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Gene silencing is essential for regulating cell fate in eukaryotes. Altered chromatin architectures contribute to maintaining the silenced state in a variety of species. The silent information regulator (Sir) proteins regulate mating type in Saccharomyces cerevisiae. One of these proteins, Sir3, interacts directly with the nucleosome to help generate silenced domains. We determined the crystal structure of a complex of the yeast Sir3 BAH (bromo-associated homology) domain and the nucleosome core particle at 3.0 angstrom resolution. We see multiple molecular interactions between the protein surfaces of the nucleosome and the BAH domain that explain numerous genetic mutations. These interactions are accompanied by structural rearrangements in both the nucleosome and the BAH domain. The structure explains how covalent modifications on H4K16 and H3K79 regulate formation of a silencing complex that contains the nucleosome as a central component.

The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins 1 – 4 . Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex 5 – 9 at 3.8-Å resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 β-barrel channels and α-helical transmembrane subunits, providing insight into critical features that are conserved in all eukaryotes 1 – 3 . Each Tom40 β-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer. The high-resolution cryo-electron microscopy structure of the yeast translocase of the outer mitochondrial membrane reveals key features of mitochondrial protein import that are conserved in all eukaryotes.

Why do certain proteins evolve much slower than others? We compared not only rates per protein, but also rates per position within individual proteins. For ∼90% of proteins, the distribution of positional rates exhibits three peaks: a peak of slow evolving residues, with average log2[normalized rate], log2μ, of ca. -2, corresponding primarily to core residues; a peak of fast evolving residues (log2μ ∼ 0.5) largely corresponding to surface residues; and a very fast peak (log2μ ∼ 2) associated with disordered segments. However, a unique fraction of proteins that evolve very slowly exhibit not only a negligible fast peak, but also a peak with a log2μ ∼ -4, rather than the standard core peak of -2. Thus, a \"freeze\" of a protein's surface seems to stop core evolution as well. We also observed a much higher fraction of substitutions in potentially interacting residues than expected by chance, including substitutions in pairs of contacting surface-core residues. Overall, the data suggest that accumulation of surface substitutions enables the acceptance of substitutions in core positions. The underlying reason for slow evolution might therefore be a highly constrained surface due to protein—protein interactions or the need to prevent misfolding or aggregation. If the surface is inaccessible to substitutions, so becomes the core, thus resulting in very slow overall rates.

The endoplasmic reticulum (ER) membrane complex (EMC) cooperates with the Sec61 translocon to co-translationally insert a transmembrane helix (TMH) of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMH of some tail-anchored proteins 1 – 3 . How EMC accomplishes this feat has been unclear. Here we report the first, to our knowledge, cryo-electron microscopy structure of the eukaryotic EMC. We found that the Saccharomyces cerevisiae EMC contains eight subunits (Emc1–6, Emc7 and Emc10), has a large lumenal region and a smaller cytosolic region, and has a transmembrane region formed by Emc4, Emc5 and Emc6 plus the transmembrane domains of Emc1 and Emc3. We identified a five-TMH fold centred around Emc3 that resembles the prokaryotic YidC insertase and that delineates a largely hydrophilic client protein pocket. The transmembrane domain of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. Mutational studies demonstrated that the flexibility of Emc4 and the hydrophilicity of the client pocket are required for EMC function. The EMC structure reveals notable evolutionary conservation with the prokaryotic insertases 4 , 5 , suggests that eukaryotic TMH insertion involves a similar mechanism, and provides a framework for detailed understanding of membrane insertion for numerous eukaryotic integral membrane proteins and tail-anchored proteins. The cryo-electron microscopy structure of the ER membrane complex provides insight into its overall architecture, evolution and function in co-translational protein insertion.

Many biomolecules undergo liquid–liquid phase separation to form liquid-like condensates that mediate diverse cellular functions 1 , 2 . Autophagy is able to degrade such condensates using autophagosomes—double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast 3 – 5 . Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein–protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS. The pre-autophagosomal structure in yeast is a liquid-like condensate of Atg proteins whose phase separation may have a critical, active role in autophagy.

The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9’s ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.Cryo-EM and liposome assays reveal that Atg9 functions as a lipid scramblase, transporting phospholipids between inner and outer liposome leaflets. Analyses of mutants in yeast support a role for this activity in autophagy.

Mitochondria import nearly all of their approximately 1,000–2,000 constituent proteins from the cytosol across their double-membrane envelope 1 – 5 . Genetic and biochemical studies have shown that the conserved protein translocase, termed the TIM23 complex, mediates import of presequence-containing proteins (preproteins) into the mitochondrial matrix and inner membrane. Among about ten different subunits of the TIM23 complex, the essential multipass membrane protein Tim23, together with the evolutionarily related protein Tim17, has long been postulated to form a protein-conducting channel 6 – 11 . However, the mechanism by which these subunits form a translocation path in the membrane and enable the import process remains unclear due to a lack of structural information. Here we determined the cryo-electron microscopy structure of the core TIM23 complex (heterotrimeric Tim17–Tim23–Tim44) from Saccharomyces cerevisiae . Contrary to the prevailing model, Tim23 and Tim17 themselves do not form a water-filled channel, but instead have separate, lipid-exposed concave cavities that face in opposite directions. Our structural and biochemical analyses show that the cavity of Tim17, but not Tim23, forms the protein translocation path, whereas Tim23 probably has a structural role. The results further suggest that, during translocation of substrate polypeptides, the nonessential subunit Mgr2 seals the lateral opening of the Tim17 cavity to facilitate the translocation process. We propose a new model for the TIM23-mediated protein import and sorting mechanism, a central pathway in mitochondrial biogenesis. The cryo-electron microscopy structure of the mitochondrial TIM23 complex from Saccharomyces cerevisiae shows that Tim17 forms a protein translocation path.

For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC–core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the ‘E-bridge’ helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC–core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II. Cryo-electron microscopy structures of the yeast pre-initiation complex (PIC) and its complex with core Mediator provide insights into the opening of promoter DNA and the initiation of transcription. TFIIH in the transcription pre-initiation complex To initiate gene transcription, RNA polymerase (Pol) II assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here, Patrick Cramer and colleagues describe cryo-electron microscopy structures of the yeast PIC and the PIC bound to the core Mediator (cMed) complex. The latter structure with the general coactivator Mediator has 46 factors, including all those that are essential for transcription initiation in yeast. The structures reveal the architecture of transcription factor IIH (TFIIH) and suggest how its 'core' and 'kinase' modules might function in promoter opening and Pol II phosphorylation, respectively.

Chromatin remodellers are helicase-like, ATP-dependent enzymes that alter chromatin structure and nucleosome positions to allow regulatory proteins access to DNA. Here we report the cryo-electron microscopy structure of chromatin remodeller Switch/sucrose non-fermentable (SWI2/SNF2) from Saccharomyces cerevisiae bound to the nucleosome. The structure shows that the two core domains of Snf2 are realigned upon nucleosome binding, suggesting activation of the enzyme. The core domains contact each other through two induced Brace helices, which are crucial for coupling ATP hydrolysis to chromatin remodelling. Snf2 binds to the phosphate backbones of one DNA gyre of the nucleosome mainly through its helicase motifs within the major domain cleft, suggesting a conserved mechanism of substrate engagement across different remodellers. Snf2 contacts the second DNA gyre via a positively charged surface, providing a mechanism to anchor the remodeller at a fixed position of the nucleosome. Snf2 locally deforms nucleosomal DNA at the site of binding, priming the substrate for the remodelling reaction. Together, these findings provide mechanistic insights into chromatin remodelling. The cryo-electron microscopy structure of Saccharomyces cerevisiae Snf2 chromatin remodeller bound to a nucleosome and a proposed mechanism for DNA translocation by Snf2 are presented. Structure of a chromatin remodeller Chromatin remodellers are helicase-like, ATP-dependent enzymes that alter chromatin structure and nucleosome positions in order to allow regulatory proteins access to DNA. Here, Zhucheng Chen and colleagues report the cryo-electron microscopy structure of the budding yeast Snf2 chromatin remodeller bound to a nucleosome and propose a mechanism for DNA translocation by Snf2. The remodeller interacts with the nucleosome mainly using helicase motifs and binds the phosphate backbones along the DNA minor groove. Most of the DNA-binding elements within Snf2 are conserved across other families of remodelling enzymes, indicating that this mode of nucleosome engagement is likely to be widespread.

Background Interspecific hybridization has proven to be a potentially valuable technique for generating de novo lager yeast strains that possess diverse and improved traits compared to their parent strains. To further enhance the value of hybridization for strain development, it would be desirable to combine phenotypic traits from more than two parent strains, as well as remove unwanted traits from hybrids. One such trait, that has limited the industrial use of de novo lager yeast hybrids, is their inherent tendency to produce phenolic off-flavours; an undesirable trait inherited from the Saccharomyces eubayanus parent. Trait removal and the addition of traits from a third strain could be achieved through sporulation and meiotic recombination or further mating. However, interspecies hybrids tend to be sterile, which impedes this opportunity. Results Here we generated a set of five hybrids from three different parent strains, two of which contained DNA from all three parent strains. These hybrids were constructed with fertile allotetraploid intermediates, which were capable of efficient sporulation. We used these eight brewing strains to examine two brewing-relevant phenotypes: stress tolerance and phenolic off-flavour formation. Lipidomics and multivariate analysis revealed links between several lipid species and the ability to ferment in low temperatures and high ethanol concentrations. Unsaturated fatty acids, such as oleic acid, and ergosterol were shown to positively influence growth at high ethanol concentrations. The ability to produce phenolic off-flavours was also successfully removed from one of the hybrids, Hybrid T2, through meiotic segregation. The potential application of these strains in industrial fermentations was demonstrated in wort fermentations, which revealed that the meiotic segregant Hybrid T2 not only didn’t produce any phenolic off-flavours, but also reached the highest ethanol concentration and consumed the most maltotriose. Conclusions Our study demonstrates the possibility of constructing complex yeast hybrids that possess traits that are relevant to industrial lager beer fermentation and that are derived from several parent strains. Yeast lipid composition was also shown to have a central role in determining ethanol and cold tolerance in brewing strains.