Latency of transcription factor Stp1 depends on a modular regulatory motif that functions as cytoplasmic retention determinant and nuclear degron.
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.
Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden.Show MeSH
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.
Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden.