Structural basis of exoribonuclease-mediated mRNA transcription termination.

Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II)1-5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.

Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

Proteasome condensate formation is driven by multivalent interactions with shuttle factors and ubiquitin chains.

Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1, show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant. We propose a model where the long K48-linked ubiquitin chains function as a scaffold for the ubiquitin-binding domains of the shuttle factors and the proteasome, allowing for the multivalent interactions that further drive condensate formation. Indeed, we determined different intrinsic ubiquitin receptors of the proteasome-Rpn1, Rpn10, and Rpn13-and the Ubl domains of Rad23 and Dsk2 are critical under different condensate-inducing conditions. In all, our data support a model where the cellular accumulation of substrates with long ubiquitin chains, potentially due to reduced cellular energy, allows for proteasome condensate formation. This suggests that proteasome condensates are not simply for proteasome storage, but function to sequester soluble ubiquitinated substrates together with inactive proteasomes.

An evolutionarily conserved phosphoserine-arginine salt bridge in the interface between ribosomal proteins uS4 and uS5 regulates translational accuracy in Saccharomyces cerevisiae.

Protein-protein and protein-rRNA interactions at the interface between ribosomal proteins uS4 and uS5 are thought to maintain the accuracy of protein synthesis by increasing selection of cognate aminoacyl-tRNAs. Selection involves a major conformational change-domain closure-that stabilizes aminoacyl-tRNA in the ribosomal acceptor (A) site. This has been thought a constitutive function of the ribosome ensuring consistent accuracy. Recently, the Saccharomyces cerevisiae Ctk1 cyclin-dependent kinase was demonstrated to ensure translational accuracy and Ser238 of uS5 proposed as its target. Surprisingly, Ser238 is outside the uS4-uS5 interface and no obvious mechanism has been proposed to explain its role. We show that the true target of Ctk1 regulation is another uS5 residue, Ser176, which lies in the interface opposite to Arg57 of uS4. Based on site specific mutagenesis, we propose that phospho-Ser176 forms a salt bridge with Arg57, which should increase selectivity by strengthening the interface. Genetic data show that Ctk1 regulates accuracy indirectly; the data suggest that the kinase Ypk2 directly phosphorylates Ser176. A second kinase pathway involving TORC1 and Pkc1 can inhibit this effect. The level of accuracy appears to depend on competitive action of these two pathways to regulate the level of Ser176 phosphorylation.

Lysine acetylation of histone acetyltransferase adaptor protein ADA2 is a mechanism of metabolic control of chromatin modification in plants.

Histone acetylation is a predominant active chromatin mark deposited by histone acetyltransferases (HATs) that transfer the acetyl group from acetyl coenzyme A (acetyl-CoA) to lysine ε-amino groups in histones. GENERAL CONTROL NON-REPRESSED PROTEIN 5 (GCN5) is one of the best-characterized HATs and functions in association with several adaptor proteins such as ADA2 within multiprotein HAT complexes. ADA2-GCN5 interaction increases GCN5 binding to acetyl-CoA and stimulates its HAT activity. It remains unclear whether the HAT activity of GCN5 (which acetylates not only histones but also cellular proteins) is regulated by acetyl-CoA levels, which vary greatly in cells under different metabolic and nutrition conditions. Here we show that the ADA2 protein itself is acetylated by GCN5 in rice cells. Lysine acetylation exposes ADA2 to a specific E3 ubiquitin ligase and reduces its protein stability. In rice plants, ADA2 protein accumulation reversely parallels its lysine acetylation and acetyl-CoA levels, both of which are dynamically regulated under varying growth conditions. Stress-induced ADA2 accumulation could stimulate GCN5 HAT activity to compensate for the reduced acetyl-CoA levels for histone acetylation. These results indicate that ADA2 lysine acetylation that senses cellular acetyl-CoA variations is a mechanism to regulate HAT activity and histone acetylation homeostasis in plants under changing environments.

Methylation of elongation factor 1A by yeast Efm4 or human eEF1A-KMT2 involves a beta-hairpin recognition motif and crosstalks with phosphorylation.

Translation elongation factor 1A (eEF1A) is an essential and highly conserved protein required for protein synthesis in eukaryotes. In both Saccharomyces cerevisiae and human, five different methyltransferases methylate specific residues on eEF1A, making eEF1A the eukaryotic protein targeted by the highest number of dedicated methyltransferases after histone H3. eEF1A methyltransferases are highly selective enzymes, only targeting eEF1A and each targeting just one or two specific residues in eEF1A. However, the mechanism of this selectivity remains poorly understood. To reveal how S. cerevisiae elongation factor methyltransferase 4 (Efm4) specifically methylates eEF1A at K316, we have used AlphaFold-Multimer modeling in combination with crosslinking mass spectrometry (XL-MS) and enzyme mutagenesis. We find that a unique beta-hairpin motif, which extends out from the core methyltransferase fold, is important for the methylation of eEF1A K316 in vitro. An alanine mutation of a single residue on this beta-hairpin, F212, significantly reduces Efm4 activity in vitro and in yeast cells. We show that the equivalent residue in human eEF1A-KMT2 (METTL10), F220, is also important for its activity towards eEF1A in vitro. We further show that the eEF1A guanine nucleotide exchange factor, eEF1Bα, inhibits Efm4 methylation of eEF1A in vitro, likely due to competitive binding. Lastly, we find that phosphorylation of eEF1A at S314 negatively crosstalks with Efm4-mediated methylation of K316. Our findings demonstrate how protein methyltransferases can be highly selective towards a single residue on a single protein in the cell.

The Hydrophilic C-terminus of Yeast Plasma-membrane Na+/H+ Antiporters Impacts Their Ability to Transport K.

Yeast plasma-membrane Na+/H+ antiporters (Nha/Sod) ensure the optimal intracellular level of alkali-metal cations and protons in cells. They are predicted to consist of 13 transmembrane segments (TMSs) and a large hydrophilic C-terminal cytoplasmic part with seven conserved domains. The substrate specificity, specifically the ability to recognize and transport K+ cations in addition to Na+ and Li+, differs among homologs. In this work, we reveal that the composition of the C-terminus impacts the ability of antiporters to transport particular cations. In the osmotolerant yeast Zygosaccharomyces rouxii, the Sod2-22 antiporter only efficiently exports Na+ and Li+, but not K+. The introduction of a negative charge or removal of a positive charge in one of the C-terminal conserved regions (C3) enabled ZrSod2-22 to transport K+. The same mutations rescued the low level of activity and purely Li+ specificity of ZrSod2-22 with the A179T mutation in TMS6, suggesting a possible interaction between this TMS and the C-terminus. The truncation or replacement of the C-terminal part of ZrSod2-22 with the C-terminus of a K+-transporting Nha/Sod antiporter (Saccharomyces cerevisiae Nha1 or Z. rouxii Nha1) also resulted in an antiporter with the capacity to export K+. In addition, in ScNha1, the replacement of three positively charged arginine residues 539-541 in the C3 region with alanine caused its inability to provide cells with tolerance to Li+. All our results demonstrate that the physiological functions of yeast Nha/Sod antiporters, either in salt tolerance or in K+ homeostasis, depend on the composition of their C-terminal parts.

The Efg1-Bud22 dimer associates with the U14 snoRNP contacting the 5' rRNA domain of an early 90S pre-ribosomal particle.

The DEAD-box helicase Dbp4 plays an essential role during the early assembly of the 40S ribosome, which is only poorly understood to date. By applying the yeast two-hybrid method and biochemical approaches, we discovered that Dbp4 interacts with the Efg1-Bud22 dimer. Both factors associate with early pre-90S particles and smaller complexes, each characterized by a high presence of the U14 snoRNA. A crosslink analysis of Bud22 revealed its contact to the U14 snoRNA and the 5' domain of the nascent 18S rRNA, close to its U14 snoRNA hybridization site. Moreover, depletion of Bud22 or Efg1 specifically affects U14 snoRNA association with pre-ribosomal complexes. Accordingly, we concluded that the role of the Efg1-Bud22 dimer is linked to the U14 snoRNA function on early 90S ribosome intermediates chaperoning the 5' domain of the nascent 18S rRNA. The successful rRNA folding of the 5' domain and the release of Efg1, Bud22, Dpb4, U14 snoRNA and associated snoRNP factors allows the subsequent recruitment of the Kre33-Bfr2-Enp2-Lcp5 module towards the 90S pre-ribosome.

Structural basis for the role of C-terminus acidic tail of Saccharomyces cerevisiae ubiquitin-conjugating enzyme (Rad6) in E3 ligase (Bre1) mediated recognition of histones.

Ubiquitination of histone H2B on chromatin is key to gene regulation. E3 ligase Bre1 and E2 Rad6 in Saccharomyces cerevisiae associate together to catalyze mono-ubiquitination at histone H2BK123. Prior studies identified the role of a highly dynamic C-terminal acidic tail of Rad6 indispensable for H2BK123 mono-ubiquitination. However, the mechanistic basis for the Rad6-acidic tail role remained elusive. Using different structural and biophysical approaches, this study for the first time uncovers the direct role of Rad6-acidic tail in interaction with the Bre1 Rad6-Binding Domain (RBD) and recognition of histones surface to facilitate histone H2B mono-ubiquitination. A combination of NMR, SAXS, ITC, site-directed mutagenesis and molecular dynamics studies reveal that RBD domain of Bre1 interacts with Rad6 to stabilize the dynamics of acidic tail. This Bre1-RBD mediated stability in acidic tail of Rad6 could be one of the key factors for facilitating correct recognition of histone surface and ubiquitin-transfer at H2BK123. We provide biophysical evidence that Rad6-acidic tail and a positivity charged surface on histone H2B are involved in recognition of E2:Histones. Taken together, this study uncovers the mechanistic basis for the role of Rad6-acidic in Bre1-RBD mediated recognition of histone surface that ensure the histone H2B mono-ubiquitination.

Intrinsically disordered regions of the Msn2 transcription factor encode multiple functions using interwoven sequence grammars.

Intrinsically disordered regions (IDRs) are abundant in eukaryotic proteins, but their sequence-function relationship remains poorly understood. IDRs of transcription factors (TFs) can direct promoter selection and recruit coactivators, as shown for the budding yeast TF Msn2. To examine how IDRs encode both these functions, we compared genomic binding specificity, coactivator recruitment, and gene induction amongst a large set of designed Msn2-IDR mutants. We find that both functions depend on multiple regions across the > 600AA IDR. Yet, transcription activity was readily disrupted by mutations that showed no effect on the Msn2 binding specificity. Our data attribute this differential sensitivity to the integration of a relaxed, composition-based code directing binding specificity with a more stringent, motif-based code controlling the recruitment of coactivators and transcription activity. Therefore, Msn2 utilizes interwoven sequence grammars for encoding multiple functions, suggesting a new IDR design paradigm of potentially general use.

Palmitoylation of CYSTM (CYSPD) proteins in yeast.

A superfamily of proteins called cysteine transmembrane is widely distributed across eukaryotes. These small proteins are characterized by the presence of a conserved motif at the C-terminal region, rich in cysteines, that has been annotated as a transmembrane domain. Orthologs of these proteins have been involved in resistance to pathogens and metal detoxification. The yeast members of the family are YBR016W, YDL012C, YDR034W-B, and YDR210W. Here, we begin the characterization of these proteins at the molecular level and show that Ybr016w, Ydr034w-b, and Ydr210w are palmitoylated proteins. Protein S-acylation or palmitoylation, is a posttranslational modification that consists of the addition of long-chain fatty acids to cysteine residues. We provide evidence that Ybr016w, Ydr210w, and Ydr034w-b are localized to the plasma membrane and exhibit varying degrees of polarity toward the daughter cell, which is dependent on endocytosis and recycling. We suggest the names CPP1, CPP2, and CPP3 (C terminally palmitoylated protein) for YBR016W, YDR210W, and YDR034W-B, respectively. We show that palmitoylation is responsible for the binding of these proteins to the membrane indicating that the cysteine transmembrane on these proteins is not a transmembrane domain. We propose renaming the C-terminal cysteine-rich domain as cysteine-rich palmitoylated domain. Loss of the palmitoyltransferase Erf2 leads to partial degradation of Ybr016w (Cpp1), whereas in the absence of the palmitoyltransferase Akr1, members of this family are completely degraded. For Cpp1, we show that this degradation occurs via the proteasome in an Rsp5-dependent manner, but is not exclusively due to a lack of Cpp1 palmitoylation.

Structural mechanism of outer kinetochore Dam1-Ndc80 complex assembly on microtubules.

Kinetochores couple chromosomes to the mitotic spindle to segregate the genome during cell division. An error correction mechanism drives the turnover of kinetochore-microtubule attachments until biorientation is achieved. The structural basis for how kinetochore-mediated chromosome segregation is accomplished and regulated remains an outstanding question. In this work, we describe the cryo-electron microscopy structure of the budding yeast outer kinetochore Ndc80 and Dam1 ring complexes assembled onto microtubules. Complex assembly occurs through multiple interfaces, and a staple within Dam1 aids ring assembly. Perturbation of key interfaces suppresses yeast viability. Force-rupture assays indicated that this is a consequence of impaired kinetochore-microtubule attachment. The presence of error correction phosphorylation sites at Ndc80-Dam1 ring complex interfaces and the Dam1 staple explains how kinetochore-microtubule attachments are destabilized and reset.

Diversity in Biological Function and Mechanism of the tRNA Methyltransferase Trm10.

ConspectusTransfer ribonucleic acid (tRNA) is the most highly modified RNA species in the cell, and loss of tRNA modifications can lead to growth defects in yeast as well as metabolic, neurological, and mitochondrial disorders in humans. Significant progress has been made toward identifying the enzymes that are responsible for installing diverse modifications in tRNA, revealing a landscape of fascinating biological and mechanistic diversity that remains to be fully explored. Most early discoveries of tRNA modification enzymes were in model systems, where many enzymes were not strictly required for viability, an observation somewhat at odds with the extreme conservation of many of the same enzymes throughout multiple domains of life. Moreover, many tRNA modification enzymes act on more than one type of tRNA substrate, which is not necessarily surprising given the similar overall secondary and tertiary structures of tRNA, yet biochemical characterization has revealed interesting patterns of substrate specificity that can be challenging to rationalize on a molecular level. Questions about how many enzymes efficiently select a precise set of target tRNAs from among a structurally similar pool of molecules persist.The tRNA methyltransferase Trm10 provides an exciting paradigm to study the biological and mechanistic questions surrounding tRNA modifications. Even though the enzyme was originally characterized in Saccharomyces cerevisiae where its deletion causes no detectable phenotype under standard lab conditions, several more recently identified phenotypes provide insight into the requirement for this modification in the overall quality control of the tRNA pool. Studies of Trm10 in yeast also revealed another characteristic feature that has turned out to be a conserved feature of enzymes throughout the Trm10 family tree. We were initially surprised to see that purified S. cerevisiae Trm10 was capable of modifying tRNA substrates that were not detectably modified by the enzyme in vivo in yeast. This pattern has continued to emerge as we and others have studied Trm10 orthologs from Archaea and Eukarya, with enzymes exhibiting in vitro substrate specificities that can differ significantly from in vivo patterns of modification. While this feature complicates efforts to predict substrate specificities of Trm10 enzymes in the absence of appropriate genetic systems, it also provides an exciting opportunity for studying how enzyme activities can be regulated to achieve dynamic patterns of biological tRNA modification, which have been shown to be increasingly important for stress responses and human disease. Finally, the intriguing diversity in target nucleotide modification that has been revealed among Trm10 orthologs is distinctive among known tRNA modifying enzymes and necessitates unusual and likely novel catalytic strategies for methylation that are being revealed by biochemical and structural studies directed toward various family members. These efforts will no doubt yield more surprising discoveries in terms of tRNA modification enzymology.

The social and structural architecture of the yeast protein interactome.

Cellular functions are mediated by protein-protein interactions, and mapping the interactome provides fundamental insights into biological systems. Affinity purification coupled to mass spectrometry is an ideal tool for such mapping, but it has been difficult to identify low copy number complexes, membrane complexes and complexes that are disrupted by protein tagging. As a result, our current knowledge of the interactome is far from complete, and assessing the reliability of reported interactions is challenging. Here we develop a sensitive high-throughput method using highly reproducible affinity enrichment coupled to mass spectrometry combined with a quantitative two-dimensional analysis strategy to comprehensively map the interactome of Saccharomyces cerevisiae. Thousand-fold reduced volumes in 96-well format enabled replicate analysis of the endogenous GFP-tagged library covering the entire expressed yeast proteome1. The 4,159 pull-downs generated a highly structured network of 3,927 proteins connected by 31,004 interactions, doubling the number of proteins and tripling the number of reliable interactions compared with existing interactome maps2. This includes very-low-abundance epigenetic complexes, organellar membrane complexes and non-taggable complexes inferred by abundance correlation. This nearly saturated interactome reveals that the vast majority of yeast proteins are highly connected, with an average of 16 interactors. Similar to social networks between humans, the average shortest distance between proteins is 4.2 interactions. AlphaFold-Multimer provided novel insights into the functional roles of previously uncharacterized proteins in complexes. Our web portal ( www.yeast-interactome.org ) enables extensive exploration of the interactome dataset.

The leucine zipper domain of the transcriptional repressor Opi1 underlies a signal transduction mechanism regulating lipid synthesis.

In Saccharomyces cerevisiae, the transcriptional repressor Opi1 regulates the expression of genes involved in phospholipid synthesis responding to the abundance of the phospholipid precursor phosphatidic acid at the endoplasmic reticulum. We report here the identification of the conserved leucine zipper (LZ) domain of Opi1 as a hot spot for gain of function mutations and the characterization of the strongest variant identified, Opi1N150D. LZ modeling posits asparagine 150 embedded on the hydrophobic surface of the zipper and specifying dynamic parallel homodimerization by allowing electrostatic bonding across the hydrophobic dimerization interface. Opi1 variants carrying any of the other three ionic residues at amino acid 150 were also repressing. Genetic analyses showed that Opi1N150D variant is dominant, and its phenotype is attenuated when loss of function mutations identified in the other two conserved domains are present in cis. We build on the notion that membrane binding facilitates LZ dimerization to antagonize an intramolecular interaction of the zipper necessary for repression. Dissecting Opi1 protein in three polypeptides containing each conserved region, we performed in vitro analyses to explore interdomain interactions. An Opi11-190 probe interacted with Opi1291-404, the C terminus that bears the activator interacting domain (AID). LZ or AID loss of function mutations attenuated the interaction of the probes but was unaffected by the N150D mutation. We propose a model for Opi1 signal transduction whereby synergy between membrane-binding events and LZ dimerization antagonizes intramolecular LZ-AID interaction and transcriptional repression.

The Saccharomyces cerevisiae SR protein Npl3 interacts with hyperphosphorylated CTD of RNA Polymerase II.

The catalytic subunit of RNA Polymerase II contains a highly conserved carboxy terminal domain (CTD) composed of multiple tandem heptad sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. The non-proline residues in CTD undergo posttranslational modifications, with Ser5 phosphorylation (Ser5P) predominating at the start of the transcription cycle and Ser2P at the end, while other phosphorylation levels are high all throughout. The differentially phosphorylated CTD is recognized by regulatory proteins, helpful during mRNA transcription and export. One such protein Npl3 is composed of two RNA binding domains and a C-terminus RGG/SR domain. The Ser411 of Npl3 is reported to make direct contact with Ser2P of CTD for its recruitment and function, while the Npl3 lacking of C-terminal 25 amino acids (Npl3Δ389-414) showed no apparent defects in mRNA synthesis. Here, we report that the RNA binding domains of Npl3 are separate folding units and interact also with the CTD. The interaction between Npl3 and CTD appears to involve not just Ser2P, but also the Ser5P and Ser7P. The Arg126 of the first RNA binding domain interacts with Ser2P whereas the Arg235 of the second RNA binding domain interacts with either Ser7P or Ser5P of another heptad. The finding provides new insight of Npl3 function for mRNA transcription.

Insights into the role of the conserved GTPase domain residues T62 and S277 in yeast Dnm1.

Mitochondrial division is a highly regulated process. The master regulator of this process is the multi-domain, conserved protein called Dnm1 in yeast. In this study, we systematically analyzed two residues, T62 and S277, reported to be putatively phosphorylated in the GTPase domain of the protein. These residues lie in the G2 and G5 motifs of the GTPase domain. Both residues are important for the function of the protein, as evident from in vivo and in vitro analysis of the non-phosphorylatable and phosphomimetic variants. Dnm1T62A/D and Dnm1S277A/D showed differences with respect to the protein localization and puncta dynamics in vivo, albeit both were non-functional as assessed by mitochondrial morphology and GTPase activity. Overall, the secondary structure of the protein variants was unaltered, but local conformational changes were observed. Interestingly, both Dnm1T62A/D and Dnm1S277A/D exhibited dominant-negative behavior when expressed in cells containing endogenous Dnm1. To our knowledge, we report for the first time a single residue (S277) change that does not alter the localization of Dnm1 but makes it non-functional in a dominant-negative manner. Intriguingly, the two residues analyzed in this study are present in the same domain but exhibit variable effects when mutated to alanine or aspartic acid.

MARCKS Effector Domain, a reversible lipid ligand, illuminates late stages of membrane fusion.

Yeast vacuolar HOPS tethers membranes, catalyzes trans-SNARE assembly between R- and Q-SNAREs, and shepherds SNAREs past early inhibition by Sec17. After partial SNARE zippering, fusion is driven slowly by either completion of SNARE zippering or by Sec17/Sec18, but rapid fusion needs zippering and Sec17/Sec18. Using reconstituted-vacuolar fusion, we find that MARCKS Effector Domain (MED) peptide, a lipid ligand, blocks fusion reversibly at a late reaction stage. The MED fusion blockade is overcome by either salt extraction, inactivation with the MED ligand calmodulin, or addition of Sec17/Sec18. During incubation with MED, SNAREs assemble stable complexes in trans and fusion becomes resistant to antibody to the Qa SNARE. When Q-SNAREs are preassembled, a synthetic tether can replace HOPS for fusion. With a synthetic tether, fusion needs both complete SNARE zippering and Sec17/Sec18 to overcome a MED block. In contrast, when SNARE domains are only two-third zippered, only HOPS will support Sec17/Sec18 driven fusion without needing complete zippering. HOPS thus remains engaged with SNAREs during zippering. MED facilitates the study of distinct fusion stages: tethering, initial trans-SNARE assembly and its sensitivity to Sec17, SNARE zippering, Sec17/Sec18 engagement, and lipid and lumenal mixing.

Structural basis of nucleosome deacetylation and DNA linker tightening by Rpd3S histone deacetylase complex.

In Saccharomyces cerevisiae, cryptic transcription at the coding region is prevented by the activity of Sin3 histone deacetylase (HDAC) complex Rpd3S, which is carried by the transcribing RNA polymerase II (RNAPII) to deacetylate and stabilize chromatin. Despite its fundamental importance, the mechanisms by which Rpd3S deacetylates nucleosomes and regulates chromatin dynamics remain elusive. Here, we determined several cryo-EM structures of Rpd3S in complex with nucleosome core particles (NCPs), including the H3/H4 deacetylation states, the alternative deacetylation state, the linker tightening state, and a state in which Rpd3S co-exists with the Hho1 linker histone on NCP. These structures suggest that Rpd3S utilizes a conserved Sin3 basic surface to navigate through the nucleosomal DNA, guided by its interactions with H3K36 methylation and the extra-nucleosomal DNA linkers, to target acetylated H3K9 and sample other histone tails. Furthermore, our structures illustrate that Rpd3S reconfigures the DNA linkers and acts in concert with Hho1 to engage the NCP, potentially unraveling how Rpd3S and Hho1 work in tandem for gene silencing.

Structure of the peroxisomal Pex1/Pex6 ATPase complex bound to a substrate.

The double-ring AAA+ ATPase Pex1/Pex6 is required for peroxisomal receptor recycling and is essential for peroxisome formation. Pex1/Pex6 mutations cause severe peroxisome associated developmental disorders. Despite its pathophysiological importance, mechanistic details of the heterohexamer are not yet available. Here, we report cryoEM structures of Pex1/Pex6 from Saccharomyces cerevisiae, with an endogenous protein substrate trapped in the central pore of the catalytically active second ring (D2). Pairs of Pex1/Pex6(D2) subdomains engage the substrate via a staircase of pore-1 loops with distinct properties. The first ring (D1) is catalytically inactive but undergoes significant conformational changes resulting in alternate widening and narrowing of its pore. These events are fueled by ATP hydrolysis in the D2 ring and disengagement of a "twin-seam" Pex1/Pex6(D2) heterodimer from the staircase. Mechanical forces are propagated in a unique manner along Pex1/Pex6 interfaces that are not available in homo-oligomeric AAA-ATPases. Our structural analysis reveals the mechanisms of how Pex1 and Pex6 coordinate to achieve substrate translocation.