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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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The N-terminal regulatory domain of Stp1 functions as modular cytoplasmic retention determinant. The Stp11-125-Htb2-GFP fusion protein is schematically presented. Immunoblot (left) of extracts prepared from and microscopic analysis (right) of strain CAY1259 (HTB2-ymCherry) carrying pDO39 (STP11-125-HTB2-GFP-HA) grown in SD media and at the indicated time points after leucine induction. For microscopic analysis, cells were immobilized on concanavalin A–coated (1 mg/ml) microscopy dishes and observed by fluorescence microscopy (Zeiss Axiovert 200M, 63× objective, differential interference contrast, GFP, and red fluorescent protein filters). Images were acquired using SlideBook software at 0, 30, and 60 min after cells were induced with leucine.
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Figure 1: The N-terminal regulatory domain of Stp1 functions as modular cytoplasmic retention determinant. The Stp11-125-Htb2-GFP fusion protein is schematically presented. Immunoblot (left) of extracts prepared from and microscopic analysis (right) of strain CAY1259 (HTB2-ymCherry) carrying pDO39 (STP11-125-HTB2-GFP-HA) grown in SD media and at the indicated time points after leucine induction. For microscopic analysis, cells were immobilized on concanavalin A–coated (1 mg/ml) microscopy dishes and observed by fluorescence microscopy (Zeiss Axiovert 200M, 63× objective, differential interference contrast, GFP, and red fluorescent protein filters). Images were acquired using SlideBook software at 0, 30, and 60 min after cells were induced with leucine.

Mentions: To test the efficacy of the Stp1 N-terminal REG domain, we examined whether it could control the localization of histone Htb2, a protein that normally efficiently targets to the nucleus. We posited that if the REG domain placed the chimeric protein under SPS-sensing pathway control, we could differentiate between the possibilities that the RI motif in the REG domain functions as a cytoplasmic retention determinant, or alternatively, functions as a nuclear export sequence (NES) that mediates efficient nuclear export. The N-terminal REG domain of Stp1 was fused to the N-terminus of histone Htb2 carrying a C-terminal green fluorescent protein (GFP)–hemagglutinin (HA) tag (Figure 1). A plasmid directing the expression of this chimera was introduced into a strain expressing chromosomal Htb2 fused with ymCherry. The Stp11-125Htb2-GFP construct was rapidly processed upon leucine induction (Figure 1, immunoblot), clearly demonstrating that the chimeric protein is subject to SPS-sensor control. The intracellular distribution of both the GFP- and Cherry-tagged Htb2 was assessed by fluorescence microscopy and followed over time after leucine induction. In contrast to the Htb2-ymCherry, which localized to distinct foci marking the nucleus, the unprocessed Stp11-125Htb2-GFP chimera displayed diffuse fluorescence in the absence of leucine induction (Figure 1, t = 0). On leucine induction, correlating precisely with SPS-sensor dependent processing, the GFP fluorescence associated with the shorter processed form of the chimera rapidly concentrated in foci that overlapped with the nuclear staining of the Htb2-ymCherry (Figure 1, merge). These results confirm and extend our previous finding that the N-terminal regulatory domain of Stp1 is modular and transferable (Andréasson and Ljungdahl, 2004). The fact that the RI-containing REG domain is able to prevent the nuclear localization of a histone protein with a high affinity for binding DNA diminishes the likelihood that RI functions as a NES mediating nucleocytoplasmic shuttling. Instead, the data suggest that the RI motif functions as a nuclear exclusion determinant that interacts and associates with cytoplasmic components.


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)

The N-terminal regulatory domain of Stp1 functions as modular cytoplasmic retention determinant. The Stp11-125-Htb2-GFP fusion protein is schematically presented. Immunoblot (left) of extracts prepared from and microscopic analysis (right) of strain CAY1259 (HTB2-ymCherry) carrying pDO39 (STP11-125-HTB2-GFP-HA) grown in SD media and at the indicated time points after leucine induction. For microscopic analysis, cells were immobilized on concanavalin A–coated (1 mg/ml) microscopy dishes and observed by fluorescence microscopy (Zeiss Axiovert 200M, 63× objective, differential interference contrast, GFP, and red fluorescent protein filters). Images were acquired using SlideBook software at 0, 30, and 60 min after cells were induced with leucine.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: The N-terminal regulatory domain of Stp1 functions as modular cytoplasmic retention determinant. The Stp11-125-Htb2-GFP fusion protein is schematically presented. Immunoblot (left) of extracts prepared from and microscopic analysis (right) of strain CAY1259 (HTB2-ymCherry) carrying pDO39 (STP11-125-HTB2-GFP-HA) grown in SD media and at the indicated time points after leucine induction. For microscopic analysis, cells were immobilized on concanavalin A–coated (1 mg/ml) microscopy dishes and observed by fluorescence microscopy (Zeiss Axiovert 200M, 63× objective, differential interference contrast, GFP, and red fluorescent protein filters). Images were acquired using SlideBook software at 0, 30, and 60 min after cells were induced with leucine.
Mentions: To test the efficacy of the Stp1 N-terminal REG domain, we examined whether it could control the localization of histone Htb2, a protein that normally efficiently targets to the nucleus. We posited that if the REG domain placed the chimeric protein under SPS-sensing pathway control, we could differentiate between the possibilities that the RI motif in the REG domain functions as a cytoplasmic retention determinant, or alternatively, functions as a nuclear export sequence (NES) that mediates efficient nuclear export. The N-terminal REG domain of Stp1 was fused to the N-terminus of histone Htb2 carrying a C-terminal green fluorescent protein (GFP)–hemagglutinin (HA) tag (Figure 1). A plasmid directing the expression of this chimera was introduced into a strain expressing chromosomal Htb2 fused with ymCherry. The Stp11-125Htb2-GFP construct was rapidly processed upon leucine induction (Figure 1, immunoblot), clearly demonstrating that the chimeric protein is subject to SPS-sensor control. The intracellular distribution of both the GFP- and Cherry-tagged Htb2 was assessed by fluorescence microscopy and followed over time after leucine induction. In contrast to the Htb2-ymCherry, which localized to distinct foci marking the nucleus, the unprocessed Stp11-125Htb2-GFP chimera displayed diffuse fluorescence in the absence of leucine induction (Figure 1, t = 0). On leucine induction, correlating precisely with SPS-sensor dependent processing, the GFP fluorescence associated with the shorter processed form of the chimera rapidly concentrated in foci that overlapped with the nuclear staining of the Htb2-ymCherry (Figure 1, merge). These results confirm and extend our previous finding that the N-terminal regulatory domain of Stp1 is modular and transferable (Andréasson and Ljungdahl, 2004). The fact that the RI-containing REG domain is able to prevent the nuclear localization of a histone protein with a high affinity for binding DNA diminishes the likelihood that RI functions as a NES mediating nucleocytoplasmic shuttling. Instead, the data suggest that the RI motif functions as a nuclear exclusion determinant that interacts and associates with cytoplasmic components.

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
Related in: MedlinePlus