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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 within the N-terminal domain of Stp1 is a conserved sequence motif required for latency. (A) Schematic representation of Stp1 and its N-terminal REG domain containing two conserved sequence motifs designated RI (aa 16–35) and RII (aa 65–97). Clustal X comparison of RI sequences of Stp1, corresponding to residues 10 and 40, and Stp2 of S. cerevisiae and the indicated fungal Stp1/Stp2 orthologues; the level of similarity is color coded as follows: conservative (blue) and similar (green) residues are highlighted; residues with weak (green text) or no similarity (black) are indicated. The RI motif is predicted to attain an amphipathic α-helix structure (Garnier et al., 1996); helical wheel projection of residues 16–33, created using Armstrong and Zidovetzki’s helical wheel script (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). Schematic representation of the Stp1 mutant protein constructs Stp1ΔRI, Stp1-133, Stp1-134, and Stp1-135. (B) Immunoblot analysis of extracts prepared from CAY123 (stp1Δ stp2Δ) carrying plasmid pCA047 (STP1) or pDO248 (ΔR1) grown in SD medium and harvested 30 min after induction by leucine as indicated; immunoreactive forms of Stp1 species are indicated at their corresponding positions of migration (top left). Immunoblot analysis of extracts prepared from MBY93 (ssy5Δ stp1Δ stp2Δ) carrying plasmids pCA047 (STP1), pDO248 (ΔR1) grown in SD medium (top right). Growth of strains on YPD and YPD + MM; 10-fold dilutions of cultures grown in SD were spotted onto plates and incubated at 30°C (bottom). (C) Growth of MBY93 (ssy5Δ stp1Δ stp2Δ) and MBY102 (ssy5Δ stp1Δ stp2Δ asi1Δ) carrying plasmid pCA047 (STP1), pCA120 (STP1-133), pDO141 (STP1-134), or pDO142 (STP1-135) on YPD and YPD + MM; 10-fold dilutions of cultures grown in SD were spotted onto plates and incubated at 30°C (top). Immunoblot analysis of Stp1 protein levels in extracts prepared from cells grown in SD medium (bottom).
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Figure 3: RI within the N-terminal domain of Stp1 is a conserved sequence motif required for latency. (A) Schematic representation of Stp1 and its N-terminal REG domain containing two conserved sequence motifs designated RI (aa 16–35) and RII (aa 65–97). Clustal X comparison of RI sequences of Stp1, corresponding to residues 10 and 40, and Stp2 of S. cerevisiae and the indicated fungal Stp1/Stp2 orthologues; the level of similarity is color coded as follows: conservative (blue) and similar (green) residues are highlighted; residues with weak (green text) or no similarity (black) are indicated. The RI motif is predicted to attain an amphipathic α-helix structure (Garnier et al., 1996); helical wheel projection of residues 16–33, created using Armstrong and Zidovetzki’s helical wheel script (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). Schematic representation of the Stp1 mutant protein constructs Stp1ΔRI, Stp1-133, Stp1-134, and Stp1-135. (B) Immunoblot analysis of extracts prepared from CAY123 (stp1Δ stp2Δ) carrying plasmid pCA047 (STP1) or pDO248 (ΔR1) grown in SD medium and harvested 30 min after induction by leucine as indicated; immunoreactive forms of Stp1 species are indicated at their corresponding positions of migration (top left). Immunoblot analysis of extracts prepared from MBY93 (ssy5Δ stp1Δ stp2Δ) carrying plasmids pCA047 (STP1), pDO248 (ΔR1) grown in SD medium (top right). Growth of strains on YPD and YPD + MM; 10-fold dilutions of cultures grown in SD were spotted onto plates and incubated at 30°C (bottom). (C) Growth of MBY93 (ssy5Δ stp1Δ stp2Δ) and MBY102 (ssy5Δ stp1Δ stp2Δ asi1Δ) carrying plasmid pCA047 (STP1), pCA120 (STP1-133), pDO141 (STP1-134), or pDO142 (STP1-135) on YPD and YPD + MM; 10-fold dilutions of cultures grown in SD were spotted onto plates and incubated at 30°C (top). Immunoblot analysis of Stp1 protein levels in extracts prepared from cells grown in SD medium (bottom).

Mentions: To directly test whether RI mediates Stp1 latency, we constructed a mutant allele in the context of a fully functional C-terminal HA epitope–tagged STP1, which encoded a protein lacking residues 16–35 (STP1ΔRI) (Figure 3A, schematic). On leucine induction, the Stp1ΔRI protein was processed as efficiently as wild type (Figure 3B, compare lanes 1a-b and 2a-b). The ability of Stp1ΔRI to induce transcription was assessed by monitoring growth on yeast extract/peptone/dextrose (YPD) medium containing 2-{[({[(4-methoxy-6-methyl)-1,3,5-triazin-2-yl]-amino}carbonyl)amino]-sulfonyl}-benzoic acid (MM). MM is an inhibitor of branched-chain amino acid synthesis, and growth on YPD plus MM requires SPS sensor–induced, Stp1/Stp2-dependent expression of high-affinity permeases for leucine, isoleucine, and valine (Jørgensen et al., 1998). Cells expressing STP1ΔRI grew as well as cells expressing wild-type STP1 on YPD plus MM (Figure 3B, dilutions 1 and 2), indicating that Stp1ΔRI is capable of activating SPS sensor–regulated genes. Of importance, in cells lacking a functional SPS sensor (ssy5Δ), expression of wild-type STP1 did not activate amino acid permease expression. By contrast, ssy5Δ cells expressing STP1ΔRI exhibited robust growth (Figure 3B, dilutions 3 and 4). In conclusion, deletion of amino acid residues 16–35 bypasses the requirement of SPS sensor– dependent proteolytic processing, indicating that RI is essential for maintaining the latent properties of Stp1 in the absence of amino acid induction.


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 within the N-terminal domain of Stp1 is a conserved sequence motif required for latency. (A) Schematic representation of Stp1 and its N-terminal REG domain containing two conserved sequence motifs designated RI (aa 16–35) and RII (aa 65–97). Clustal X comparison of RI sequences of Stp1, corresponding to residues 10 and 40, and Stp2 of S. cerevisiae and the indicated fungal Stp1/Stp2 orthologues; the level of similarity is color coded as follows: conservative (blue) and similar (green) residues are highlighted; residues with weak (green text) or no similarity (black) are indicated. The RI motif is predicted to attain an amphipathic α-helix structure (Garnier et al., 1996); helical wheel projection of residues 16–33, created using Armstrong and Zidovetzki’s helical wheel script (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). Schematic representation of the Stp1 mutant protein constructs Stp1ΔRI, Stp1-133, Stp1-134, and Stp1-135. (B) Immunoblot analysis of extracts prepared from CAY123 (stp1Δ stp2Δ) carrying plasmid pCA047 (STP1) or pDO248 (ΔR1) grown in SD medium and harvested 30 min after induction by leucine as indicated; immunoreactive forms of Stp1 species are indicated at their corresponding positions of migration (top left). Immunoblot analysis of extracts prepared from MBY93 (ssy5Δ stp1Δ stp2Δ) carrying plasmids pCA047 (STP1), pDO248 (ΔR1) grown in SD medium (top right). Growth of strains on YPD and YPD + MM; 10-fold dilutions of cultures grown in SD were spotted onto plates and incubated at 30°C (bottom). (C) Growth of MBY93 (ssy5Δ stp1Δ stp2Δ) and MBY102 (ssy5Δ stp1Δ stp2Δ asi1Δ) carrying plasmid pCA047 (STP1), pCA120 (STP1-133), pDO141 (STP1-134), or pDO142 (STP1-135) on YPD and YPD + MM; 10-fold dilutions of cultures grown in SD were spotted onto plates and incubated at 30°C (top). Immunoblot analysis of Stp1 protein levels in extracts prepared from cells grown in SD medium (bottom).
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Figure 3: RI within the N-terminal domain of Stp1 is a conserved sequence motif required for latency. (A) Schematic representation of Stp1 and its N-terminal REG domain containing two conserved sequence motifs designated RI (aa 16–35) and RII (aa 65–97). Clustal X comparison of RI sequences of Stp1, corresponding to residues 10 and 40, and Stp2 of S. cerevisiae and the indicated fungal Stp1/Stp2 orthologues; the level of similarity is color coded as follows: conservative (blue) and similar (green) residues are highlighted; residues with weak (green text) or no similarity (black) are indicated. The RI motif is predicted to attain an amphipathic α-helix structure (Garnier et al., 1996); helical wheel projection of residues 16–33, created using Armstrong and Zidovetzki’s helical wheel script (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). Schematic representation of the Stp1 mutant protein constructs Stp1ΔRI, Stp1-133, Stp1-134, and Stp1-135. (B) Immunoblot analysis of extracts prepared from CAY123 (stp1Δ stp2Δ) carrying plasmid pCA047 (STP1) or pDO248 (ΔR1) grown in SD medium and harvested 30 min after induction by leucine as indicated; immunoreactive forms of Stp1 species are indicated at their corresponding positions of migration (top left). Immunoblot analysis of extracts prepared from MBY93 (ssy5Δ stp1Δ stp2Δ) carrying plasmids pCA047 (STP1), pDO248 (ΔR1) grown in SD medium (top right). Growth of strains on YPD and YPD + MM; 10-fold dilutions of cultures grown in SD were spotted onto plates and incubated at 30°C (bottom). (C) Growth of MBY93 (ssy5Δ stp1Δ stp2Δ) and MBY102 (ssy5Δ stp1Δ stp2Δ asi1Δ) carrying plasmid pCA047 (STP1), pCA120 (STP1-133), pDO141 (STP1-134), or pDO142 (STP1-135) on YPD and YPD + MM; 10-fold dilutions of cultures grown in SD were spotted onto plates and incubated at 30°C (top). Immunoblot analysis of Stp1 protein levels in extracts prepared from cells grown in SD medium (bottom).
Mentions: To directly test whether RI mediates Stp1 latency, we constructed a mutant allele in the context of a fully functional C-terminal HA epitope–tagged STP1, which encoded a protein lacking residues 16–35 (STP1ΔRI) (Figure 3A, schematic). On leucine induction, the Stp1ΔRI protein was processed as efficiently as wild type (Figure 3B, compare lanes 1a-b and 2a-b). The ability of Stp1ΔRI to induce transcription was assessed by monitoring growth on yeast extract/peptone/dextrose (YPD) medium containing 2-{[({[(4-methoxy-6-methyl)-1,3,5-triazin-2-yl]-amino}carbonyl)amino]-sulfonyl}-benzoic acid (MM). MM is an inhibitor of branched-chain amino acid synthesis, and growth on YPD plus MM requires SPS sensor–induced, Stp1/Stp2-dependent expression of high-affinity permeases for leucine, isoleucine, and valine (Jørgensen et al., 1998). Cells expressing STP1ΔRI grew as well as cells expressing wild-type STP1 on YPD plus MM (Figure 3B, dilutions 1 and 2), indicating that Stp1ΔRI is capable of activating SPS sensor–regulated genes. Of importance, in cells lacking a functional SPS sensor (ssy5Δ), expression of wild-type STP1 did not activate amino acid permease expression. By contrast, ssy5Δ cells expressing STP1ΔRI exhibited robust growth (Figure 3B, dilutions 3 and 4). In conclusion, deletion of amino acid residues 16–35 bypasses the requirement of SPS sensor– dependent proteolytic processing, indicating that RI is essential for maintaining the latent properties of Stp1 in the absence of amino acid induction.

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