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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 of Stp1 suffices to direct hSOS to the plasma membrane. Growth of (A) CAY187 (cdc25-2) carrying pCA094 (ø), pCA104 (hSOS-Stp12-158), or pDO196 (hSOS-Stp12-158(Δ16-21)) and of (B) CAY187 (cdc25-2) carrying pCA094 (ø), pCA104 (hSOS-Stp12-158), pDO203 (hSOS-Stp12-64), pDO205 (hSOS-Stp116-33), pDO230 (hSOS-Stp116-37), pDO231 (hSOS-Stp116-50), or pDO221 (hSOS-Stp116-64). Plates were incubated at 25 and 37°C as indicated. Right, hSOS-REG fusion constructs.
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Figure 2: RI of Stp1 suffices to direct hSOS to the plasma membrane. Growth of (A) CAY187 (cdc25-2) carrying pCA094 (ø), pCA104 (hSOS-Stp12-158), or pDO196 (hSOS-Stp12-158(Δ16-21)) and of (B) CAY187 (cdc25-2) carrying pCA094 (ø), pCA104 (hSOS-Stp12-158), pDO203 (hSOS-Stp12-64), pDO205 (hSOS-Stp116-33), pDO230 (hSOS-Stp116-37), pDO231 (hSOS-Stp116-50), or pDO221 (hSOS-Stp116-64). Plates were incubated at 25 and 37°C as indicated. Right, hSOS-REG fusion constructs.

Mentions: To examine the notion that RI mediates an interaction with a cytoplasmic component, we used the SOS recruitment system (SRS). The SRS exploits the fact that the human Cdc25 homologue SOS (hSOS) can suppress the temperature-sensitive growth defect of a cdc25-2 strain when it is localized in close proximity to the plasma membrane, a property that is dependent on hSOS being fused to a plasma membrane–associated partner (Aronheim et al., 1997). We previously showed that the N-terminal REG domain of Stp1, amino acids 2–158 (hSOS-REG2-158), can direct hSOS to the plasma membrane (Andréasson and Ljungdahl, 2002). To examine specifically whether RI mediates the plasma membrane interaction of the hSOS-REG2-158 fusion protein, we constructed hSOS-REG2-158(Δ16-21) lacking the first six amino acid (aa) residues of RI (aa 16–21 of Stp1; Figure 2A). We introduced hSOS alone (ø), hSOS-REG2-158, and hSOS-REG2-158(Δ16-21) into a temperature-sensitive cdc25-2 reporter strain and assayed growth at permissive (25°C) and nonpermissive (37°C) temperatures. All strains grew equally well at 25°C; however, only the full-length hSOS-REG2-158 construct enabled growth at 37°C (Figure 2A; compare dilution 2 with 1 and 3). The hSOS-REG2-158(Δ16-21) mutant did not suppress the growth defect of the cdc25-2 strain, indicating that in the absence of RI, the REG domain is unable to associate with the plasma membrane.


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 of Stp1 suffices to direct hSOS to the plasma membrane. Growth of (A) CAY187 (cdc25-2) carrying pCA094 (ø), pCA104 (hSOS-Stp12-158), or pDO196 (hSOS-Stp12-158(Δ16-21)) and of (B) CAY187 (cdc25-2) carrying pCA094 (ø), pCA104 (hSOS-Stp12-158), pDO203 (hSOS-Stp12-64), pDO205 (hSOS-Stp116-33), pDO230 (hSOS-Stp116-37), pDO231 (hSOS-Stp116-50), or pDO221 (hSOS-Stp116-64). Plates were incubated at 25 and 37°C as indicated. Right, hSOS-REG fusion constructs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: RI of Stp1 suffices to direct hSOS to the plasma membrane. Growth of (A) CAY187 (cdc25-2) carrying pCA094 (ø), pCA104 (hSOS-Stp12-158), or pDO196 (hSOS-Stp12-158(Δ16-21)) and of (B) CAY187 (cdc25-2) carrying pCA094 (ø), pCA104 (hSOS-Stp12-158), pDO203 (hSOS-Stp12-64), pDO205 (hSOS-Stp116-33), pDO230 (hSOS-Stp116-37), pDO231 (hSOS-Stp116-50), or pDO221 (hSOS-Stp116-64). Plates were incubated at 25 and 37°C as indicated. Right, hSOS-REG fusion constructs.
Mentions: To examine the notion that RI mediates an interaction with a cytoplasmic component, we used the SOS recruitment system (SRS). The SRS exploits the fact that the human Cdc25 homologue SOS (hSOS) can suppress the temperature-sensitive growth defect of a cdc25-2 strain when it is localized in close proximity to the plasma membrane, a property that is dependent on hSOS being fused to a plasma membrane–associated partner (Aronheim et al., 1997). We previously showed that the N-terminal REG domain of Stp1, amino acids 2–158 (hSOS-REG2-158), can direct hSOS to the plasma membrane (Andréasson and Ljungdahl, 2002). To examine specifically whether RI mediates the plasma membrane interaction of the hSOS-REG2-158 fusion protein, we constructed hSOS-REG2-158(Δ16-21) lacking the first six amino acid (aa) residues of RI (aa 16–21 of Stp1; Figure 2A). We introduced hSOS alone (ø), hSOS-REG2-158, and hSOS-REG2-158(Δ16-21) into a temperature-sensitive cdc25-2 reporter strain and assayed growth at permissive (25°C) and nonpermissive (37°C) temperatures. All strains grew equally well at 25°C; however, only the full-length hSOS-REG2-158 construct enabled growth at 37°C (Figure 2A; compare dilution 2 with 1 and 3). The hSOS-REG2-158(Δ16-21) mutant did not suppress the growth defect of the cdc25-2 strain, indicating that in the absence of RI, the REG domain is unable to associate with the plasma membrane.

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