Arf1-dependent LRBA recruitment to Rab4 endosomes is required for endolysosome homeostasis.

Deleterious mutations in the lipopolysaccharide responsive beige-like anchor protein (LRBA) gene cause severe childhood immune dysregulation. The complexity of the symptoms involving multiple organs and the broad range of unpredictable clinical manifestations of LRBA deficiency complicate the choice of therapeutic interventions. Although LRBA has been linked to Rab11-dependent trafficking of the immune checkpoint protein CTLA-4, its precise cellular role remains elusive. We show that LRBA, however, only slightly colocalizes with Rab11. Instead, LRBA is recruited by members of the small GTPase Arf protein family to the TGN and to Rab4+ endosomes, where it controls intracellular traffic. In patient-derived fibroblasts, loss of LRBA led to defects in the endosomal pathway promoting the accumulation of enlarged endolysosomes and lysosome secretion. Thus, LRBA appears to regulate flow through the endosomal system on Rab4+ endosomes. Our data strongly suggest functions of LRBA beyond CTLA-4 trafficking and provide a conceptual framework to develop new therapies for LRBA deficiency.

Ribosomal protein RPL39L is an efficiency factor in the cotranslational folding of a subset of proteins with alpha helical domains.

Increasingly many studies reveal how ribosome composition can be tuned to optimally translate the transcriptome of individual cell types. In this study, we investigated the expression pattern, structure within the ribosome and effect on protein synthesis of the ribosomal protein paralog 39L (RPL39L). With a novel mass spectrometric approach we revealed the expression of RPL39L protein beyond mouse germ cells, in human pluripotent cells, cancer cell lines and tissue samples. We generated RPL39L knock-out mouse embryonic stem cell (mESC) lines and demonstrated that RPL39L impacts the dynamics of translation, to support the pluripotency and differentiation, spontaneous and along the germ cell lineage. Most differences in protein abundance between WT and RPL39L KO lines were explained by widespread autophagy. By CryoEM analysis of purified RPL39 and RPL39L-containing ribosomes we found that, unlike RPL39, RPL39L has two distinct conformations in the exposed segment of the nascent peptide exit tunnel, creating a distinct hydrophobic patch that has been predicted to support the efficient co-translational folding of alpha helices. Our study shows that ribosomal protein paralogs provide switchable modular components that can tune translation to the protein production needs of individual cell types.

The ER-SURF pathway uses ER-mitochondria contact sites for protein targeting to mitochondria.

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria in a post-translational reaction. Mitochondrial precursor proteins which use the ER-SURF pathway employ the surface of the endoplasmic reticulum (ER) as an important sorting platform. How they reach the mitochondrial import machinery from the ER is not known. Here we show that mitochondrial contact sites play a crucial role in the ER-to-mitochondria transfer of precursor proteins. The ER mitochondria encounter structure (ERMES) and Tom70, together with Djp1 and Lam6, are part of two parallel and partially redundant ER-to-mitochondria delivery routes. When ER-to-mitochondria transfer is prevented by loss of these two contact sites, many precursors of mitochondrial inner membrane proteins are left stranded on the ER membrane, resulting in mitochondrial dysfunction. Our observations support an active role of the ER in mitochondrial protein biogenesis.

The protein tyrosine phosphatase PPH-7 is required for fertility and embryonic development in C. elegans at elevated temperatures.

Post-translational modifications are key in the regulation of activity, structure, localization, and stability of most proteins in eukaryotes. Phosphorylation is potentially the most studied post-translational modification, also due to its reversibility and thereby the regulatory role this modification often plays. While most research attention was focused on kinases in the past, phosphatases remain understudied, most probably because the addition and presence of the modification is more easily studied than its removal and absence. Here, we report the identification of an uncharacterized protein tyrosine phosphatase PPH-7 in C. elegans, a member of the evolutionary conserved PTPN family of phosphatases. Lack of PPH-7 function led to reduction of fertility and embryonic lethality at elevated temperatures. Proteomics revealed changes in the regulation of targets of the von Hippel-Lindau (VHL) E3 ligase, suggesting a potential role for PPH-7 in the regulation of VHL.

Functional interplay of lipid droplets and mitochondria.

Our body stores energy mostly in form of fatty acids (FAs) in lipid droplets (LDs). From there the FAs can be mobilized and transferred to peroxisomes and mitochondria. This transfer is dependent on close opposition of LDs and mitochondria and peroxisomes and happens at membrane contact sites. However, the composition and the dynamics of these contact sites is not well understood, which is in part due to the dependence on the metabolic state of the cell and on the cell- and tissue-type. Here, we summarize the current knowledge on the contacts between lipid droplets and mitochondria both in mammals and in the yeast Saccharomyces cerevisiae, in which various contact sites are well studied. We discuss possible functions of the contact site and their implication in disease.

Arf1 coordinates fatty acid metabolism and mitochondrial homeostasis.

Lipid mobilization through fatty acid ß-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where ß-oxidation products enter mitochondria and fuel the tricarboxylic acid cycle. Little is known about the physical and metabolic cooperation between these organelles. Here we found that expression of fatty acid transporters and of the rate-limiting enzyme involved in ß-oxidation is decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. Consequently, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although ß-oxidation occurs in both mitochondria and peroxisomes in mammals, Arf1's role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact sites.

Protein sorting from endosomes to the TGN.

Retrograde transport from endosomes to the trans-Golgi network is essential for recycling of protein and lipid cargoes to counterbalance anterograde membrane traffic. Protein cargo subjected to retrograde traffic include lysosomal acid-hydrolase receptors, SNARE proteins, processing enzymes, nutrient transporters, a variety of other transmembrane proteins, and some extracellular non-host proteins such as viral, plant, and bacterial toxins. Efficient delivery of these protein cargo molecules depends on sorting machineries selectively recognizing and concentrating them for their directed retrograde transport from endosomal compartments. In this review, we outline the different retrograde transport pathways governed by various sorting machineries involved in endosome-to-TGN transport. In addition, we discuss how this transport route can be analyzed experimentally.

Membrane tethers at a glance.

Cargo delivery from one compartment to the next relies on the fusion of vesicles with different cellular organelles in a process that requires the concerted action of tethering factors. Although all tethers act to bridge vesicle membranes to mediate fusion, they form very diverse groups as they differ in composition, and in their overall architecture and size, as well as their protein interactome. However, their conserved function relies on a common design. Recent data on class C Vps complexes indicates that tethers play a significant role in membrane fusion beyond vesicle capturing. Furthermore, these studies provide additional mechanistic insights into membrane fusion events and reveal that tethers should be considered as key players of the fusion machinery. Moreover, the discovery of the novel tether FERARI complex has changed our understanding of cargo transport in the endosomal system as it has been shown to mediate 'kiss-and-run' vesicle-target membrane interactions. In this Cell Science at a Glance and the accompanying poster, we compare the structure of the coiled-coil and the multisubunit CATCHR and class C Vps tether families on the basis of their functional analogy. We discuss the mechanism of membrane fusion, and summarize how tethers capture vesicles, mediate membrane fusion at different cellular compartments and regulate cargo traffic.

Sorting of cargo in the tubular endosomal network.

Intercellular communication is an essential process in all multicellular organisms. During this process, molecules secreted by one cell will bind to a receptor on the cognate cell leading to the subsequent uptake of the receptor-ligand complex. Once inside, the cell then determines the fate of the receptor-ligand complex and any other proteins that were endocytosed together. Approximately 80% of endocytosed material is recycled back to the plasma membrane either directly or indirectly via the Golgi apparatus and the remaining 20% is delivered to the lysosome for degradation. Although most pathways have been identified, we still lack understanding on how specificity in sorting of recycling cargos into different pathways is achieved, and how the cell reaches high accuracy of these processes in the absence of clear sorting signals in the bulk of the client proteins. In this review, we will summarize our current understanding of the mechanism behind recycling cargo sorting and propose a model of differential affinities between cargo and cargo receptors/adaptors with regards to iterative sorting in endosomes.

FERARI and cargo adaptors coordinate cargo flow through sorting endosomes.

Cellular organization, compartmentalization and cell-to-cell communication are crucially dependent on endosomal pathways. Sorting endosomes provide a transit point for various trafficking pathways and decide the fate of proteins: recycling, secretion or degradation. FERARI (Factors for Endosome Recycling and Rab Interactions) play a key role in shaping these compartments and coordinate Rab GTPase function with membrane fusion and fission of vesicles through a kiss-and-run mechanism. Here, we show that FERARI also mediate kiss-and-run of Rab5-positive vesicles with sorting endosomes. During these encounters, cargo flows from Rab5-positive vesicles into sorting endosomes and from there in Rab11-positive vesicles. Cargo flow from sorting endosomes into Rab11 structures relies on the cargo adaptor SNX6, while cargo retention in the Rab11 compartment is dependent on AP1. The available cargo amount appears to regulate the duration of kisses. We propose that FERARI, together with cargo adaptors, coordinate the vectorial flow of cargo through sorting endosomes.

The Caenorhabditis elegans Patched domain protein PTR-4 is required for proper organization of the precuticular apical extracellular matrix.

The Patched-related superfamily of transmembrane proteins can transport lipids or other hydrophobic molecules across cell membranes. While the Hedgehog receptor Patched has been intensively studied, much less is known about the biological roles of other Patched-related family members. Caenorhabditis elegans has a large number of Patched-related proteins, despite lacking a canonical Hedgehog pathway. Here, we show that PTR-4 promotes the assembly of the precuticle apical extracellular matrix, a transient and molecularly distinct matrix that precedes and patterns the later collagenous cuticle or exoskeleton. ptr-4 mutants share many phenotypes with precuticle mutants, including defects in eggshell dissolution, tube shaping, alae (cuticle ridge) structure, molting, and cuticle barrier function. PTR-4 localizes to the apical side of a subset of outward-facing epithelia, in a cyclical manner that peaks when precuticle matrix is present. Finally, PTR-4 is required to limit the accumulation of the lipocalin LPR-3 and to properly localize the Zona Pellucida domain protein LET-653 within the precuticle. We propose that PTR-4 transports lipids or other hydrophobic components that help to organize the precuticle and that the cuticle and molting defects seen in ptr-4 mutants result at least in part from earlier disorganization of the precuticle.

A novel live-cell imaging assay reveals regulation of endosome maturation.

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

The mRNA decapping complex is buffered by nuclear localization.

mRNA decay is a key step in regulating the cellular proteome. Processing bodies (P-bodies) are thought to be sites of mRNA decay and/or storage. P-body units assemble into P-body granules under stress conditions. How this assembly is regulated, however, remains poorly understood. Here, we show, in the yeast Saccharomyces cerevisiae, that the translational repressor Scd6 and the decapping stimulator Edc3 act partially redundantly in P-body assembly by sequestering the Dcp1-Dcp2 (denoted Dcp1/2) decapping complex in the cytoplasm and preventing it from becoming imported into the nucleus by the karyopherin ß protein Kap95. One of two nuclear localization signals in Dcp2 overlaps with the RNA-binding site, suggesting an additional mechanism to regulate Dcp1/2 localization. Nuclear Dcp1/2 does not drive mRNA decay and might be stored there as a readily releasable pool, indicating a dynamic equilibrium between cytoplasmic and nuclear Dcp1/2. Cytoplasmic Dcp1/2 is linked to Dhh1 via Edc3. Functional P-bodies are present at the endoplasmic reticulum where Dcp2 potentially acts to increase the local concentration of Dhh1 through interaction with Edc3 to drive phase separation and hence P-body formation.

The RNA-binding protein Puf5 contributes to buffering of mRNA upon chromatin-mediated changes in nascent transcription.

Gene expression involves regulation of chromatin structure and transcription, as well as processing of the transcribed mRNA. While there are feedback mechanisms, it is not clear whether these include crosstalk between chromatin architecture and mRNA decay. To address this, we performed a genome-wide genetic screen using a Saccharomyces cerevisiae strain harbouring the H3K56A mutation, which is known to perturb chromatin structure and nascent transcription. We identified Puf5 (also known as Mpt5) as essential in an H3K56A background. Depletion of Puf5 in this background leads to downregulation of Puf5 targets. We suggest that Puf5 plays a role in post-transcriptional buffering of mRNAs, and support this by transcriptional shutoff experiments in which Puf5 mRNA targets are degraded slower in H3K56A cells compared to wild-type cells. Finally, we show that post-transcriptional buffering of Puf5 targets is widespread and does not occur only in an H3K56A mutant, but also in an H3K4R background, which leads to a global increase in nascent transcription. Our data suggest that Puf5 determines the fate of its mRNA targets in a context-dependent manner acting as an mRNA surveillance hub balancing deregulated nascent transcription to maintain physiological mRNA levels.

Patched regulates lipid homeostasis by controlling cellular cholesterol levels.

Hedgehog (Hh) signaling is essential during development and in organ physiology. In the canonical pathway, Hh binding to Patched (PTCH) relieves the inhibition of Smoothened (SMO). Yet, PTCH may also perform SMO-independent functions. While the PTCH homolog PTC-3 is essential in C. elegans, worms lack SMO, providing an excellent model to probe non-canonical PTCH function. Here, we show that PTC-3 is a cholesterol transporter. ptc-3(RNAi) leads to accumulation of intracellular cholesterol and defects in ER structure and lipid droplet formation. These phenotypes were accompanied by a reduction in acyl chain (FA) length and desaturation. ptc-3(RNAi)-induced lethality, fat content and ER morphology defects were rescued by reducing dietary cholesterol. We provide evidence that cholesterol accumulation modulates the function of nuclear hormone receptors such as of the PPARα homolog NHR-49 and NHR-181, and affects FA composition. Our data uncover a role for PTCH in organelle structure maintenance and fat metabolism.

CHIMs are versatile cholesterol analogs mimicking and visualizing cholesterol behavior in lipid bilayers and cells.

Cholesterol is an essential component of cellular membranes regulating the structural integrity and fluidity of biological bilayers and cellular processes such as signal transduction and membrane trafficking. However, tools to investigate the role and dynamics of cholesterol in live cells are still scarce and often show limited applicability. To address this, we previously developed a class of imidazolium-based cholesterol analogs, CHIMs. Here we confirm that CHIM membrane integration characteristics largely mimic those of cholesterol. Computational studies in simulated phospholipid bilayers and biophysical analyses of model membranes reveal that in biologically relevant systems CHIMs behave similarly to natural cholesterol. Importantly, the analogs can functionally replace cholesterol in membranes, can be readily labeled by click chemistry and follow trafficking pathways of cholesterol in live cells. Thus, CHIMs represent chemically versatile cholesterol analogs that can serve as a flexible toolbox to study cholesterol behavior and function in live cells and organisms.

Means of intracellular communication: touching, kissing, fusing.

Eukaryotic cells are complicated factories that need ensure productivity and functionality on the cellular level as well as being able to communicate with their environment. In order to do so cells developed intracellular communication systems. For a long time, research focused mainly on the secretory/biosynthetic and endocytic routes for communication, leaving the communication with other organelles apart. In the last decade, this view has changed dramatically and a more holistic view of intracellular communication is emerging. We are still at the tip of the iceberg, but a common theme of touching, kissing, fusing is emerging as general principles of communication.