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Ribosomes comprise a large (LSU) and a small subunit (SSU) which are synthesized independently in the nucleolus before being exported into the cytoplasm, where they assemble into functional ribosomes. Individual maturation steps have been analyzed in detail using biochemical methods, light microscopy and conventional electron microscopy (EM). In recent years, single particle analysis (SPA) has yielded molecular resolution structures of several pre-ribosomal intermediates. It falls short, however, of revealing the spatiotemporal sequence of ribosome biogenesis in the cellular context. Here, we present our study on native nucleoli in Chlamydomonas reinhardtii, in which we follow the formation of LSU and SSU precursors by in situ cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). By combining both positional and molecular data, we reveal gradients of ribosome maturation within the granular component (GC), offering a new perspective on how the liquid-liquid-phase separation of the nucleolus supports ribosome biogenesis. The large and small subunits of the ribosome are synthesized independently within the nucleolus — a membrane-less compartment within the nucleus — before being exported into the cytoplasm. Here, the authors use in situ cryo-ET to observe ribosome maturation and reveal the native organization of the nucleolus.

The ability to selectively degrade cellular components ranging from proteins to large complexes and organelles is essential for cellular quality control. Failure leads to accumulation of unwanted material that gives rise to neurodegeneration, cancer, and infectious diseases (1). Two main degradative pathways have evolved: the ubiquitin–proteasome system (UPS) and autophagy. While the selective degradation of soluble proteins is normally conducted by the UPS, which needs unfolding of its substrates to traverse through the narrow openings of the proteasome, macroautophagy (hereafter termed autophagy) is able to sequester cytosolic cargo into a newly synthesized double-membrane compartment, called the autophagosome, that later fuses with the vacuole/lysosome for degradation. This empowers autophagy to degrade bulky cargo such as invading pathogens, protein aggregates, or damaged/disused organelles. Both pathways are essential components of the proteostasis network. In PNAS, Fu et al. (2) uncover a role of the protein p62 in orchestrating proteasomal protein turnover in the nucleus (Fig. 1). Fig. 1. Depiction of the functional roles of p62 within the cell. The cytoplasmic pool of p62 serves as a cargo receptor for the recognition of ubiquitylated protein aggregates by selective autophagy. Here, p62 targets ubiquitylated misfolded proteins and phase separates into condensates through multivalent interactions established with multiple Ubs linked in chains. The nuclear pool of p62 also forms condensates in a similar fashion as its cytosolic counterpart but recruits the UPS for efficient removal of nuclear proteins. The local p62-dependent concentration of the UPS members facilitates efficient removal of its substrates. The multifunctional protein p62/SQSTM1 plays an important role in targeting ubiquitin (Ub)-modified proteins to either the proteasome or the autophagy machinery (3). Ubiquitylated proteins are captured by binding to the C-terminal Ub-associated (UBA) domain of p62 (4). To couple cargo recognition with autophagosome biogenesis, p62 interacts with lipidated LC3 via its LC3 interacting region (LIR). … [↵][1]1To whom correspondence may be addressed. Email: florian.wilfling{at}biophys.mpg.de. [1]: #xref-corresp-1-1

Cell cycle tags allow to restrict target protein expression to specific cell cycle phases. Here, we present an advanced toolbox of cell cycle tag constructs in budding yeast with defined and compatible peak expression that allow comparison of protein functionality at different cell cycle phases. We apply this technology to the question of how and when Mus81-Mms4 and Yen1 nucleases act on DNA replication or recombination structures. Restriction of Mus81-Mms4 to M phase but not S phase allows a wildtype response to various forms of replication perturbation and DNA damage in S phase, suggesting it acts as a post-replicative resolvase. Moreover, we use cell cycle tags to reinstall cell cycle control to a deregulated version of Yen1, showing that its premature activation interferes with the response to perturbed replication. Curbing resolvase activity and establishing a hierarchy of resolution mechanisms are therefore the principal reasons underlying resolvase cell cycle regulation., eLife, 9, ISSN:2050-084X

SummaryAutophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargoes to the membrane bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we discover a selective autophagy pathway for protein condensates formed by endocytic proteins. In this pathway, the endocytic yeast protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis, does not involve other known selective autophagy receptors, but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates – at the plasma membrane and in autophagic bodies – shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy pathway. Our data suggest a model for autophagic degradation of membraneless compartments by the action of intrinsic autophagy receptors.HighlightsEde1 is a selective autophagy receptor for aberrant CME protein assembliesAberrant CME assemblies form by liquid-liquid phase separationCore autophagy machinery and Ede1 are important for degradation of CME condensatesUltrastrucural view of a LLPS compartment at the PM and within autophagic bodies

Gross and Graef preview two studies (Lee et al. and Tomioka et al.) describing the targeted degradation of nuclear pore complexes by selective autophagy., Lee et al. (2020. Nat. Cell Biol. https://doi.org/10.1038/s41556-019-0459-2) and, in this issue, Tomioka et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.201910063) describe the targeted degradation of nuclear pore complexes (NPCs) by selective autophagy, providing insight into the mechanisms of turnover for individual nucleoporins and entire NPCs.

Intracellular trafficking between organelles is achieved by coat protein complexes, coat protomers, that bud vesicles from bilayer membranes. Lipid droplets are protected by a monolayer and thus seem unsuitable targets for coatomers. Unexpectedly, coat protein complex I (COPI) is required for lipid droplet targeting of some proteins, suggesting a possible direct interaction between COPI and lipid droplets. Here, we find that COPI coat components can bud 60-nm triacylglycerol nanodroplets from artificial lipid droplet (LD) interfaces. This budding decreases phospholipid packing of the monolayer decorating the mother LD. As a result, hydrophobic triacylglycerol molecules become more exposed to the aqueous environment, increasing LD surface tension. In vivo, this surface tension increase may prime lipid droplets for reactions with neighboring proteins or membranes. It provides a mechanism fundamentally different from transport vesicle formation by COPI, likely responsible for the diverse lipid droplet phenotypes associated with depletion of COPI subunits.

SummaryLipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis.

SummaryLipid droplets (LDs) are cellular storage organelles for neutral lipids that vary in size and abundance according to cellular needs. Physiological conditions that promote lipid storage rapidly and markedly increase LD volume and surface. How the need for surface phospholipids is sensed and balanced during this process is unknown. Here, we show that phosphatidylcholine (PC) acts as a surfactant to prevent LD coalescence, which otherwise yields large, lipolysis-resistant LDs and triglyceride (TG) accumulation. The need for additional PC to coat the enlarging surface during LD expansion is provided by the Kennedy pathway, which is activated by reversible targeting of the rate-limiting enzyme, CTP:phosphocholine cytidylyltransferase (CCT), to growing LD surfaces. The requirement, targeting, and activation of CCT to growing LDs were similar in cells of Drosophila and mice. Our results reveal a mechanism to maintain PC homeostasis at the expanding LD monolayer through targeted activation of a key PC synthesis enzyme.

SummaryNuclear pore complexes (NPCs) mediate exchange across the nuclear envelope. They consist of hundreds of proteins called nucleoporins (Nups) that assemble in multiple copies to fuse the inner and outer nuclear membranes. Elucidating the molecular function and architecture of NPCs imposes a formidable challenge and requires the convergence of in vitro and in situ approaches. How exactly NPC architecture accommodates processes such as mRNA export or NPC assembly and turnover inside of cells remains poorly understood. Here we combine integrated in situ structural biology, correlative light and electron microscopy with yeast genetics to structurally analyze NPCs within the native context of Saccharomyces cerevisiae cells under conditions of starvation and exponential growth. We find an unanticipated in situ layout of nucleoporins with respect to overall dimensions and conformation of the NPC scaffold that could not have been predicted from previous in vitro analysis. Particularly striking is the configuration of the Nup159 complex, which appears critical to spatially accommodate not only mRNA export but also NPC turnover by selective autophagy. We capture structural snapshots of NPC turnover, revealing that it occurs through nuclear envelope herniae and NPC-containing nuclear vesicles. Our study provides the basis for understanding the various membrane remodeling events that happen at the interface of the nuclear envelope with the autophagy apparatus and emphasizes the need of investigating macromolecular complexes in their cellular context.