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Zervopoulos et al. (2022) propose a non-canonical nuclear import pathway for the functional mitochondrial pyruvate dehydrogenase complex (PDC), facilitated by dynamic MFN2-mediated tethering of mitochondria to the nuclear envelope upon exposure to proliferative stimuli.

Protein degradation, a major eukaryotic response to cellular signals, is subject to numerous layers of regulation. In yeast, the evolutionarily conserved GID E3 ligase mediates glucose-induced degradation of fructose-1,6-bisphosphatase (Fbp1), malate dehydrogenase (Mdh2), and other gluconeogenic enzymes. “GID” is a collection of E3 ligase complexes; a core scaffold, RING-type catalytic core, and a supramolecular assembly module together with interchangeable substrate receptors select targets for ubiquitylation. However, knowledge of additional cellular factors directly regulating GID-type E3s remains rudimentary. Here, we structurally and biochemically characterize Gid12 as a modulator of the GID E3 ligase complex. Our collection of cryo-EM reconstructions shows that Gid12 forms an extensive interface sealing the substrate receptor Gid4 onto the scaffold, and remodeling the degron binding site. Gid12 also sterically blocks a recruited Fbp1 or Mdh2 from the ubiquitylation active sites. Our analysis of the role of Gid12 establishes principles that may more generally underlie E3 ligase regulation.

This protocol describes how to plunge-freeze yeast on EM grids and how to target autophagic structures by combining cryo confocal fluorescence data to FIB-milling and tomogram acquisition

During macroautophagy, phagophores establish multiple membrane contact sites (MCSs) with other organelles that are pivotal for proper phagophore assembly and growth. In S. cerevisiae, phagophore contacts have been observed with the vacuole, the ER, and lipid droplets. In situ imaging studies have greatly advanced our understanding of the structure and function of these sites. Here, we discuss how in situ structural methods like cryo-CLEM can give unprecedented insights into MCSs, and how they help to elucidate the structural arrangements of MCSs within cells. We further summarize the current knowledge of the contact sites in autophagy, focusing on autophagosome biogenesis in the model organism S. cerevisiae.

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

The ubiquitin-proteasome-system (UPS) fulfills an essential role in regulating protein homeostasis by spatially and temporally controlling proteolysis in an ATP- and ubiquitin-dependent manner. However, the localization of proteasomes is highly variable under diverse cellular conditions. In yeast, newly synthesized proteasomes are primarily localized to the nucleus during cell proliferation. Yeast proteasomes are transported into the nucleus through the nuclear pore either as immature subcomplexes or as mature enzymes via adaptor proteins Sts1 and Blm10, while in mammalian cells, post-mitotic uptake of proteasomes into the nucleus is mediated by AKIRIN2, an adaptor protein essentially required for nuclear protein degradation. Stressful growth conditions and the reversible halt of proliferation, i.e. quiescence, are associated with a decline in ATP and the re-organization of proteasome localization. Cellular stress leads to proteasome accumulation in membraneless granules either in the nucleus or in the cytoplasm. In quiescence, yeast proteasomes are sequestered in a ubiquitin-dependent manner into motile and reversible proteasome storage granules (PSGs) in the cytoplasm. In cancer cells upon amino acid deprivation, heat shock, osmotic stress, oxidative stress, or the inhibition of either proteasome activity or nuclear export, reversible proteasome foci containing poly-ubiquitinated substrates are formed by liquid-liquid phase separation in the nucleus. In this review, we summarize recent literature revealing new links between nuclear transport, ubiquitin signaling and the intracellular organization of proteasomes during cellular stress conditions.

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

Selective disposal of a wide range of cellular entities by macroautophagy/autophagy is achieved through a special class of proteins called autophagy receptors, which link corresponding cargo to the membrane-bound autophagosomal protein Atg8/LC3. In pursuit of novel autophagy receptors and their cargo, we uncovered a previously undescribed autophagy pathway for removal of aberrant clathrin-mediated endocytosis (CME) protein condensates in S. cerevisiae. Of these CME proteins, Ede1 functions as an autophagy receptor, harboring distinct Atg8-binding domains and driving phase separation into condensates. The aberrant CME condensates at the plasma membrane (PM) exhibit a drop-like structure surrounded by a fenestrated ER, which are engulfed in pieces in an Ede1-dependent manner by autophagy. Thus, our work suggests that aberrant CME is a target for autophagic degradation, with the scaffold protein Ede1 serving as a built-in autophagy receptor that monitors the assembly status of the CME machinery.

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.

Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues.

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.