Sumoylation is Largely Dispensable for Normal Growth but Facilitates Heat Tolerance in Yeast

Abstract

Numerous proteins are sumoylated in normally growing yeast and SUMO conjugation levels rise upon exposure to several stress conditions. We observe high levels of sumoylation also during early exponential growth and when nutrient-rich medium is used. However, we find that reduced sumoylation (∼75% less than normal) is remarkably well-tolerated, with no apparent growth defects under nonstress conditions or under osmotic, oxidative, or ethanol stresses. In contrast, strains with reduced activity of Ubc9, the sole SUMO conjugase, are temperature-sensitive, implicating sumoylation in the heat stress response, specifically. Aligned with this, a mild heat shock triggers increased sumoylation which requires functional levels of Ubc9, but likely also depends on decreased desumoylation, since heat shock reduces protein levels of Ulp1, the major SUMO protease. Furthermore, we find that a ubc9 mutant strain with only ∼5% of normal sumoylation levels shows a modest growth defect, has abnormal genomic distribution of RNA polymerase II (RNAPII), and displays a greatly expanded redistribution of RNAPII after heat shock. Together, our data implies that SUMO conjugations are largely dispensable under normal conditions, but a threshold level of Ubc9 activity is needed to maintain transcriptional control and to modulate the redistribution of RNAPII and promote survival when temperatures rise.

Keywords: Ubc9; Ulp1; heat shock; sumoylation; transcription; yeast.

FIG 1

Many proteins are dynamically sumoylated in nonstressed yeast. (A) W303a and 8HIS-Smt3 yeast strains were grown in SC medium at the standard temperature (30 °C) and cell lysates were prepared under nondenaturing conditions, in the presence of NEM, except where indicated. Lysates were analyzed by SUMO, GAPDH, and Ubc9 immunoblots under conditions that facilitate detection of smaller proteins (see Experimental Procedures). The positions of SUMO conjugates, Ubc9-SUMO (“Ubc9-S”), unconjugated SUMO (“Free SUMO”), and unmodified Ubc9 are indicated. (B) Lysates from W303a and 8HIS-Smt3 strains were prepared in sample buffer containing the indicated concentrations of β-mercaptoethanol (β-me), then analyzed by PAGE and SUMO and GAPDH immunoblots. (C) Aliquots of a W303a culture grown in SC medium at 30 °C were collected at six intervals over 15 h and protein extracts, approximately normalized by cell numbers, were prepared and analyzed by immunoblot with antibodies for SUMO, GAPDH, ribosomal protein RPL3, and RNAPII subunit Rpb3. The culture density (cells/mL) at each time point is indicated at top and depicted in the curve below. The inset at the right of the SUMO immunoblot (lane 7; “1.0 norm.”) is from lysate of a culture grown to ∼1.0 × 10⁷ cells/mL in which the exposure (signal intensity) of the SUMO immunoblot was adjusted to approximately match the signal level of the sample in lane 6. (D) The indicated common lab yeast strains, including haploid and diploid W303 (“W303a” and “W303 dip.,” respectively), were grown in either SC or YPD medium to exponential phase, then protein extracts were generated and analyzed by SUMO, Ubc9, and GAPDH immunoblots.

FIG 2

Yeast display very high tolerance for reduced sumoylation levels in nonstress growth conditions. (A) Cultures of the Ubc9 anchor away strain, Ubc9-AA, and its parent, Parent-AA, were left untreated or treated with rapamycin (“+Rap.”) for 30 min, then extracts were prepared and analyzed by SUMO and GAPDH immunoblots. SUMO conjugation levels were quantified by densitometry and the average and standard deviations (error bars) from three experiments are plotted at right. (B) Lysates were prepared from cultures of W303a, Parent-AA, Ubc9-AA, or from an unrelated control anchor away strain, Rpb1-AA, and analyzed by immunoblots with antibodies that recognize Ubc9 or a subunit of RNA Polymerase II, Rpb3, which serves as a loading control. (C) Chromatin fractionation was performed with the Ubc9-AA strain, and whole cell extract (WCE), soluble (i.e., nonchromatin), and chromatin fractions (Chrom.) were analyzed by immunoblots with antibodies for SUMO, GAPDH, which is predominantly cytoplasmic, and H3, which is chromatin-bound. (D) The Ubc9-AA strain was grown in the indicated conditions (see Experimental Procedures for details), then lysates were prepared and analyzed by SUMO and GAPDH immunoblot. I A spot assay was performed with the Ubc9-AA and Parent-AA strains, on either SC medium, or SC supplemented with rapamycin. Plates were imaged after 2 days of growth. (F) Cultures of the Ubc9-AA and Parent-AA strains were prepared in SC medium, with or without rapamycin, at an absorbance (595 nm) of ∼0.2, then grown for 20 h while absorbance (culture density) measurements were made every 15 min. The two Parent-AA curves are virtually indistinguishable and are therefore marked with “overlap.” Triplicate cultures were grown for each sample, and the average values were plotted to create the growth curves shown. Values for average absorbance measurements and standard deviations for each triplicate set are listed in Table S5.

FIG 3

Yeast strains with constitutively low levels of Ubc9 activity cannot survive elevated temperatures. (A) Protein extracts were prepared from cultures of the indicated strains grown in untreated SC medium and analyzed by SUMO and GAPDH immunoblots. (B) Spot assays were performed with the indicated strains, onto either SC medium, or SC supplemented with the indicated stressors (see Experimental Procedures) and incubated at the standard growth temperature of 30 °C, or at the elevated temperature of 37 °C. Lower panel shows spot assay for a series of mutant strains, grouped with their respective wild-type parent strains, as positive controls for the various stress conditions. (C) Growth curves were generated, as in Fig. 2F, for the indicated strains and conditions. Curves that overlap significantly and are indistinguishable are marked with “overlap.” Individual graphs include two curves except for the Parent-AA and Ubc9-AA set at 37 °C, which includes untreated and rapamycin-treated samples for both strains. Graph data are in Table S5.

FIG 4

Induced reduction of Ubc9 levels also sensitizes yeast to high temperatures. (A) Cultures of a strain expressing the UBC9 gene from a tetracycline-repressible promoter, Ubc9-TO, and its isogenic parent strain, Parent-TO, were exposed to the indicated concentrations of the tetracycline analog doxycycline for several hours, then lysates were prepared and examined by SUMO, Ubc9, and H3 immunoblots. (B) Growth curves were generated, as in Fig. 2F, for the Ubc9-TO and Parent-TO strains grown in the absence or presence of doxycycline at the indicated temperatures. (C) Spot assays were performed with the Ubc9-TO and Parent-TO strains, onto either SC medium, or SC supplemented with doxycycline. Plates were incubated at the indicated temperatures and were imaged after 2 days of growth. As none of the strains grow in the presence of doxycycline at 39.5 °C on solid medium, a temperature of 38.5 °C was used as the highest temperature for this experiment. (D) A spot assay was performed comparing growth of the indicated background and parental yeast strains at 30 °C, 37 °C, or 39.5 °C, for the indicated number of days.

FIG 5

Mild heat shock elevates global sumoylation levels and reduces Ulp1 protein abundance. (A) Lysates were prepared from W303a yeast cultures that were heat shocked at 37 °C for the indicated durations, followed by SUMO and GAPDH immunoblots. Quantification of sumoylation levels of three independent analyses was performed, with average sumoylation levels graphed and standard deviations shown. Student’s t test analysis indicated that the 30- and 60-min time-point samples are significantly elevated compared to the 0-min control (P < 0.05; asterisks). (B) Cultures of the Ubc9-AA, ubc9-6, and ubc9-1 strains, and their respective parental strains, were heat-shocked at 37 °C for 30 min (+) or untreated (−). Lysates were prepared and analyzed by SUMO, Ubc9, and GAPDH immunoblots. (C) Spot assays were performed with the ULP1 and ulp1-mt strains on SC medium at 30 °C or 37 °C, or under the indicate stress conditions, and imaged after 4 days of growth. (D) Cultures of the ulp1-mt strain, its isogenic parental strain (ULP1), and the wild-type lab strain BY4741 were heat-shocked or untreated, then analyzed by SUMO and GAPDH immunoblots. The analysis was also performed using MG132-treated ulp1-mt cultures (inset; see Experimental Procedures). Quantification is shown at right with average relative sumoylation levels graphed with standard deviations. Student’s t test analysis indicates significantly elevated sumoylation levels in the wild-type strains after heat shock, and a significant but modest reduction in the ulp1-mt strain (asterisks). (E) Cultures of strains expressing HA-tagged versions of Ulp1 or Ulp2, or their parental untagged strain, YPH499, were heat-shocked or untreated. A strain expressing an HA-tagged version of an unrelated protein, Sko1, was used as a control. Lysates were analyzed by HA and GAPDH immunoblots. Quantification of HA-tagged protein levels is shown at right. Student’s t tests support that heat shock significantly reduces Ulp1-HA levels (asterisk) but does not affect Ulp2-HA or Sko1-HA levels. (F) HA and GAPDH immunoblots of extracts prepared using denaturing conditions (TCA precipitation; see Experimental Procedures) from the Ulp1-HA-expressing strain. (G) Cultures of the Ulp1-HA strain were treated with MG132 (+) or DMSO (−), as indicated in Experimental Procedures, then heat-shocked or left at 30 °C. Lysates were prepared and analyzed by HA and GAPDH immunoblots. (H) Cultures of the ULP1 or ulp1-mt strains were grown and aliquots were collected at the indicated culture densities. Lysates were prepared and analyzed by SUMO and GAPDH immunoblots. (I) Aliquots of a Ulp1-HA culture were collected at the indicated culture densities, then lysates were prepared and analyzed by HA and GAPDH immunoblots.

FIG 6

Constitutively reduced sumoylation alters gene expression patterns and expands the heat shock-induced genomic redistribution of RNAPII. (A) Scatterplot comparing RNAPII levels at all protein-coding genes in WT and ubc9-6 strains, as determined by our previously reported ChIP-seq (see Table S1). (B) GO term analysis was performed on genes showing elevated or reduced RNAPII levels across their ORFs in ubc9-6 (see Experimental Procedures and Table S2 for details). (C) Comparison of global steady-state mRNA levels in ubc9-6 and WT strains as determined by RNA-seq (see Tables 2 and S3). (D) Scatterplot comparing ubc9-6-mediated changes in mRNA abundance and RNAPII occupancy for all protein-coding genes based on the RNA-seq and RNAPII ChIP-seq analyses. Genes showing significantly altered levels of both RNAPII occupancy and mRNA abundance are shown in red or blue, as indicated (see Table S4). The four quadrants (Qd1 to Qd4) refer to subsets of genes that show correlated or anticorrelated effects in the RNA-seq and RNAPII ChIP-seq studies (see Table S4). (E) Scatterplots comparing heat shock-induced changes to RNAPII occupancy levels across protein-coding genes in WT and ubc9-6 strains. RNAPII ChIP-seq was performed in both strains in untreated (NT) or heat-shocked cultures (HS; 37 °C for 12 min) as previously described. Genes showing significantly elevated or reduced RNAPII occupancy in the heat shock samples are shown in red or blue, respectively (see Table S1). (F) Doughnut plots showing the proportion of protein-coding genes that display altered RNAPII levels after heat shock in WT and ubc9-6 strains. (G) Venn diagram comparing the number of genes showing significantly reduced or elevated RNAPII levels after heat shock in the WT and ubc9-6 strains. The number of genes in each set is shown above the Venn diagram, whereas the numbers of genes that overlap different sets is indicated on the diagram itself.