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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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Overexpression of STP1 leads to constitutive promoter induction. (A) Amino acid sequences of Stp1 and Stp2 of S. cerevisiae and fungal Stp1 orthologues as in Figure 3 compared using Clustal X; the level of similarity of the aligned protein sequences is plotted. Schematic presentations of Stp1 and mutant proteins lacking discrete conserved domains. Immunoblot analysis of extracts from MBY93 (ssy5Δ stp1Δ stp2Δ) and carrying pCA047 (STP1), pDO243 (Δ94-113), pDO237 (Δ127-150), pDO244 (Δ288-306), pDO246 (Δ365-420), or pDO247 (Δ443-471) treated with CHX as in Figure 5. (B) Immunoblot analysis of extracts from MBY93 (ssy5Δ stp1Δ stp2Δ) and CAY123 (stp1Δ stp2Δ) carrying pCA072 (pRS316-FLAG-STP1-HA; CEN) or pCA078 (pRS202-FLAG-STP1-HA; 2μ). Cells were grown in SD medium and harvested 30 min after induction by leucine as indicated. Levels of Pgk1 serve as internal control for protein loading. (C) Growth of strains in B on YPD and YPD + MM.
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Figure 6: Overexpression of STP1 leads to constitutive promoter induction. (A) Amino acid sequences of Stp1 and Stp2 of S. cerevisiae and fungal Stp1 orthologues as in Figure 3 compared using Clustal X; the level of similarity of the aligned protein sequences is plotted. Schematic presentations of Stp1 and mutant proteins lacking discrete conserved domains. Immunoblot analysis of extracts from MBY93 (ssy5Δ stp1Δ stp2Δ) and carrying pCA047 (STP1), pDO243 (Δ94-113), pDO237 (Δ127-150), pDO244 (Δ288-306), pDO246 (Δ365-420), or pDO247 (Δ443-471) treated with CHX as in Figure 5. (B) Immunoblot analysis of extracts from MBY93 (ssy5Δ stp1Δ stp2Δ) and CAY123 (stp1Δ stp2Δ) carrying pCA072 (pRS316-FLAG-STP1-HA; CEN) or pCA078 (pRS202-FLAG-STP1-HA; 2μ). Cells were grown in SD medium and harvested 30 min after induction by leucine as indicated. Levels of Pgk1 serve as internal control for protein loading. (C) Growth of strains in B on YPD and YPD + MM.

Mentions: To analyze further whether turnover plays a role in Stp1 regulation, we sought to assess whether increased protein levels would lead to constitutive activity. To achieve higher steady-state levels of Stp1, we used two approaches. Initially, to effect a decrease in degradation, that is, to experimentally stabilize Stp1, we attempted to mutate potential degron sequences of Stp1. We examined the Stp1 amino acid sequence for putative conserved PEST motifs, that is, sequence motifs rich in proline, glutamic acid, serine, and threonine residues that are implicated in conferring protein instability (Rechsteiner and Rogers, 1996). We found five regions of sequence conservation that were rich in P, E, S, and T residues (Figure 6A). We performed a systematic deletion analysis targeting these conserved sequences. The stability of the mutant proteins was assessed in CHX chase assays, followed by immunoblotting; however, none of the conserved regions appeared to significantly affect Stp1 stability (Figure 6A, bottom). To elevate the protein pool by other means, we then expressed Stp1 from a 2μm plasmid, which led to increased steady-state levels of the protein (Figure 6B, compare lane 2 with lane 1). Of interest, we found that overexpression of Stp1 in ssy5Δ cells led to constitutive, processing-independent amino acid permease gene expression as judged by resistance to MM (Figure 6C, compare dilution 2 with dilution 1). This finding indicates that the mechanisms regulating RI-dependent Stp1 latency are saturable and thus sensitive to protein levels.


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)

Overexpression of STP1 leads to constitutive promoter induction. (A) Amino acid sequences of Stp1 and Stp2 of S. cerevisiae and fungal Stp1 orthologues as in Figure 3 compared using Clustal X; the level of similarity of the aligned protein sequences is plotted. Schematic presentations of Stp1 and mutant proteins lacking discrete conserved domains. Immunoblot analysis of extracts from MBY93 (ssy5Δ stp1Δ stp2Δ) and carrying pCA047 (STP1), pDO243 (Δ94-113), pDO237 (Δ127-150), pDO244 (Δ288-306), pDO246 (Δ365-420), or pDO247 (Δ443-471) treated with CHX as in Figure 5. (B) Immunoblot analysis of extracts from MBY93 (ssy5Δ stp1Δ stp2Δ) and CAY123 (stp1Δ stp2Δ) carrying pCA072 (pRS316-FLAG-STP1-HA; CEN) or pCA078 (pRS202-FLAG-STP1-HA; 2μ). Cells were grown in SD medium and harvested 30 min after induction by leucine as indicated. Levels of Pgk1 serve as internal control for protein loading. (C) Growth of strains in B on YPD and YPD + MM.
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Related In: Results  -  Collection

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Figure 6: Overexpression of STP1 leads to constitutive promoter induction. (A) Amino acid sequences of Stp1 and Stp2 of S. cerevisiae and fungal Stp1 orthologues as in Figure 3 compared using Clustal X; the level of similarity of the aligned protein sequences is plotted. Schematic presentations of Stp1 and mutant proteins lacking discrete conserved domains. Immunoblot analysis of extracts from MBY93 (ssy5Δ stp1Δ stp2Δ) and carrying pCA047 (STP1), pDO243 (Δ94-113), pDO237 (Δ127-150), pDO244 (Δ288-306), pDO246 (Δ365-420), or pDO247 (Δ443-471) treated with CHX as in Figure 5. (B) Immunoblot analysis of extracts from MBY93 (ssy5Δ stp1Δ stp2Δ) and CAY123 (stp1Δ stp2Δ) carrying pCA072 (pRS316-FLAG-STP1-HA; CEN) or pCA078 (pRS202-FLAG-STP1-HA; 2μ). Cells were grown in SD medium and harvested 30 min after induction by leucine as indicated. Levels of Pgk1 serve as internal control for protein loading. (C) Growth of strains in B on YPD and YPD + MM.
Mentions: To analyze further whether turnover plays a role in Stp1 regulation, we sought to assess whether increased protein levels would lead to constitutive activity. To achieve higher steady-state levels of Stp1, we used two approaches. Initially, to effect a decrease in degradation, that is, to experimentally stabilize Stp1, we attempted to mutate potential degron sequences of Stp1. We examined the Stp1 amino acid sequence for putative conserved PEST motifs, that is, sequence motifs rich in proline, glutamic acid, serine, and threonine residues that are implicated in conferring protein instability (Rechsteiner and Rogers, 1996). We found five regions of sequence conservation that were rich in P, E, S, and T residues (Figure 6A). We performed a systematic deletion analysis targeting these conserved sequences. The stability of the mutant proteins was assessed in CHX chase assays, followed by immunoblotting; however, none of the conserved regions appeared to significantly affect Stp1 stability (Figure 6A, bottom). To elevate the protein pool by other means, we then expressed Stp1 from a 2μm plasmid, which led to increased steady-state levels of the protein (Figure 6B, compare lane 2 with lane 1). Of interest, we found that overexpression of Stp1 in ssy5Δ cells led to constitutive, processing-independent amino acid permease gene expression as judged by resistance to MM (Figure 6C, compare dilution 2 with dilution 1). This finding indicates that the mechanisms regulating RI-dependent Stp1 latency are saturable and thus sensitive to protein levels.

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