Novel E3 Ubiquitin Ligases That Regulate Histone Protein Levels in the Budding Yeast Saccharomyces cerevisiae

Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus) domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene) finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs) YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases) respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3 ubiquitin ligases to ensure excess histone degradation and thus contribute to the maintenance of genomic stability

The Cdc48 Complex Alleviates the Cytotoxicity of Misfolded Proteins by Regulating Ubiquitin Homeostasis

The accumulation of misfolded proteins is associated with multiple neurodegenerative disorders, but it remains poorly defined how this accumulation causes cytotoxicity. Here, we demonstrate that the Cdc48/p97 segregase machinery drives the clearance of ubiquitinated model misfolded protein Huntingtin (Htt103QP) and limits its aggregation. Nuclear ubiquitin ligase San1 acts upstream of Cdc48 to ubiquitinate Htt103QP. Unexpectedly, deletion of SAN1 and/or its cytosolic counterpart UBR1 rescues the toxicity associated with Cdc48 deficiency, suggesting that ubiquitin depletion, rather than compromised proteolysis of misfolded proteins, causes the growth defect in cells with Cdc48 deficiency. Indeed, Cdc48 deficiency leads to elevated protein ubiquitination levels and decreased free ubiquitin, which depends on San1/Ubr1. Furthermore, enhancing free ubiquitin levels rescues the toxicity in various Cdc48 pathway mutants and restores normal turnover of a known Cdc48-independent substrate. Our work highlights a previously unappreciated function for Cdc48 in ensuring the regeneration of monoubiquitin that is critical for normal cellular function

Yeast Fin1-PP1 dephosphorylates an Ipl1 substrate, Ndc80, to remove Bub1-Bub3 checkpoint proteins from the kinetochore during anaphase.

The spindle assembly checkpoint (SAC) prevents anaphase onset in response to chromosome attachment defects, and SAC silencing is essential for anaphase onset. Following anaphase onset, activated Cdc14 phosphatase dephosphorylates the substrates of cyclin-dependent kinase to facilitate anaphase progression and mitotic exit. In budding yeast, Cdc14 dephosphorylates Fin1, a regulatory subunit of protein phosphatase 1 (PP1), to enable kinetochore localization of Fin1-PP1. We previously showed that kinetochore-localized Fin1-PP1 promotes the removal of the SAC protein Bub1 from the kinetochore during anaphase. We report here that Fin1-PP1 also promotes kinetochore removal of Bub3, the Bub1 partner, but has no effect on another SAC protein Mad1. Moreover, the kinetochore localization of Bub1-Bub3 during anaphase requires Aurora B/Ipl1 kinase activity. We further showed that Fin1-PP1 facilitates the dephosphorylation of kinetochore protein Ndc80, a known Ipl1 substrate. This dephosphorylation reduces kinetochore association of Bub1-Bub3 during anaphase. In addition, we found that untimely Ndc80 dephosphorylation causes viability loss in response to tensionless chromosome attachments. These results suggest that timely localization of Fin1-PP1 to the kinetochore controls the functional window of SAC and is therefore critical for faithful chromosome segregation

Neurodegenerative disease-associated inclusion bodies are cleared by selective autophagy in budding yeast

Protein misfolding, aggregation, and accumulation cause neurodegenerative disorders. One such disorder, Huntington’s disease, is caused by an increased number of glutamine-encoding trinucleotide repeats CAG in the first exon of the huntingtin (HTT) gene. Mutant proteins of Htt exon 1 with polyglutamine expansion are prone to aggregation and form pathological inclusion bodies in neurons. Extensive studies have shown that misfolded proteins are cleared by the ubiquitin-proteasome system or autophagy to alleviate their cytotoxicity. Misfolded proteins can form small soluble aggregates or large insoluble inclusion bodies. Previous works have elucidated the role of autophagy in the clearance of misfolded protein aggregates, but autophagic clearance of inclusion bodies remains poorly characterised. Here we use mutant Htt exon 1 with 103 polyglutamine (Htt103QP) as a model substrate to study the autophagic clearance of inclusion bodies in budding yeast. We found that the core autophagy-related proteins were required for Htt103QP inclusion body autophagy. Moreover, our evidence indicates that the autophagy of Htt103QP inclusion bodies is selective. Interestingly, Cue5/Tollip, a known autophagy receptor for aggrephagy, is dispensable for this inclusion body autophagy. From the known selective autophagy receptors in budding yeast, we identified three that are essential for inclusion body autophagy. Amyloid beta peptide (Aβ42) is a major component of amyloid plaques found in Alzheimer’s disease brains. Interestingly, a similar selective autophagy pathway contributes to the clearance of Aβ42 inclusion bodies in budding yeast. Therefore, our results reveal a novel autophagic pathway specific for inclusion bodies associated with neurodegenerative diseases, which we have termed IBophagy

The absence of specific yeast heat-shock proteins leads to abnormal aggregation and compromised autophagic clearance of mutant Huntingtin proteins

<div><p>The functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding and aggregation. Heat shock proteins (HSPs) function as molecular chaperones that aid in protein folding and the degradation of misfolded proteins. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (<i>HTT</i>) results in the expression of misfolded Huntingtin protein (Htt), which contributes to the development of Huntington’s disease. We previously found that the degradation of mutated Htt with polyQ expansion (Htt103QP) depends on both ubiquitin proteasome system and autophagy. However, the role of heat shock proteins in the clearance of mutated Htt remains poorly understood. Here, we report that cytosolic Hsp70 (Ssa family), its nucleotide exchange factors (Sse1 and Fes1), and a Hsp40 co-chaperone (Ydj1) are required for inclusion body formation of Htt103QP proteins and their clearance via autophagy. Extended induction of Htt103QP-GFP leads to the formation of a single inclusion body in wild-type yeast cells, but mutant cells lacking these HSPs exhibit increased number of Htt103QP aggregates. Most notably, we detected more aggregated forms of Htt103QP in <i>sse1</i>Δ mutant cells using an agarose gel assay. Increased protein aggregates are also observed in these HSP mutants even in the absence Htt103QP overexpression. Importantly, these HSPs are required for autophagy-mediated Htt103QP clearance, but are less critical for proteasome-dependent degradation. These findings suggest a chaperone network that facilitates inclusion body formation of misfolded proteins and the subsequent autophagic clearance.</p></div

The autophagic degradation of Htt103QP is compromised in <i>sse1</i>Δ and <i>fes1</i>Δ mutants.

<p>(A) The autophagic disposal of Htt103QP in WT and <i>sse1</i>Δ mutant cells. <i>pep4</i>Δ <i>VPH1-mApple</i> (WT) and <i>sse1</i>Δ <i>pep4</i>Δ <i>VPH1-mApple</i> (<i>sse1</i>Δ) cells containing Flag-Htt103QP-GFP were grown in galactose medium (YEPG) at 30°C for 16 hr. Glucose was then added to shut off Htt103QP expression, and 100 mM hydroxyurea (HU) was added to block cell cycle progression. Representative fluorescent images are shown for the localization of Htt103QP-GFP and Vph1-mApple before and after addition of glucose and HU. Vph1-mApple was used to mark vacuole structure. Pep4 is a vacuolar protease, and <i>pep4</i>Δ mutant was used to slow degradation inside the vacuole to enhance visualization. (B) Htt103QP autophagic disposal in WT and <i>fes1</i>Δ mutant cells. A similar protocol was used to examine the vacuolar localization of Htt103QP-GFP. (C and D) Quantitation of cells with Htt103QP IB or aggregates localized outside the vacuole over time. The cells from (A) and (B) are used for the quantitation. The quantified results (three independent repeats) were obtained by counting the number of cells containing at least one IB or aggregate (n = 100). All microscopy in this figure was performed on the EVOS microscope.</p

Cytosolic Hsp70 nucleotide exchange factors Sse1 and Fes1 are required for Htt103QP inclusion body (IB) formation.

<p>(A) Htt103QP overexpression and the growth of WT and <i>sse1</i>Δ cells. Cells containing <i>P</i>_<i>GAL</i><i>Flag-Htt103QP-GFP</i> were grown to saturation, 10-fold diluted, and spotted onto glucose (YPD) or galactose (YEPG) (yeast extract peptone and galactose) plates. The plates were incubated at 30°C for 2 days. (B) Confocal DIC and fluorescent images showing Htt103QP IB formation. WT and <i>sse1</i>Δ cells with <i>P</i>_<i>GAL</i><i>Flag-Htt103QP-GFP</i> were incubated at 30°C in YEPG medium for 16 hrs to induce Htt103QP-GFP expression. The cells were fixed by paraformaldehyde for 5 min and then resuspended in PBS buffer for confocal microscopy. Scale bar = 5μm. <b>(C)</b> 2.5-D analysis showing the intensity of fluorescent GFP peaks of the images from (B). Software used was Zen Blue from Zeiss. (D) Htt103QP IB formation in yeast mutants lacking Hsp70 nucleotide exchange factors (NEFs). Cells with the indicated genotypes were grown and treated as described above and then examined using an EVOS fluorescence microscope. Representative fluorescent images are shown for WT, <i>snl1</i>Δ, <i>sse1</i>Δ, <i>sse2</i>Δ, <i>fes1</i>Δ, and <i>sse1</i>Δ <i>fes1</i>Δ cells expressing Htt103QP-GFP. Scale bar = 5μm. (E) Cells from (D) were quantified for the number of aggregates in each cell: 1, 2 or >2 (n = 100 cells). The results are the average of three independent experiments. (F) The average number of aggregates in WT, <i>sse1</i>Δ, <i>fes1</i>Δ, and <i>sse1</i>Δ <i>fes1</i>Δ cells from (D) were quantified (n > 40 cells). The results are the average of three independent experiments. * indicates statistical comparison between WT and mutants. p < .0001 in all instances.</p

Hsp104-GFP positive aggregates are increased in <i>sse1</i>Δ and <i>ydj1-151</i> mutants.

<p>(A) Hsp104 recruitment to Htt103QP aggregates is independent of chaperones. Cell with indicated genotypes were grown in non-inducible raffinose medium to mid-log phase then galactose was added. Images were taken after incubation for 3 hr. Representative fluorescence images show cellular localization of Hsp104-GFP and Htt103QP-mApple. DIC and merged images are also shown. Scale bar = 5μm. (B) Constitutive appearance of Hsp104-GFP positive aggregates in <i>sse1</i>Δ and <i>ydj1-151</i> mutants. Cells with indicated genotypes were grown in raffinose or galactose containing medium to mid-log phase. Representative fluorescence images are shown. WT cells show predominantly nuclear localized Hsp104-GFP in the absence of Htt103QP overexpression. Scale bar = 5μm. (C) Cells from (B) were quantified based on the presence of Hsp104-GFP positive aggregate(s). Results are the average of three independent experiments (n = 100 cells). All images in this figure was obtained using the EVOS microscope.</p

Sse1 and Fes1 are required for efficient recognition of Htt103QP by autophagy machinery.

<p>(A) The co-localization of Htt103QP and Atg8 in WT, <i>sse1</i>Δ, and <i>fes1</i>Δ cells. Cells containing <i>GFP-ATG8</i> and <i>P</i>_<i>GAL</i><i>Flag-Htt103QP-mApple</i> were grown in galactose medium YEPG for 16 hrs and fixed for microscopy. Representative fluorescence images show the localization of Htt103QP-mApple and GFP-Atg8. Scale bar = 5μm. (B) Quantitation of total number of Htt103QP aggregates colocalized with GFP-Atg8 in WT, <i>sse1</i>Δ, and <i>fes1</i>Δ cells (n = 100 IB/aggregates). Quantities indicate the average of three independent experiments. * indicates statistical comparison. p < .001. (C) Quantitation of the number of GFP-Atg8 autophagosomes present in WT, <i>sse1</i>Δ and <i>fes1</i>Δ cells incubated in glucose or galactose medium (n = 100 cells). Quantities indicate the average of three independent experiments. (D) Sse1 and Fes1 are not required for starvation-induced autophagy. WT, <i>sse1</i>Δ, and <i>fes1</i>Δ cells with <i>GFP-ATG8</i> were grown in SC -TRP medium, and then shifted to nitrogen-deficient medium (SD-N) for 24 hr. The cells were collected to detect the presence of GFP-Atg8 and the cleavage product, monomeric GFP, using anti-GFP antibody. Pgk1: loading control. The EVOS microscope was used to obtain the images in this figure.</p

Hsp40 co-chaperone Ydj1 is required for efficient autophagic degradation of Htt103QP.

<p>(A) Htt103QP autophagy in WT and <i>ydj1-151</i> mutant cells. <i>pep4</i>Δ <i>VPH1-mApple</i> (WT) and <i>ydj1-151 pep4</i>Δ <i>VPH1-mApple</i> (<i>ydj1-151</i>) cells containing <i>P</i>_<i>GAL</i><i>Flag-Htt103QP-GFP</i> were grown in galactose medium (YEPG) at 25°C for 16 hrs. Cells were then shifted to semi-permissive temperatures (32°C) for 10 min prior to glucose and hydroxyurea (HU) addition. Representative fluorescent images are shown for the localization of Htt103QP-GFP and Vph1-mApple (vacuolar marker) before and after addition of glucose and HU. Scale bar = 5μm. (B) Quantitation of cells with Htt103QP IB or aggregates localized outside the vacuole over time. Results from three independent experiments were used for the quantitation. The quantified results were obtained by counting the number of cells containing at least one IB or aggregate (n = 100). (C) The co-localization of Htt103QP and Atg8 in WT and <i>ydj1-151</i> cells. Cells containing <i>GFP-ATG8</i> and <i>P</i>_<i>GAL</i><i>Flag-Htt103QP-mApple</i> were grown in galactose medium YEPG for 16 hrs at 32°C and fixed for microscopy. Representative fluorescence images show the localization of Htt103QP-mApple and GFP-Atg8. (D) Quantitation of total number of Htt103QP aggregates colocalized with GFP-Atg8 in WT and <i>ydj1-151</i> cells (n = 100 IB/aggregates). Quantities indicate the average of three independent experiments. * indicates statistical comparison. p < .005. (E) Quantitation of the number of GFP-Atg8 autophagosomes present in WT and <i>ydj1-151</i> cells incubated in glucose or galactose medium (n = 100 cells). Quantities indicate the average of three independent experiments. All images in this figure were acquired using the EVOS microscope.</p