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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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Turnover of nuclear-localized Stp1 is Asi dependent. (A) Immunoblot analysis of extracts from MBY89 (ssy5Δ; WT), MBY106 (ssy5Δ asi1Δ), MBY108 (ssy5Δ asi2Δ), and MBY110 (ssy5Δ asi3Δ) carrying pAB27 (RI15-35) or pDO74 (RI17-33) as indicated. (B) Immunoblot analysis of cell extracts from MBY93 (ssy5Δ stp1Δ stp2Δ), MBY102 (ssy5Δ stp1Δ stp2Δ asi1Δ), CAY86 (grr1Δ), and CAY109 (grr1Δ asi1Δ) carrying pCA047 (STP1), pAB27 (RI15-35), pCA120 (STP1-133), or pDO248 (STP1ΔR1) treated with CHX. Cells were pregrown in SD; samples were taken 0 and 10 min after cultures received an aliquot of CHX (final concentration, 100 µg/ml). Levels of Pgk1 serve as internal control for protein loading.
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Figure 5: Turnover of nuclear-localized Stp1 is Asi dependent. (A) Immunoblot analysis of extracts from MBY89 (ssy5Δ; WT), MBY106 (ssy5Δ asi1Δ), MBY108 (ssy5Δ asi2Δ), and MBY110 (ssy5Δ asi3Δ) carrying pAB27 (RI15-35) or pDO74 (RI17-33) as indicated. (B) Immunoblot analysis of cell extracts from MBY93 (ssy5Δ stp1Δ stp2Δ), MBY102 (ssy5Δ stp1Δ stp2Δ asi1Δ), CAY86 (grr1Δ), and CAY109 (grr1Δ asi1Δ) carrying pCA047 (STP1), pAB27 (RI15-35), pCA120 (STP1-133), or pDO248 (STP1ΔR1) treated with CHX. Cells were pregrown in SD; samples were taken 0 and 10 min after cultures received an aliquot of CHX (final concentration, 100 µg/ml). Levels of Pgk1 serve as internal control for protein loading.

Mentions: Next we asked whether the increased steady-state levels of RI15-35 and RI17-33 in asi1Δ cells—the consequence of enhanced stability in the absence of Asi1—could also be observed in cells lacking ASI2 or ASI3. Cells carrying deletions of ASI2 or ASI3 exhibit the same levels of Stp1/2-dependent promoter induction as asi1Δ mutants, and no additive effects are observed in cells carrying all possible double- and triple- mutant combinations (Zargari et al., 2007). We analyzed the steady-state amount of proteins in asi2Δ and asi3Δ cells in comparison to those in asi1Δ and ASI wild-type cells. Indeed, RI15-35 and RI17-33 proteins accumulated to the same extent in cells lacking any of the ASI genes compared with the amount detected in ASI wild-type cells (Figure 5A, compare lanes 2–4 with lane 1 and lanes 6–8 with lane 5). In conclusion, deletion of ASI1, ASI2, or ASI3 results in an identical phenotype; consequently, Asi1, Asi2, and Asi3 appear to contribute equally to maintaining Stp1 latency and do so by affecting levels of Stp1 in a RI-dependent manner.


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)

Turnover of nuclear-localized Stp1 is Asi dependent. (A) Immunoblot analysis of extracts from MBY89 (ssy5Δ; WT), MBY106 (ssy5Δ asi1Δ), MBY108 (ssy5Δ asi2Δ), and MBY110 (ssy5Δ asi3Δ) carrying pAB27 (RI15-35) or pDO74 (RI17-33) as indicated. (B) Immunoblot analysis of cell extracts from MBY93 (ssy5Δ stp1Δ stp2Δ), MBY102 (ssy5Δ stp1Δ stp2Δ asi1Δ), CAY86 (grr1Δ), and CAY109 (grr1Δ asi1Δ) carrying pCA047 (STP1), pAB27 (RI15-35), pCA120 (STP1-133), or pDO248 (STP1ΔR1) treated with CHX. Cells were pregrown in SD; samples were taken 0 and 10 min after cultures received an aliquot of CHX (final concentration, 100 µg/ml). Levels of Pgk1 serve as internal control for protein loading.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Turnover of nuclear-localized Stp1 is Asi dependent. (A) Immunoblot analysis of extracts from MBY89 (ssy5Δ; WT), MBY106 (ssy5Δ asi1Δ), MBY108 (ssy5Δ asi2Δ), and MBY110 (ssy5Δ asi3Δ) carrying pAB27 (RI15-35) or pDO74 (RI17-33) as indicated. (B) Immunoblot analysis of cell extracts from MBY93 (ssy5Δ stp1Δ stp2Δ), MBY102 (ssy5Δ stp1Δ stp2Δ asi1Δ), CAY86 (grr1Δ), and CAY109 (grr1Δ asi1Δ) carrying pCA047 (STP1), pAB27 (RI15-35), pCA120 (STP1-133), or pDO248 (STP1ΔR1) treated with CHX. Cells were pregrown in SD; samples were taken 0 and 10 min after cultures received an aliquot of CHX (final concentration, 100 µg/ml). Levels of Pgk1 serve as internal control for protein loading.
Mentions: Next we asked whether the increased steady-state levels of RI15-35 and RI17-33 in asi1Δ cells—the consequence of enhanced stability in the absence of Asi1—could also be observed in cells lacking ASI2 or ASI3. Cells carrying deletions of ASI2 or ASI3 exhibit the same levels of Stp1/2-dependent promoter induction as asi1Δ mutants, and no additive effects are observed in cells carrying all possible double- and triple- mutant combinations (Zargari et al., 2007). We analyzed the steady-state amount of proteins in asi2Δ and asi3Δ cells in comparison to those in asi1Δ and ASI wild-type cells. Indeed, RI15-35 and RI17-33 proteins accumulated to the same extent in cells lacking any of the ASI genes compared with the amount detected in ASI wild-type cells (Figure 5A, compare lanes 2–4 with lane 1 and lanes 6–8 with lane 5). In conclusion, deletion of ASI1, ASI2, or ASI3 results in an identical phenotype; consequently, Asi1, Asi2, and Asi3 appear to contribute equally to maintaining Stp1 latency and do so by affecting levels of Stp1 in a RI-dependent manner.

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