Deletions of the E3 ligase Not4, and the deadenylase subunits Ccr4 and Caf1, have different phenotypes.

<p>The indicated strains were grown to exponential phase and diluted to the same OD₆₀₀ of 0.5. 10-fold serial dilutions were spotted on the YPD plates containing, when indicated, HygB 0.1 mg/ml; CHX 0.05 µg/ml; AZC 0.5 mg/ml, and left to grow for 4 days (A, except 16°C), for 17 days (A, 16°C) or for 6 days (B).</p

The deletion of Not4, but not the deletion of the deadenylase, stabilizes the proteasomal substrate CPY*.

<p><b>A.</b> CPY*-HA was expressed from an episome under control of a copper dependent promoter in wild-type (WT) and <i>not4Δ</i> cells. Cells were exponentially grown without induction to OD₆₀₀ of 0.6 (time 0). 0.1 mM CuSO₄ was added to the media and cells were collected at indicated time points (2, 4 and 11 h) and analyzed by SDS-PAGE and western blot with antibodies against HA, to see CPY*-HA levels, and against Egd2 as a loading control. The positions of CPY*-HA and ubiquitinated CPY*-HA (CPY*-HA-Ub) are indicated on the right. The molecular weight markers are indicated on the left. <b>B.</b> Stability of CPY*-HA was analyzed in wild-type, <i>caf1Δ</i>, <i>ccr4Δ</i>, and <i>not4Δ</i> cells. Cells were grown exponentially and treated (+CHX) or not (−CHX) with CHX. Samples were collected at indicated time points and analyzed as in A. Since CPY*-HA expression was different in mutant strains (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086218#pone.0086218.s002" target="_blank">Fig. S2A</a>) 2 times less material was loaded on the gel in the case of <i>not4Δ</i> samples compared to wild type, and 4 times less material was loaded on the gel in the case of the <i>ccr4Δ</i> and <i>caf1Δ</i> samples compared to wild type. <b>C.</b> Stability of CPY*-HA was analyzed in <i>ubr1Δ</i>, <i>ubr2Δ</i>, <i>ltn1Δ</i>, and <i>san1Δ</i> cells as in B. <b>D.</b> Stability of CPY*-HA was analyzed in <i>cim3-1</i> and <i>pre1-1</i> cells as in B.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Proteasome was defective in <i>not4Δ</i> cells but not in <i>ccr4Δ</i> cells.

<p><b>A.</b> Total cellular extracts were prepared from wild-type, <i>caf1Δ</i>, <i>ccr4Δ</i>, and <i>not4Δ</i> cells and loaded on 3.5% native gels. After electrophoresis gels were incubated with Suc-LLVY-AMC to analyze the proteasome activity in the absence (-SDS) and then in the presence (+SDS) of 0.02% SDS to detect the latent CP activity. The positions of double (RP₂-CP) and single (RP₁-CP) capped proteasomes and CP alone are indicated on the left. <b>B.</b> RPs were purified from wild-type, <i>caf1Δ</i>, <i>ccr4Δ</i>, and <i>not4Δ</i> cells, loaded on a gradient 3–12% native gel and then analyzed for activity (upper panel). The same purified material was analyzed by SDS-PAGE and western blot with antibodies against the RP subunit (Rpt1) and with antibodies against CP subunits (α1-7) (lower panel).</p

Not4 accumulates in polysomes in response to AZC and high temperature.

<p><b>A.</b> Polysome profiles from the cells expressing Not4-ProteinA. Cells were exponentially grown on YPD media at 30°C or 37°C, as indicated, and collected at OD₆₀₀ of 1.0. When indicated, cells were treated with 0.4 mg/ml of AZC. AZC was added at OD₆₀₀ of 0.15 and cells were grown till OD₆₀₀ of 1.0 and collected. Extracts, containing 3 mg of total proteins, were subjected to 7–47% sucrose gradient centrifugation and analyzed by UV reading at 254 nm. Fraction numbers and the positions of 40S, 60S, 80S, and polysomes are indicated. <b>B.</b> Fractions were collected and analyzed by western blot with PAP and Rpl35 antibodies. <b>C.</b> Not4 content in polysomes was quantified. For this the Not4 signal in polysomes (fraction 10) was quantified with ImageQuant TL software (GE Healthcare) and normalized on the Rpl35 signal.</p

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

<div><p>Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome.</p></div

The Not4 deletion caused accumulation of aggregated and polyubiquitinated newly synthesized proteins.

<p><b>A.</b> Aggregates were isolated from the indicated cells and analyzed by SDS-PAGE and Coomassie staining (upper panel), or western blot with antibodies against ubiquitin (middle panel), or against Ssa1, Ssb1, Egd2, Rpn8, and Rpl35 (lower panel). <b>B.</b> Aggregates were isolated from the same cells treated with S³⁵-Met for 5 min and analyzed by SDS-PAGE and radioisotope imaging (upper panel). Images were quantified (lower panel). “au” is a ratio of the signal observed in the aggregates to the signal observed in the total protein fraction.</p

Assembly of Rpb1 with the R2TP Hsp90 co-chaperone is reduced and Rpb1 aggregates in <i>not5Δ</i>.

<p><b>A.</b> Total extracts and protein aggregates from the indicated strains were analyzed on SDS-PAGE followed by western blotting with antibodies against Rpb1. <b>B</b>. Rvb1-TT or Rvb2-TT were purified from wild-type and <i>not5Δ</i> cells. Equal amounts of the total extract (Input), and the purified proteins (Purified Rvb1 or 2) were analyzed by western blotting with anti-CBP or anti-Rpb1 antibodies. <b>C</b>. Rpb1 was immunoprecipitated from wild-type or <i>not5Δ</i> and the level of Rpb1 and Hsp90 proteins in the total extract (Input) immunoprecipitate (Ip) was evaluated by western blotting. <b>D</b>. Total extracts (TE) or protein aggregates (A) from wild-type cells (WT) or <i>not5Δ</i> cells or from <i>not5Δ</i> cells expressing NLS-Not5 as indicated were separated on SDS-PAGE and tested by western blotting for the levels of Hsp90 or Rpb1. <b>E</b>. Total extracts from wild-type or <i>not5Δ</i> cells grown in galactose expressing Rpb11-TT or not, and expressing or not NLS-Not5 or LexA-Rpb4, as indicated, were separated on native gels and analyzed by western blotting with anti- CBP antibodies (left panel). The same extracts were separated on sucrose gradients and the polysome profiles are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004569#pgen.1004569.s001" target="_blank">Fig. S1A</a>, whereas the distribution of LexA-Rpb4 along the sucrose gradient is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004569#pgen.1004569.s001" target="_blank">Fig. S1B</a>. Total extracts from <i>not5Δ</i> cells expressing Rpb11-TT and expressing either Myc-Not5 (Not5) or Myc-Not5-NES (NES) from episomes were separated by Native-PAGE and analyzed by western blotting with anti-CBP antibodies (upper panel) or by SDS-PAGE and analyzed by western blotting with anti-Rpb1 antibodies (lower panel). (<b>F</b>). Total extracts from wild-type or <i>not5Δ</i> cells expressing Rvb1-TT, Rvb2-TT or Hsp82-TT were analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004569#pgen-1004569-g001" target="_blank">Fig. 1C</a>. The polysome profiles for these experiments are available in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004569#pgen.1004569.s015" target="_blank">Fig. S15</a>. <b>G</b>. Total extracts from WT, <i>not5Δ</i> or <i>rpb4Δ</i> cells expressing Rpb9-TT from cells were separated on native gels and analyzed by western blotting with anti-CBP antibodies.</p

Polymerase sub-complexes lacking Rpb1 accumulate in <i>not5Δ</i>.

<p><b>A</b> and <b>B</b>. Total extracts from cells expressing the indicated Tap-tagged (TT) polymerase subunits were separated on native gels (upper panels) or SDS-PAGE (lower panels) and analyzed by western blotting with anti- CBP antibodies. <b>C</b>. Rpb9-TT was purified by single step affinity and the purified proteins were analyzed on native gels (upper panels) or SDS-PAGE (lower panels) and western blotting with anti-CBP antibodies (left panel) or anti-Rpb1 antibodies (right panel). <b>D</b>. Total extracts from cells expressing Rpb11-TT were either untreated (-) or treated with DNase or RNase as indicated and separated by Native-PAGE, and analyzed by western blotting with PAP antibodies.</p

Presence of Rpb7 in polysomes or in the cytoplasm is not affected by Not5.

<p><b>A</b>. Wild-type or mutant cells expressing Tap-tagged Rpb7 as indicated were analyzed on sucrose gradients as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004569#pgen-1004569-g001" target="_blank">Fig. 1C</a>. The polysome profiles and protein loading for these experiments are available in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004569#pgen.1004569.s015" target="_blank">Fig. S15</a>. <b>B</b>. Wild-type and <i>not5Δ</i> cells expressing Rpb7-TT were grown exponentially and stained with anti-CBP antibodies or DAPI as for Fig. 1G. The merged pictures are displayed.</p