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 ultimately test whether RI suffices to mediate cytoplasmic retention and constitutes a transferable Asi-dependent degron, we fused residues 16–50 of Stp1 containing RI to an artificial transcription factor composed of the bacterial DNA-binding protein lexA (lexA) and the strong viral VP16 transcription activation domain (AD). We used lexA operator–driven LYS2 and LEU2 genes as reporters for DNA binding of lexA-AD (Figure 7A). We then assessed cell growth in the absence of leucine and lysine as a functional readout for nuclear accumulation. When expressed in wild-type cells, the artificial transcription factor (lexA-AD) induced the reporter genes to facilitate growth in the absence of leucine and lysine (Figure 7B, dilution 1). In contrast, the fusion of lexA to Stp116-50 abolished promoter induction; no growth was observed (Figure 7B, compare dilution 2 with dilution 1). However, when expressed in cells lacking Asi1, robust growth was detected (Figure 7B, dilution 3). Of note, as judged by immunoblotting, the steady-state expression level of Stp116-50-lexA-AD was elevated in cells lacking Asi1 (Figure 7B, bottom, compare lane 3 with lane 2). Next we performed a CHX chase over a time course of 60 min to assess protein stability in wild-type and asi1Δ cells (Figure 7C). Strikingly, Stp116-50-lexA-AD was completely stable in asi1Δ cells (Figure 7C, lanes 6–10), whereas it efficiently turned over in wild-type cells (Figure 7C, lanes 1–5). Together these results show that the RI-containing domain of Stp1 comprising amino acids 16–50 contains the necessary information for Asi-dependent promoter exclusion, presumably by mediating efficient degradation.
Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden.