNA-binding protein lexA fused to the viral VP16 transcription AD. OPlexA-LEU2 and OPlexA-LYS2 reporter genes were used to assess binding to lexA operators. (B) Growth analysis of CAY235 (ASI1) and SHY016 (asi1Δ) carrying pDO211 (lexA-AD) or pDO260 (STP116-50-lexA-AD) on nonselective and selective media lacking leucine and lysine. Immunoblot of extracts prepared from strains. The immunoreactive forms of the lexA proteins present in cell extracts are schematically represented at their corresponding positions of migration. (C) Immunoblot analysis of extracts from strains CAY235 (ASI1) and SHY016 (asi1Δ) carrying pDO260 (STP116-50-lexA-AD) treated with CHX. Cells were pregrown in SD; at t = 0, the cultures received an aliquot of CHX (final concentration, 100 μg/ml), and samples were taken at the indicated time points. Levels of Pgk1 serve as internal control for protein loading.
Mentions: To ultimately test whether RI suffices to mediate cytoplasmic retention and constitutes a transferable Asi-dependent degron, we fused residues 16–50 of Stp1 containing RI to an artificial transcription factor composed of the bacterial DNA-binding protein lexA (lexA) and the strong viral VP16 transcription activation domain (AD). We used lexA operator–driven LYS2 and LEU2 genes as reporters for DNA binding of lexA-AD (Figure 7A). We then assessed cell growth in the absence of leucine and lysine as a functional readout for nuclear accumulation. When expressed in wild-type cells, the artificial transcription factor (lexA-AD) induced the reporter genes to facilitate growth in the absence of leucine and lysine (Figure 7B, dilution 1). In contrast, the fusion of lexA to Stp116-50 abolished promoter induction; no growth was observed (Figure 7B, compare dilution 2 with dilution 1). However, when expressed in cells lacking Asi1, robust growth was detected (Figure 7B, dilution 3). Of note, as judged by immunoblotting, the steady-state expression level of Stp116-50-lexA-AD was elevated in cells lacking Asi1 (Figure 7B, bottom, compare lane 3 with lane 2). Next we performed a CHX chase over a time course of 60 min to assess protein stability in wild-type and asi1Δ cells (Figure 7C). Strikingly, Stp116-50-lexA-AD was completely stable in asi1Δ cells (Figure 7C, lanes 6–10), whereas it efficiently turned over in wild-type cells (Figure 7C, lanes 1–5). Together these results show that the RI-containing domain of Stp1 comprising amino acids 16–50 contains the necessary information for Asi-dependent promoter exclusion, presumably by mediating efficient degradation.

Bottom Line: Stp1, the effector transcription factor, is synthesized as a latent cytoplasmic precursor with an N-terminal regulatory domain that restricts its nuclear accumulation.Our results indicate that RI mediates latency by two distinct activities: it functions as a cytoplasmic retention determinant and an Asi-dependent degron.These findings provide novel insights into the SPS-sensing pathway and demonstrate for the first time that the inner nuclear membrane Asi proteins function in a degradation pathway in the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden.

Show MeSH