PMC

Model for the function of Fes1L and Fes1S. See Discussion for details.

Fes1L and Fes1S are isoforms with similar NEF activity. Basal and induced rates of ADP dissociation from Ssa1. Ssa1-MABA-ADP (0.25 μM) was rapidly mixed with ATP in a stopped-flow instrument in the presence or absence of Fes1L (1 μM) or Fes1S (1 μM).

Fes1S is a cytosolic protein, and Fes1L is targeted to the nucleus by a C-terminal NLS. (A) An NLS-like sequence in the C-terminus of Fes1L and the changes in amino acid sequence of the Fes1L mutant K/R → E. (B) Western analysis of C-terminally GFP-tagged Fes1S, Fes1L, and K/R → E expressed from plasmids with the endogenous FES1 promoter in fes1Δ cells. (C) Fluorescence microscopy localization of the GFP fusions in B. The nucleus is labeled with mCherry-tagged Histone 2B (Htb2). Scale bar, 10 μM.

Fes1S is essential for ubiquitin-proteasome system–dependent degradation of misfolded proteins. (A) Western analysis of the turnover of the misfolded model protein Rpo41* after the addition of cycloheximide (CHX). Strains as in . Error bars represent SD (N = 3). (B) Western analysis as in A but of misfolded model protein Ste6* C. (C) Western analysis as in A but of the turnover of the nuclear-targeted misfolded model protein ΔssPrA. (D) Cell suspensions of the strains expressing empty vector control (VC) or Ste6* C or ΔssPrA were serially diluted 10-fold and spotted onto selective medium. Photographs were taken after 3 d of incubation at 25°C. Bottom, Western analysis of the steady-state expression of ΔssPrA.

Temperature tolerance requires Fes1S but not Fes1L activity. (A) Schematic representation of the chromosomal modifications made in the WT FES1 locus to obtain the following strains: no expression of Fes1L or Fes1S (fes1Δ), only expression of Fes1S (fes1ΔL), only expression of Fes1L (fes1ΔS), and only expression of Fes1L but at higher than WT levels (fes1ΔS,L↑). (B) Western analysis of the expression of Fes1L and Fes1S in the strains in A. Quantification of Fes1 isoform expression levels (bottom). Error bar indicates SD (N = 3). (C) Analysis of the fes1 temperature-sensitive growth phenotype of the strains in A. Cell suspensions were 10-fold serially diluted, spotted onto solid SC or SC supplemented with 1 mg/l l-canavanine (Can) medium, and incubated at the indicated temperature.

Fes1S keeps the heat shock response. (A) Heat map representing the relative expression of 157 differentially expressed genes over triplicate RNA-Seq samples from WT, fes1Δ, fes1ΔL, and fes1ΔS strains. The dendrogram displays how the samples cluster based on the differential expression. (B) Density plots that display the expression of all 5656 genes (blue) and 78 heat shock–regulated genes (red) in fes1Δ, fes1ΔL, and fes1ΔS relative to WT cells. (C) Relative β-galactosidase activity expressed from the heat shock–responsive reporter P_HSP104-LacZ. Error bars indicate the SD of three biological replicates. t test relative to WT: *p ≤ 0.05, ***p ≤ 0.001.

Heat shock–regulated alternative splicing of FES1 transcripts results in the expression of Fes1L and Fes1S. (A) The heat shock element–promoted (HSE) FES1 locus contains sequence elements that encode two exons (I and II) separated by an intron (dashed line). Use of the promoter-proximal site of polyadenylation in the intron (▼) supports the expression of Fes1S from a transcript that encompasses exon I. Alternative splicing removes the promoter- proximal polyadenylation site in the intron and supports expression of Fes1L from exons I and II. (B) RT-PCR analysis of transcripts that encompass exon I and II, using primers F and R as indicated in A. The longer transcript (Pre) contains the intron, and the shorter transcript is splicing (Spliced) and supports the expression of Fes1L. (C) Western analysis of protein extracts from WT and fes1Δ with rabbit serum raised against recombinant Fes1S. Both Fes1S (32.6 kDa) and Fes1L (34.2 kDa) are expressed from the endogenous FES1 locus. Parallel blotting against Pgk1 functions as a loading control. (D) Western analysis as in C of fes1Δ cells transformed with vector control (VC) or derivatives that carry either the WT FES1 locus or a version with a 9.4- kDa 6×HA (HA) fused in-frame to the 3′ end of exon II. (E) Western analysis as in C of WT and splicing-impaired mutants prp18Δ and mud2Δ. (F) Western analysis as in C of fes1Δ cells transformed with VC or derivatives that carry either WT FES1 locus or a derivative with a T-to-C substitution mutation at the polyadenylation site indicated in A. (G) Quantification of FES1 transcript variants from Illumina 1G Analyzer reads under growth of WT yeast at 22°C in YPD medium and after transient heat shock (HS); 37°C, 15 min. Data are presented as reads per lane per kilobase for mRNA. Error bars indicate SD of data from two biological replicates. (H) Quantification of Fes1L and Fes1S expression levels using Western analysis under the same conditions as in G. Error bars indicate SD (N = 3). *p < 0.05, **p < 0.01, ns > 0.05.