The Yeast Ess1 Prolyl Isomerase Controls Swi6 and Whi5 Nuclear Localization

Abstract

The Ess1 prolyl isomerase from Saccharomyces cerevisiae and its human ortholog, Pin1, play critical roles in transcription by regulating RNA polymerase II. In human cells, Pin1 also regulates a variety of signaling proteins, and Pin1 misexpression is linked to several human diseases. To gain insight into Ess1/Pin1 function, we carried out a synthetic genetic array screen to identify novel targets of Ess1 in yeast. We identified potential targets of Ess1 in transcription, stress, and cell-cycle pathways. We focused on the cell-cycle regulators Swi6 and Whi5, both of which show highly regulated nucleocytoplasmic shuttling during the cell cycle. Surprisingly, Ess1 did not control their transcription but instead was necessary for their nuclear localization. Ess1 associated with Swi6 and Whi5 in vivo and bound directly to peptides corresponding to their nuclear localization sequences in vitro. Binding by Ess1 was significant only if the Swi6 and Whi5 peptides were phosphorylated at Ser-Pro motifs, the target sites of cyclin-dependent kinases. On the basis of these results, we propose a model in which Ess1 induces a conformational switch (cis-trans isomerization) at phospho-Ser-Pro sites within the nuclear targeting sequences of Swi6 and Whi5. This switch would promote nuclear entry and/or retention during late M and G1 phases and might work by stimulating dephosphorylation at these sites by the Cdc14 phosphatase. This is the first study to identify targets of Ess1 in yeast other than RNA polymerase II.

The Ess1 prolyl isomerase: Traffic cop of the RNA polymerase II transcription cycle

“…Binding to proline-rich peptides or proteins can be measured using a variety of general techniques including filter immunoblots [84], two-hybrid analysis [85], GST-pulldown [86], fluorescence anisotropy [68], circular dichroism (CD) [87], NMR [88], and most recently by biolayer interferometry (BLI) [89]. Only apparent dissociation constants (K app ), however, can be determined for intact Ess1/Pin1 proteins because of the dual-binding mode.…”

“…Using a different method (BLI), the K app of intact Ess1 for a Ser5 phosphorylated peptide was estimated at 2.1–2.6 μM [89]. Binding affinities of the isolated WW-domain of Ess1 were measured using CD to be ∼70 μM for pSer2 peptides, 80–100 μM for pSer5 peptides and ∼20 μM for doubly-phosphorylated peptides [87].…”

“…In a large-scale synthetic genetic array screen, Ess1 was linked to a number of transcription regulators including the cell cycle transcription factors Swi6 and Whi5 [89]. Surprisingly, the expression of the genes encoding these proteins was not affected.…”

“…Instead their nuclear-cytoplasmic shuttling was defective in ess1 mutant cells. Using GFP-tagged proteins and in vitro binding assays, it was shown that Ess1 is required for nuclear localization of Swi6 and Whi5, and that Ess1 binds peptides corresponding to the nuclear localization sequence (NLS) of Swi6, and the NLS and nuclear export sequences (NES) of Whi5 [89]. The NLS and NES sequences each contain between one and three Ser-Pro motifs, and Ess1 binding as assayed using biolayer interferometry, was dependent upon phosphorylation of these serines.…”

Prolyl isomerases in gene transcription

“…In yeast, genetic studies linked the Ess1 isomerase to cell cycle transcription factors Swi6 and Whi5 [70], whose nuclear-cytoplasmic shuttling is regulated by phosphorylation [71–74]. Ess1 is required for nuclear localization of Swi6 and Whi5, and Ess1 binds specifically to phosphorylated peptides corresponding to the NLS of Swi6, and to the NLS and nuclear export sequences (NES) of Whi5 [70], which contain between one and three Ser-Pro binding motifs.…”