Selective autophagy is a quality control pathway through which cellular components are sequestered into double-membrane vesicles and delivered to specific intracellular compartments. This process requires autophagy receptors that link cargo to growing autophagosomal membranes. Selective autophagy is also implicated in various membrane trafficking events. Here we discuss the current view on how cargo selection and transport are achieved during selective autophagy, and point out molecular mechanisms that are congruent between autophagy and vesicle trafficking pathways.

This is an accepted version of a paper published in Nature. This paper has been peer-reviewed but does not include the final publisher proof-corrections or journal pagination.Citation for the published paper: von Heijne, G., Contreras, X., Ernst, A., Haberkant, P., Björkholm, P. et al. (2012) "Molecular recognition of a single sphingolipid species by a protein's transmembrane domain" Nature, 481 (7382): 525-529 URL: http://dx

The ubiquitin system regulates virtually all aspects of cellular function. We report a method to target the myriad enzymes that govern ubiquitination of protein substrates. We used massively diverse combinatorial libraries of ubiquitin variants to develop inhibitors of four deubiquitinases (DUBs) and analyzed the DUB-inhibitor complexes with crystallography. We extended the selection strategy to the ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes and found that ubiquitin variants can also enhance enzyme activity. Last, we showed that ubiquitin variants can bind selectively to ubiquitin-binding domains. Ubiquitin variants exhibit selective function in cells and thus enable orthogonal modulation of specific enzymatic steps in the ubiquitin system.

Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR)1–3. However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ)4. We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells4, 5 and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants6 for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

Different modes of cell death regulate immunity. Whereas necrotic (necroptotic, pyroptotic) cell death triggers inflammation, apoptosis contributes to its resolution. Interleukin-1 (IL-1) family cytokines are key players in this interaction. A number of IL-1 family cytokines are produced by necrotic cells to induce sterile inflammation. However, release of IL-1 family proteins from apoptotic cells to regulate inflammation was not described. Here we show that IL-38, a poorly characterized IL-1 family cytokine, is produced selectively by human apoptotic cells to limit inflammation. Depletion of IL-38 in apoptotic cells provoked enhanced IL-6 and IL-8 levels and AP1 activation in co-cultured human primary macrophages, subsequently inducing Th17 cell expansion at the expense of IL-10-producing T cells. IL-38 was N-terminally processed in apoptotic cells to generate a mature cytokine with distinct properties. Both full-length and truncated IL-38 bound to X-linked interleukin-1 receptor accessory protein-like 1 (IL1RAPL1). However, whereas the IL-38 precursor induced an increase in IL-6 production by human macrophages, truncated IL-38 reduced IL-6 production by attenuating the JNK/AP1 pathway downstream of IL1RAPL1. In conclusion, we identified a mechanism of apoptotic cell-dependent immune regulation requiring IL-38 processing and secretion, which might be relevant in resolution of inflammation, autoimmunity, and cancer.

Our concept of biological membranes has markedly changed, from the fluid mosaic model to the current model that lipids and proteins have the ability to separate into microdomains, differing in their protein and lipid compositions. Since the breakthrough in crystallizing membrane proteins, the most powerful method to define lipid-binding sites on proteins has been X-ray and electron crystallography. More recently, chemical biology approaches have been developed to analyze protein-lipid interactions. Such methods have the advantage of providing highly specific cellular probes. With the advent of novel tools to study functions of individual lipid species in membranes together with structural analysis and simulations at the atomistic resolution, a growing number of specific protein-lipid complexes are defined and their functions explored. In the present article, we discuss the various modes of intramembrane protein -lipid interactions in cellular membranes, including examples for both annular and nonannular bound lipids. Furthermore, we will discuss possible functional roles of such specific protein-lipid interactions as well as roles of lipids as chaperones in protein folding and transport.

Maintaining a fluid bilayer is essential for cell signaling and survival. Lipid saturation is a key factor determining lipid packing and membrane fluidity, and it must be tightly controlled to guarantee organelle function and identity. A dedicated eukaryotic mechanism of lipid saturation sensing, however, remains elusive. Here we show that Mga2, a transcription factor conserved among fungi, acts as a lipid-packing sensor in the ER membrane to control the production of unsaturated fatty acids. Systematic mutagenesis, molecular dynamics simulations, and electron paramagnetic resonance spectroscopy identify a pivotal role of the oligomeric transmembrane helix (TMH) of Mga2 for intra-membrane sensing, and they show that the lipid environment controls the proteolytic activation of Mga2 by stabilizing alternative rotational orientations of the TMH region. This work establishes a eukaryotic strategy of lipid saturation sensing that differs significantly from the analogous bacterial mechanism relying on hydrophobic thickness.

SUMMARY While retrograde cargo selection in the Golgi is known to depend on specific signals, it is unknown whether anterograde cargo is sorted and anterograde signals have not been identified. We suggest here that S-palmitoylation of anterograde cargo at the Golgi membrane interface is an anterograde signal, and that it results in concentration in curved regions at the Golgi rims by simple physical chemistry. The rate of transport across the Golgi of two S-palmitoylated membrane proteins is controlled by Spalmitoylation. The bulk of S-palmitoylated proteins in the Golgi behave analogously, as revealed by click chemistry-based fluorescence and electron microscopy. These palmitoylated cargo concentrate in the most highly curved regions of the Golgi membranes, including the fenestrated perimeters of cisternae and associated vesicles. A palmitoylated transmembrane domain behaves similarly in model systems.