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 ascertain the minimal sequence requirement of the RI motif, we created a series of mutant alleles encoding altered N-terminal domains of Stp1. Specifically, we deleted the sequence encoding amino acids 2–64 from STP1 and reinserted sequences encoding RI variants, that is, amino acids 15–35, 17–33, and 22–35 between minimal linker sequences, fused in-frame to the L65 codon of RII, respectively (Figure 4A). We expressed these RI constructs in ssy5Δ stp1Δ stp2Δ cells and analyzed Stp1 activity by assessing growth on YPD plus MM. Similar to wild-type Stp1, the RI15-35 and RI17-33 proteins were expressed as latent forms and did not facilitate growth (Figure 4B, compare dilutions 3 and 5 with dilution 1), indicating that amino acid residues RI comprising amino acids 15–35 or 17–33 suffice in isolation to confer latency to Stp1. However, in asi1Δ cells, the expression of RI15-35 and RI17-33 supported a more robust level of resistance to MM than that resulting from expression of wild-type Stp1 (Figure 4B, compare dilutions 4 and 6 with dilution 2). Consistent with our previous data, deletion of aa 17–21 creating the RI22-35 construct resulted in a constitutively active protein; robust growth on YPD plus MM was observed in ssy5Δ stp1Δ stp2Δ cells, and asi1Δ had no additional negative effect on latency (Figure 4B, dilutions 7 and 8).
Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden.