Proteasome granular localization is regulated through mitochondrial respiration and kinase signaling

In yeast, proteasomes are enriched in cell nuclei where they execute important cellular functions. Nutrient-stress can change this localization indicating proteasomes respond to the cell's metabolic state. However, the signals that connect these processes remain poorly understood. Carbon starvation triggers a reversible translocation of proteasomes to cytosolic condensates known as proteasome storage granules (PSGs). Surprisingly, we observed strongly reduced PSG levels when cells had active cellular respiration prior to starvation. This suggests the mitochondrial activity of cells is a determining factor in the response of proteasomes to carbon starvation. Consistent with this, upon inhibition of mitochondrial function we observed proteasomes relocalize to granules. These links between proteasomes and metabolism involve specific signaling pathways, as we identified a MAP kinase cascade that is critical to the formation of proteasome granules after respiratory growth but not following glycolytic growth. Furthermore, the yeast homolog of AMP kinase, Snf1, is important for proteasome granule formation induced by mitochondrial inhibitors, while dispensable for granule formation following carbon starvation. We propose a model where mitochondrial activity promotes proteasome nuclear localization.

Structure of the human ATM kinase and mechanism of Nbs1 binding

DNA double-strand breaks (DSBs) can lead to mutations, chromosomal rearrangements, genome instability, and ultimately cancer. Central to the sensing of DSBs are ATM (Ataxia telangiectasia mutated) kinase, which belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, and the MRN (Mre11-Rad50-Nbs1) protein complex that activates ATM. How the MRN complex recruits and activates ATM kinase is poorly understood. Previous studies indicate that the FxF/Y motif of Nbs1 directly binds to ATM kinase, and is required to retain active ATM at sites of DNA damage. Here, we report the 2.5 Å resolution cryo-EM structures of human ATM and its complex with the Nbs1 FxF/Y motif. In keeping with previous structures of ATM and its yeast homolog Tel1, the dimeric human ATM kinase adopts a symmetric, butterfly-shaped autoinhibited structure. The conformation of the ATM kinase domain is most similar to the inactive states of other PIKKs, suggesting that activation may involve an analogous realigning the N and C lobes along with relieving the blockage of the substrate-binding site. We show that the Nbs1 FxF/Y motif binds to a conserved hydrophobic cleft within the Spiral domain of ATM, suggesting an allosteric mechanism of activation. We evaluate the importance of these interactions with mutagenesis and biochemical assays.

Structure and transport mechanism of P5B-ATPases

In human cells, P5B-ATPases execute the active export of physiologically important polyamines such as spermine from lysosomes to the cytosol, a function linked to a palette of disorders. Yet, the overall shape of P5B-ATPases and the mechanisms of polyamine recognition, uptake and transport remain elusive. Here we describe a series of cryo-electron microscopy structures of a yeast homolog of human ATP13A2-5, Ypk9, determined at resolutions reaching 3.4 Å, and depicting three separate transport cycle intermediates, including spermine-bound conformations. Surprisingly, in the absence of cargo, Ypk9 rests in a phosphorylated conformation auto-inhibited by the N-terminus. Spermine uptake is accomplished through an electronegative cleft lined by transmembrane segments 2, 4 and 6. Despite the dramatically different nature of the transported cargo, these findings pinpoint shared principles of transport and regulation among the evolutionary related P4-, P5A- and P5B-ATPases. The data also provide a framework for analysis of associated maladies, such as Parkinson’s disease.

Nutrient transceptors physically interact with the yeast S6/Protein kinase B homolog, Sch9, a TOR kinase target

Multiple starvation-induced, high-affinity nutrient transporters in yeast function as receptors for activation of the Protein Kinase A (PKA) pathway upon re-addition of their substrate. We now show that these transceptors may play more extended roles in nutrient regulation. The Gap1 amino acid, Mep2 ammonium, Pho84 phosphate and Sul1 sulfate transceptors physically interact in vitro and in vivo with the PKA-related Sch9 protein kinase, the yeast homolog of mammalian S6 Protein Kinase and Protein Kinase B. Sch9 is a phosphorylation target of TOR and well-known to affect nutrient-controlled cellular processes, such as growth rate. Mapping with peptide microarrays suggests specific interaction domains in Gap1 for Sch9 binding. Mutagenesis of the major domain affects the upstart of growth upon addition of L-citrulline to nitrogen-starved cells to different extents but apparently does not affect in vitro binding. It also does not correlate with the drop in L-citrulline uptake capacity or transceptor activation of the PKA target trehalase by the Gap1 mutant forms. Our results reveal a nutrient transceptor-Sch9-TOR axis in which Sch9 accessibility for phosphorylation by TOR may be affected by nutrient transceptor-Sch9 interaction under conditions of nutrient starvation or other environmental challenges.

Phosphorylated CtIP bridges DNA to promote annealing of broken ends

The early steps of DNA double-strand break (DSB) repair in human cells involve the MRE11-RAD50-NBS1 (MRN) complex and its cofactor, phosphorylated CtIP. The roles of these proteins in nucleolytic DSB resection are well characterized, but their role in bridging the DNA ends for efficient and correct repair is much less explored. Here we study the binding of phosphorylated CtIP, which promotes the endonuclease activity of MRN, to single long (∼50 kb) DNA molecules using nanofluidic channels and compare it to the yeast homolog Sae2. CtIP bridges DNA in a manner that depends on the oligomeric state of the protein, and truncated mutants demonstrate that the bridging depends on CtIP regions distinct from those that stimulate the nuclease activity of MRN. Sae2 is a much smaller protein than CtIP, and its bridging is significantly less efficient. Our results demonstrate that the nuclease cofactor and structural functions of CtIP may depend on the same protein population, which may be crucial for CtIP functions in both homologous recombination and microhomology-mediated end-joining.

EDF1 coordinates cellular responses to ribosome collisions

Translation of aberrant mRNAs induces ribosomal collisions, thereby triggering pathways for mRNA and nascent peptide degradation and ribosomal rescue. Here we use sucrose gradient fractionation combined with quantitative proteomics to systematically identify proteins associated with collided ribosomes. This approach identified Endothelial differentiation-related factor 1 (EDF1) as a novel protein recruited to collided ribosomes during translational distress. Cryo-electron microscopic analyses of EDF1 and its yeast homolog Mbf1 revealed a conserved 40S ribosomal subunit binding site at the mRNA entry channel near the collision interface. EDF1 recruits the translational repressors GIGYF2 and EIF4E2 to collided ribosomes to initiate a negative-feedback loop that prevents new ribosomes from translating defective mRNAs. Further, EDF1 regulates an immediate-early transcriptional response to ribosomal collisions. Our results uncover mechanisms through which EDF1 coordinates multiple responses of the ribosome-mediated quality control pathway and provide novel insights into the intersection of ribosome-mediated quality control with global transcriptional regulation.

Bro1 directly stimulates Vps4 activity to promote Intralumenal Vesicle Formation during Multivesicular Body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) form intralumenal vesicles (ILVs) during the conversion of endosomes to multivesicular bodies (MVBs). The assembly and disassembly of an ESCRT-III polymer facilitates membrane remodeling and scission during this process. The ESCRT-III-associated protein Bro1 (the yeast homolog of mammalian proteins ALIX and HD-PTP) promotes ESCRT-III assembly and inhibits disassembly of ESCRT-III filaments by impeding Vps4, a AAA-ATPase that dismantles ESCRT-III polymers. Here we show that the evolutionarily conserved “V domain” of Bro1-family proteins directly stimulate Vps4 ATPase activity and this activity is enhanced by interaction with ubiquitin. Surprisingly, a carboxyl-terminal fragment of Bro1 containing the V domain supports ILV formation but not sorting of cargo into ILVs, revealing that these two processes can be uncoupled. These studies implicate Bro1 as a factor coordinating cargo sorting with direct regulation of Vps4 to modulate ESCRT-III driven ILV formation during MVB biogenesis.

The disordered regions of SETD2 govern H3K36me3 deposition by regulating its proteasome-mediated decay

ABSTRACTSETD2 is the sole methyltransferase that tri-methylates histone H3 at lysine 36 in mammals. It has an extended N-terminal region which is absent in its yeast homolog Set2. The function of this poorly characterized region in regulating SETD2 stability has been reported. However, how this region regulates SETD2 half-life and the consequences of the cellular accumulation of SETD2 is unclear. Here we show that the SETD2 N-terminal region contains disordered regions and is targeted for degradation by the proteasome. The marked increase in global H3K36me3 that occurs on the removal of the N-terminal segment results in a non-canonical distribution including reduced enrichment over gene bodies and exons. An increased SETD2 abundance leads to widespread changes in transcription and alternative splicing. Thus, the regulation of SETD2 levels through intrinsically disordered region-facilitated proteolysis is important to maintain the fidelity of transcription and splicing related processes.

Yeast Genome Maintenance by the Multifunctional PIF1 DNA Helicase Family

The two PIF1 family helicases in Saccharomyces cerevisiae, Rrm3, and ScPif1, associate with thousands of sites throughout the genome where they perform overlapping and distinct roles in telomere length maintenance, replication through non-histone proteins and G4 structures, lagging strand replication, replication fork convergence, the repair of DNA double-strand break ends, and transposable element mobility. ScPif1 and its fission yeast homolog Pfh1 also localize to mitochondria where they protect mitochondrial genome integrity. In addition to yeast serving as a model system for the rapid functional evaluation of human Pif1 variants, yeast cells lacking Rrm3 have proven useful for elucidating the cellular response to replication fork pausing at endogenous sites. Here, we review the increasingly important cellular functions of the yeast PIF1 helicases in maintaining genome integrity, and highlight recent advances in our understanding of their roles in facilitating fork progression through replisome barriers, their functional interactions with DNA repair, and replication stress response pathways.