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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. [ABSTRACT FROM AUTHOR]

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-andrun' 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, various cellular functions are compartmentalized and performed by sophisticated and specialized organelles. However, the membrane-bounded organelles need to communicate with each other and with the cytoplasm, and sense the outside through the plasma membrane to coordinate various functions and to maintain cellular homeostasis. To maintain homeostasis, the information on the cellular state must be collected and appropriate responses initiated. The endoplasmic reticulum fulfils these functions. In this review, I will discuss various aspects of how the ER senses and relays information and acts to protect the cell, in what sometimes could be interpreted as an altruistic behavior. [ABSTRACT FROM AUTHOR]

The Caenorhabditis elegans early embryo is highly polarized, requiring sequestration of cytoplasmic polarity factors at the plasma membrane. This compartmentalization aids asymmetric distribution of lipids and proteins, which is partially responsible for the fates of the daughter cells. Since most plasma membrane proteins are glycosylated, we determined the effect of attenuation of N-glycosylation on cell polarity. While polarity establishment was not perturbed, the size difference between the two cells formed in first cell division (AB and P1) was more variable in embryos with reduced N-glycosylation than in the mock-treated embryos. In addition, among other deficiencies, we observed spindle orientation defects in two-cell embryos. Moreover, cell–cell adhesion was specifically lost at the two-cell stage when N-glycosylation was reduced. This loss-of-adhesion phenotype was rescued by interfering with polarity establishment, indicating that polarity establishment enforces plasma membrane compartmentalization. Consistent with this idea, the decreased plasma membrane levels of the adhesion proteins E-cadherin and MAGI-1 in ribo-1(RNAi) embryos were restored in the absence of functional PAR-2. Our data suggest a general role for N-glycosylation in plasma membrane compartmentalization and cell polarity. [ABSTRACT FROM AUTHOR]

The trans-Golgi network functions in the distribution of cargo into different transport vesicles that are destined to endosomes, lysosomes and the plasma membrane. Over the years, it has become clear that more than one transport pathway promotes plasma membrane localization of proteins. In spite of the importance of temporal and spatial control of protein localization at the plasma membrane, the regulation of sorting into and the formation of different transport containers are still poorly understood. In this review different transport pathways, with a special emphasis on exomer-dependent transport, and concepts of regulation and sorting at the TGN are discussed. [ABSTRACT FROM AUTHOR]

Endocytosis describes the processes by which proteins, peptides and solutes, and also pathogens, enter the cell. Endocytosed material progresses to endosomes. Genetic studies in yeast, worms, flies and mammals have identified a set of universally conserved proteins that are essential for early-to-late endosome transition and lysosome biogenesis, and for endolysosomal trafficking pathways, including autophagy. The two Vps-C complexes CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting) perform diverse biochemical functions in endocytosis: they tether membranes, interact with Rab GTPases, activate and proof-read SNARE assembly to drive membrane fusion, and possibly attach endosomes to the cytoskeleton. In addition, several of the CORVET and HOPS subunits have diversified in metazoans, and probably form additional specialized complexes to accomodate the higher complexity of trafficking pathways in these cells. Recent studies offer new insights into the complex relationships between CORVET and HOPS complexes and other factors of the endolysosomal pathway. Interactions with V- ATPase, the ESCRT machinery, phosphoinositides, the cytoskeleton and the Rab switch suggest an intricate cooperative network for endosome maturation. Accumulating evidence supports the view that endosomal tethering complexes implement a regulatory logic that governs endomembrane identity and dynamics. [ABSTRACT FROM AUTHOR]

N-glycosylation of proteins is an essential process, and N-glucans serve as important beacons in protein folding and ER associated degradation. More importantly, N-glycosylation increases the structural repertoire of proteins because the addition of the N-glucan on proteins will serve as a base for further sugar additions in the Golgi apparatus, and hence complex three-dimensional structures can be build. N-glycosylation is mediated by the ER-resident OST complex, which is essential throughout eukaryotes. Partial knockdown of conserved OST complex members, such as C. elegans RIBO-1, led to an embryonic lethal phenotype. Although the ER morphology was not grossly altered in ribo-1(RNAi) oocytes and embryos, secretion of yolk and of the yolk receptor RME-2 was perturbed in those worms. Perhaps as a consequence of reduced arrival of N-glycosylated proteins at the plasma membrane, cytokinesis occurred less efficiently leading to multinuclear cells. Unexpectedly, we detected a chromosome segregation defect in ribo-1(RNAi) embryos suggesting an essential role of at least one N-glycosylated protein in metaphase-anaphase transition. [ABSTRACT FROM AUTHOR]

The DSL1 complex is a conserved tethering complex at the endoplasmic reticulum that recognizes Golgi-derived COPI vesicles and hands them over to the fusion machinery. The DSL1 complex is the simplest tethering complex of the complexes associated with tethering containing helical rods ( CATCHR) family. CATCHR tethering complexes play a role at compartments along the exocytic and endocytic pathways. In this review, different functions of the DSL1 complex are discussed, some open questions with the seemingly straightforward picture are pointed out and alternative functions of the DSL1 complex members are mentioned. [ABSTRACT FROM AUTHOR]

Abstract: Arf GAP proteins are a versatile and diverse group of proteins. They control the activity of the GTP-binding proteins of the ARF family by inducing the hydrolysis of GTP that is bound to Arf proteins. The best-studied role of Arf GAPs is in intracellular traffic. In this review, we will focus mainly on the Arf GAPs that play a role in vesicle formation, Arf GAP1, Arf GAP2 and Arf GAP3 and their yeast homologues, Gcs1p and Glo3p. We discuss the roles of Arf GAPs as regulators and effectors for Arf GTP-binding proteins. [Copyright &y& Elsevier]

Proteins are endocytosed by various pathways into the cell. All these pathways converge at the level of the early endosome. The fate of the early endosome and how proteins are sorted into recycling and late endosomes/multi-vesicular body is a matter of debate and intense research. Obviously, the transition from early to late endosome poses an interesting logistic problem and would merit attention on an intellectual level. Numerous diseases are also caused by defects in turning off/over signaling molecules or mis-sorting of proteins at the level of the early endosome. This brief review aims to discuss different molecular mechanisms whereby early-to-late endosome transition could be achieved. [ABSTRACT FROM AUTHOR]

Transport in the ER–Golgi shuttle is performed by COPII in the anterograde and by COPI in the retrograde direction. How these transport steps are regulated and which mechanisms act to balance the vesicle flow between the ER and the Golgi apparatus are the issues currently under investigation. The elucidation of the structures of COPII components and tethers for COPI and COPII vesicles, together with detailed biochemical and cell biological analysis over the past few years greatly increased our knowledge on how vesicles are formed, not only at the ER and the Golgi but also at other organelles and the plasma membrane by analogy. Moreover, recent work provided more than just a glimpse on the function of tethering proteins at arrival sites on the ER and the Golgi. Most likely, organized exit and arrival sites exist at each vesicular transport step along the exocytic and endocytic routes. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: Several different processes must be completed in order to proceed through cell division. First, the centrosomes have to be duplicated and the genomic material is replicated. The separation of the chromatin is achieved by a bipolar spindle, which in turn is organized by the two centrosomes. The last step of cell division involves the separation of cellular content and the cleavage of the cell by cytokinesis. We used RNAi to study the centrosomal component SAS-5 in the early Caenorhabditis elegans embryo. While the first cell division and the establishment of polarity of sas-5 dsRNA-treated embryos was indistinguishable from wild type, subsequent cleavages were abnormal. Time-lapse microscopy studies of worms expressing β-tubulin::GFP revealed that the absence of SAS-5 results in a failure of mitotic spindle assembly starting at the two-cell stage embryo. Furthermore, the chromatin in at least one of the two cells in the early embryo was dispersed. Yet, this dispersion did neither trigger apoptosis nor affect nuclear envelope assembly. No intrinsic size control for the nucleus seems to exist in the early embryo. [Copyright &y& Elsevier]

Directionality in intracellular trafficking is essential to ensure the correct localization of proteins along the secretory pathway. Here, we found evidence for an active mechanism that prohibited back-fusion of de novo-generated vesicles with their donor compartment. Tip20p is a peripheral membrane protein implicated in consumption of COPI vesicles at the endoplasmic reticulum. However, a specific mutant of TIP20 did not interfere with COPII vesicle generation but allowed these vesicles to fuse back to the endoplasmic reticulum, a process that does not occur normally in the cell. [ABSTRACT FROM AUTHOR]

Understanding the biosynthetic pathways involved in cellular transport is an important issue in cell biology. More than a hundred years after the discovery of the Golgi apparatus, we still do not understand the regulation of vesicular transport to, within and from the Golgi apparatus. Recently, however, it has become clear that cargo might not simply be a passive passenger, and that ADP-ribosylation factor (ARF) GAPs are not only GTPase-activating proteins for ARF, but might play crucial roles in regulating coat protein complex I vesicle formation. [ABSTRACT FROM AUTHOR]

Addresses the intracellular dynamics of two mannosyltransferase complexes, Mnn9p and Mnn10p. Inclusion of retrieval to the endoplasmic reticulum (ER) in the itinerary of the complexes despite the normal cis-Golgi localization and function of the proteins; Consistency of behavior with an active Golgi to ER recycling process.

Mitochondria are the powerhouse of cells as they produce the bulk of ATP which is consumed by the cell. They form a highly interconnected network that is governed by fission and fusion processes. In addition, mitochondria and the endoplasmic reticulum (ER) are found in close proximity to each other and it is thought that they maintain contact sites to exchange molecules. The regulation and the function of these contact sites need to be further explored. The small GTPase Arf1 (ADP-ribosylation factor 1), which is best known for its essential role in the generation of coatomer protein I (COPI)-coated vesicles at the Golgi complex appears to be also essential for the dynamics and maintenance of mitochondrial function, presumably at ER-mitochondrial contact sites. [ABSTRACT FROM AUTHOR]

Discusses various aspects of transport vesicle budding. Assembly of cytosolic coat proteins on a donor membrane to form a vesicle; Specific requirements in order to produce a functional vesicle; Guanosine triphosphatase activation and membrane binding; Assembly of a priming complex; Formation of a polymetric coat.

Focuses on a study which assessed function of phospholipase D (PLD) and protein cargo in coat protein complex I (COPI) buds and vesicles' formation. Details on protein transport in cells; In-depth look at the formation process; Methodology used to conduct the study; Results of the study.

Studies the reconstitution of retrograde transport from the Golgi apparatus to the endoplasmic reticulum. Retrieval of soluble reporter protein; Requirements of a full cycle of anterograde and retrograde transport; Putative retrograde carriers.

Reports on the association of the Tub4p novel tubulin with the spindle pole body(SPB) of Saccharomyces cerevisiae. Involvement in the microtubule attachments; Functions in organization; Assembly of Tub4-1 in spindle; Post-spindle formation defect; Chromosome loss in Tub4-1; Significance of mitotic checkpoint control gene BUB2 in Tub4-1's cell cycle and survival.

Focuses on the association of Tub4p with the inner and outerplaques of the yeast microtubule organizing center, the spindlepole body (SPB). Involvement of the SPB substructures in theattachment of the nuclear and cytoplasmic microtubules; Role ofTub4p in microtubule organization; Inhibition of microtubulenucleation after disruption of the essential y-tubulin genes inAspergillus nidulans.

Investigates the cell cycle-dependent localization of the Kar1 protein and its interaction with other spindle pole body (SPB) components. DNA techniques; Strain constructions; Nuclei isolation, SPB preparation, cell fractionation and cell extracts; Preparation of affinity-purified anti-Kar1p, anti-N-Nuflp, anti-beta-tubulin antibodies; Immunoelectron microscopy.

Investigates the cell cycle function of the calcium-binding protein, cell division cycle 31 (CDC31) of Saccharomyces cerevisiae. Cloning and isolation of the CDC31; Localization of the CDC31; Role of CDC31 in spindle pole body duplication; Ca(2+) binding to CDC31; Cofractionation with spindle pole bodies; Role of CDC31 in cell viability.

How are tight junctions maintained? In this issue of Cell, provide intriguing evidence for a new pathway that links polarity proteins and vesicle transport to the maintenance of tight junctions, through the control of Cdc42 by Rich1, a GTPase-activating protein. [Copyright &y& Elsevier]

Two recent structural studies reveal how cargo proteins interact with vesicle coats, which may also explain vesicle formation of retrograde transport vesicles. [ABSTRACT FROM AUTHOR]

Summary: How are SNARE proteins included into transport vesicles? One way is through the interaction with ArfGAP proteins. A recent study reports that the ArfGAP Hrb can wrap around the SNARE VAMP7, causing its endocytosis by clathrin-coated vesicles. [Copyright &y& Elsevier]

COPII vesicles transport proteins destined for secretion from the ER to the Golgi apparatus. A recent study has shown that, in budding yeast, the formation of COPII vesicles requires Yip1p, an effector protein of a Rab GTPase. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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 Patchedrelated 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. [ABSTRACT FROM AUTHOR]

Intracellular transport is largely dependent on vesicles that bud off from one compartment and fuse with the target compartment. The first contact of an incoming vesicle with the target membrane is mediated by tethering factors. The tethering factor responsible for recruiting Golgi-derived vesicles to the ER is the Dsl1 tethering complex, which is comprised of the essential proteins Dsllp, Dsl3p, and Tip20p. We investigated the role of the Tip20p subunit at the ER by analyzing two mutants, tip20-5 and tip20-8. Both mutants contained multiple mutations that were scattered throughout the TIP20 sequence. Individual mutations could not reproduce the temperature-sensitive phenotype of tip20-5 and tip20-8, indicating that the overall structure of Tip20p might be altered in the mutants. Using molecular dynamics simulations comparing Tip20p and Tip20-8p revealed that some regions, particularly the N-terminal domain and parts of the stalk region, were more flexible in the mutant protein, consistent with its increased susceptibility to proteolysis. Both Tip20-Sp and Tip20-8p mutants prevented proper ER trans-SNARE complex assembly in vitro, Moreover, Tip2p mutant proteins disturbed the interaction between Dsllp and the coatomer coat complex, indicating that the Dsllp-coatomer interaction could be stabilized or regulated by Tip20p. We provide evidence for a direct role of the Dsl1 complex, in particular Tip20p, in the formation and stabilization of ER SNARE complexes. [ABSTRACT FROM AUTHOR]

The p24 family members are transmembrane proteins assembled into heteromeric complexes that continuously cycle between the ER and the Golgi apparatus. These cargo proteins were assumed to play a structural role in COPI budding because of their major presence in mammalian COPI vesicles. However, this putative function has not been proved conclusively so far. Furthermore, deletion of all eight yeast p24 family members does not produce severe transport phenotypes, suggesting that the p24 complex is not essential for COPI function. In this paper we provide direct evidence that the yeast p24 complex plays an active role in retrograde transport from Golgi to ER by facilitating the formation of COPI-coated vesicles. Therefore, our results demonstrate that p24 proteins are important for vesicle formation instead of simply being a passive traveler, supporting the model in which cargo together with a small GTPase of the ARF superfamily and coat subunits act as primer for vesicle formation. [ABSTRACT FROM AUTHOR]

POM121 and gp210 were, until this point, the only known membrane-integral nucleoporins (Nups) of vertebrates and, thus, the only candidate anchors for nuclear pore complexes (NPCs) within the nuclear membrane. In an accompanying study (see Stavru et al. on p. 477 of this issue), we provided evidence that NPCs can exist independently of POM121 and gp210, and we predicted that vertebrate NPCs contain additional membrane-integral constituents. We identify such an additional membrane protein in the NPCs of mammals, frogs, insects, and nematodes as the orthologue to yeast Ndc1p/Cut11p. Human NDC1 (hNDC1) likely possesses six transmembrane segments, and it is located at the nuclear pore wall. Depletion of hNDC1 from human HeLa cells interferes with the assembly of phenylalanine-glycine repeat Nups into NPCs. The loss of NDC1 function in Caenorhabditis elegans also causes severe NPC defects and very high larval and embryonic mortality. However, it is not ultimately lethal. Instead, homozygous NDC1-deficient worms can be propagated. This indicates that none of the membrane-integral Nups is universally essential for NPC assembly, and suggests that NPC biogenesis is an extremely fault-tolerant process. [ABSTRACT FROM AUTHOR]

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. Matos et al characterise previously developed cholesterol analogues, named CHIMs, which can be fluorescently labelled by click-chemistry. They find that CHIMs segregate similarly to cholesterol in lipid bilayers, allow the visualisation of cholesterol dynamics in cells, and can functionally replace cholesterol in C.elegans. [ABSTRACT FROM AUTHOR]

ARF proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the actions of GTPase-activating proteins (GAPs) for their function. The in vitro GTPase activity of the Saccharo- myces cerevisiae ARF proteins Arf1 and Arf2 is stimu- lated by the yeast Gcs1 protein, and in vivo genetic interactions between arf and gcs1 mutations implicate Gcs1 in vesicular transport. However, the Gcs1 protein is dispensable, indicating that additional ARF GAP proteins exist. We show that the structurally related protein Glo3, which is also dispensable, also exhibits ARF GAP activity. Genetic and in vitro approaches reveal that Glo3 and Gcs1 have an overlapping essential function at the endoplasmic reticulum (ER)—Golgi stage of vesicular transport. Mutant cells deficient for both ARF GAPs cannot proliferate, undergo a dramatic accumulation of ER and are defective for protein transport between ER and Golgi. The glo3Δ and gcs1Δ single mutations each interact with a sec21 mutation that affects a component of COPI, which mediates vesicular transport within the ER—Golgi shuttle, while increased dosage of the BET1, BOS1 and SEC22 genes encoding members of a v-SNARE family that functions within the ER—Golgi alleviates the effects of a glo3Δ mutation. An in vitro assay indicates that efficient retrieval from the Golgi to the ER requires these two proteins. These findings suggest that Glo3 and Gcs1 ARF GAPs mediate retrograde vesicular transport from the Golgi to the ER. [ABSTRACT FROM AUTHOR]

Spatiotemporal restriction of signaling plays a critical role in animal development and tissue homeostasis. All stem and progenitor cells in newly hatched C. elegans larvae are quiescent and capable of suspending their development until sufficient food is supplied. Here, we show that ptr-18, which encodes the evolutionarily conserved patched-related (PTR)/patched domain-containing (PTCHD) protein, temporally restricts the availability of extracellular hedgehog-related protein to establish the capacity of progenitor cells to maintain quiescence. We found that neural progenitor cells exit from quiescence in ptr-18 mutant larvae even when hatched under starved conditions. This unwanted reactivation depended on the activity of a specific set of hedgehog-related grl genes including grl-7. Unexpectedly, neither PTR-18 nor GRL-7 were expressed in newly hatched wild-type larvae. Instead, at the late embryonic stage, both PTR-18 and GRL-7 proteins were first localized around the apical membrane of hypodermal and neural progenitor cells and subsequently targeted for lysosomal degradation before hatching. Loss of ptr-18 caused a significant delay in GRL-7 clearance, causing this protein to be retained in the extracellular space in newly hatched ptr-18 mutant larvae. Furthermore, the putative transporter activity of PTR-18 was shown to be required for the appropriate function of the protein. These findings not only uncover a previously undescribed role of PTR/PTCHD in the clearance of extracellular hedgehog-related proteins via endocytosis-mediated degradation but also illustrate that failure to temporally restrict intercellular signaling during embryogenesis can subsequently compromise post-embryonic progenitor cell function. Author summary: Inside the animal body, many "signals" are exchanged between tissues and cells to ensure healthy growth and physiological conditions. Too much or less signals have been known to cause various diseases such as cancer and diabetes. When developing animals are not fed enough food, their growth rates generally slow down. We found that when nematodes lacking the PTR-18 protein are hatched out of the egg in the absence of sufficient food, they do not appropriately halt their growth. Analyses at the molecular and cellular levels showed that immediately before hatching, PTR-18 re-uptakes a signaling molecule called GRL-7, which is known to promote growth, from the outside to the inside of cells, resulting in the degradation of GRL-7. As a result, worms lacking PTR-18 still contain excess GRL-7 after hatching, which results in unwanted growth even when ample food is not available. Thus, our findings uncover the role of PTR-18 in terminating a signal on time. Humans also possess a protein called PTCHD1, which is structurally similar to the worm's PTR-18 and has been proposed to cause autistic spectrum disorders and learning disabilities. Our findings may provide a clue to further understand the function of human PTCHD1. [ABSTRACT FROM AUTHOR]

The endo‐lysosome system is involved in endocytosis, protein sorting, and degradation as well as autophagy. Numerous toxins and pathogens exploit this system to enter host cells and exert their deleterious effects. Modulation of host endo‐lysosome pathway may restrict multiple toxins intoxication as well as pathogen infection. ABMA, selected from a high‐throughput screening against the cytotoxicity of ricin toxin, exhibits a broad‐spectrum antitoxin and antipathogen activity. Here, we show that ABMA selectively retains endocytosed protein and toxin to late endosomes and thus delaying their intracellular trafficking. It also impairs the autophagic flux by excessive fusion of late endosomes and autophagosomes. Its exclusive action on late endosomes and corresponding consequences on the endo‐lysosomal pathway and autophagic flux are distinct from known inhibitors such as bafilomycin A1, EGA, or chloroquine. Hence, besides being a broad‐spectrum inhibitor of endocytosed toxins and pathogens, ABMA may serve as a molecular tool to dissect endo‐lysosome system‐related cellular physiology and mechanisms of pathogenesis. [ABSTRACT FROM AUTHOR]

Mitochondrial biogenesis relies on the synthesis of hundreds of different precursor proteins in the cytosol and their subsequent import into the organelle. Recent studies suggest that the surface of the endoplasmic reticulum (ER) actively contributes to the targeting of some mitochondrial precursors. In the past, in vitro import experiments with isolated mitochondria proved to be extremely powerful to elucidate the individual reactions of the mitochondrial import machinery. However, this in vitro approach is not well suited to study the influence of non-mitochondrial membranes. In this study, we describe an in vitro system using semi-intact yeast cells to test a potential import relevance of the ER proteins Erg3, Lcb5 and Ssh1, all being required for efficient mitochondrial respiration. We optimized the conditions of this experimental test system and found that cells lacking Ssh1, a paralog of the Sec61 translocation pore, show a reduced import efficiency of mitochondrial precursor proteins. Our results suggest that Ssh1, directly or indirectly, increases the efficiency of the biogenesis of mitochondrial proteins. Our findings are compatible with a functional interdependence of the mitochondrial and the ER protein translocation systems. [ABSTRACT FROM AUTHOR]

The article presents a study in which a genome-wide screen in yeast was used to identify factors critical for the intracellular sorting of the mitochondrial inner membrane protein Oxa1 and uncovered a pathway which retrieves mitochondrial membrane proteins from the endoplasmic reticulum (ER) surface and reroutes them to mitochondria.

Cargo is transported from the trans-Golgi Network to the plasma membrane by adaptor complexes, which are pan-eukaryotic components. However, in yeast, cargo can also be exported by the exomer complex, a heterotetrameric protein complex consisting of two copies of Chs5, and any two members of four paralogous proteins (ChAPs). To understand the larger relevance of exomer, its phylogenetic distribution and function outside of yeast need to be explored. We find that the four ChAP proteins are derived from gene duplications after the divergence of Yarrowia from the remaining Saccharomycotina, with BC8 paralogues (Bch2 and Chs6) being more diverged relative to the BB8 paralogues (Bch1 and Bud7), suggesting neofunctionalization. Outside Ascomycota, a single preduplicate ChAP is present in nearly all Fungi and in diverse eukaryotes, but has been repeatedly lost. Chs5, however, is a fungal specific feature, appearing coincidentally with the loss of AP-4. In contrast, the ChAP protein is a wide-spread, yet uncharacterized, membrane-trafficking component, adding one more piece to the increasingly complex machinery deduced as being present in our ancient eukaryotic ancestor. [ABSTRACT FROM AUTHOR]

Cells respond to stress by remodeling their transcriptome through transcription and degradation. Xrn1p-dependent degradation in P-bodies is the most prevalent decay pathway, yet, P-bodies may facilitate not only decay, but also act as a storage compartment. However, which and how mRNAs are selected into different degradation pathways and what determines the fate of any given mRNA in P-bodies remain largely unknown. We devised a new method to identify both common and stress-specific mRNA subsets associated with P-bodies. mRNAs targeted for degradation to P-bodies, decayed with different kinetics. Moreover, the localization of a specific set of mRNAs to P-bodies under glucose deprivation was obligatory to prevent decay. Depending on its client mRNA, the RNA-binding protein Puf5p either promoted or inhibited decay. Furthermore, the Puf5p-dependent storage of a subset of mRNAs in P-bodies under glucose starvation may be beneficial with respect to chronological lifespan. [ABSTRACT FROM AUTHOR]

Despite its biological and medical relevance, traffic from the Golgi to the plasma membrane (PM) is one of the least understood steps of secretion. Exomer is a protein complex that mediates the trafficking of certain cargoes from the trans-Golgi network/early endosomes to the PM in budding yeast. Here, we show that in Schizosaccharomyces pombe the Cfr1 and Bch1 proteins constitute the simplest form of an exomer. Cfr1 co-immunoprecipitates with Assembly Polypeptide adaptor 1 (AP-1), AP-2, and Golgi-localized, gamma-adaptin ear domain homology, ARF-binding (GGA) subunits, and cfr1+ interacts genetically with AP-1 and GGA genes. Exomer-defective cells exhibit multiple mild defects, including alterations in the morphology of Golgi stacks and the distribution of the synaptobrevin-like Syb1 protein, carboxypeptidase missorting, and stress sensitivity. S. pombe apm1Δ cells exhibit a defect in trafficking through the early endosomes that is severely aggravated in the absence of exomer. apm1Δ cfr1Δ cells exhibit a dramatic disorganization of intracellular compartments, including massive accumulation of electron-dense tubulovesicular structures. While the trans-Golgi network/early endosomes are severely disorganized in the apm1Δ cfr1Δ strain, gga21Δ gga22Δ cfr1Δ cells exhibit a significant disturbance of the prevacuolar/vacuolar compartments. Our findings show that exomer collaborates with clathrin adaptors in trafficking through diverse cellular compartments, and that this collaboration is important to maintain their integrity. These results indicate that the effect of eliminating exomer is more pervasive than that described to date, and suggest that exomer complexes might participate in diverse steps of vesicle transport in other organisms. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER)—mitochondrial contact sites play a pivotal role in exchange of lipids and ions between the two organelles. How size and function of these contact sites are regulated remains elusive. Here we report a previously unanticipated, but conserved role of the small GTPase Sar1 in the regulation of ER-mitochondrial contact site size. Activated Sar1 introduces membrane curvature through its N-terminal amphiphatic helix at the ER-mitochondria interphase and thereby reducing contact size. Conversely, the S. cerevisiae N3-Sar1 mutant, in which curvature induction is decreased, caused an increase in ER-mitochondrial contacts. As a consequence, ER tubules are no longer able to mark the prospective scission site on mitochondria, thereby impairing mitochondrial dynamics. Consistently, blocking mitochondrial fusion partially rescued, whereas deletion of the dynamin-like protein enhanced the phenotype in the sar1D32G mutant. We conclude that Sar1 regulates the size of ER-mitochondria contact sites through its effects on membrane curvature. [ABSTRACT FROM AUTHOR]

The mammalian serum- and glucocorticoid-inducible kinase SGK1 regulates the endocytosis of ion channels. Here we report that in C. elegans sgk-1 null mutants, GFP-tagged MIG-14/Wntless, the sorting receptor of Wnt, failed to localize to the basolateral membrane of intestinal cells; instead, it was mis-sorted to lysosomes. This effect can be explained in part by altered sphingolipid levels, because reducing glucosylceramide biosynthesis restored the localization of MIG-14::GFP. Membrane traffic was not perturbed in general, as no obvious morphological defects were detected for early endosomes, the Golgi apparatus, and the endoplasmic reticulum (ER) in sgk-1 null animals. The recycling of MIG-14/Wntless through the Golgi might be partially responsible for the observed phenotype because the subcellular distribution of two plasma membrane cargoes that do not recycle through the trans-Golgi network (TGN) was affected to a lesser degree. Consistently, knockdown of the ArfGEF gbf-1 altered the distribution of SGK-1 at the basolateral membrane of intestinal cells. In addition, we found that sgk-1(RNAi) induced unfolded protein response in the ER, suggesting at least an indirect role of SGK-1 early in the secretory pathway. We propose that SGK-1 function is required for lipid homeostasis and that it acts at different intracellular trafficking steps. [ABSTRACT FROM AUTHOR]

The small GTPase Arf1 plays critical roles in membrane traffic by initiating the recruitment of coat proteins and by modulating the activity of lipid-modifying enzymes. Here, we report an unexpected but evolutionarily conserved role for Arf1 and the Arf GEF GBF1 at mitochondria. Loss of function of ARF-1 or GBF-1 impaired mitochondrial morphology and activity in Caenorhabditis elegans. Similarly, mitochondrial defects were observed in mammalian and yeast cells. In Saccharomyces cerevisiae, aberrant clusters of the mitofusin Fzo1 accumulated in arf1-11 mutants and were resolved by overexpression of Cdc48, an AAA- ATPase involved in ER and mitochondria-associated degradation processes. Yeast Arf1 co-fractionated with ER and mitochondrial membranes and interacted genetically with the contact site component Gem1. Furthermore, similar mitochondrial abnormalities resulted from knockdown of either GBF-1 or contact site components in worms, suggesting that the role of Arf1 in mitochondrial functioning is linked to ER-mitochondrial contacts. Thus, Arf1 is involved in mitochondrial homeostasis and dynamics, independent of its role in vesicular traffic. [ABSTRACT FROM AUTHOR]

Numerous mRNAs are degraded in processing bodies (P bodies) in Saccharomyces cerevisiae. In logarithmically growing cells, only 0-1 P bodies per cell are detectable. However, the number and appearance of P bodies change once the cell encounters stress. Here, we show that the polysome-associated mRNA-binding protein Scp160 interacts with P body components, such as the decapping protein Dcp2 and the scaffold protein Pat1, presumably, on polysomes. Loss of either Scp160 or its interaction partner Bfr1 caused the formation of Dcp2-positive structures. These Dcp2-positive foci contained mRNA, because their formation was inhibited by the presence of cycloheximide. In addition, Scp160 was required for proper P body formation because only a subset of bona fide P body components could assemble into the Dcp2-positive foci in Δscp160 cells. In either Abfr1 or Ascp160 cells, P body formation was uncoupled from translational attenuation as the polysome profile remained unchanged. Collectively, our data suggest that Bfr1 and Scp160 prevent P body formation under normal growth conditions. [ABSTRACT FROM AUTHOR]

Chs3, the catalytic subunit of chitin synthase III in Saccharomyces cerevisiae, is a complex polytopic membrane protein whose plasma membrane expression is tightly controlled: export from the ER requires interaction with Chs7; exit from the Golgi is dependent on the exomer complex, and precise bud neck localization relies on endocytosis. Moreover, Chs3 is efficiently recycled from endosomes to the TGN in an AP-1-dependent manner. Here we show that the export of Chs3 requires the cargo receptor Erv14, in a step that is independent of Chs7. Chs3 oligomerized in the ER through its N-terminal cytosolic region. However, the truncated Δ126 Chs3 was still exported by Erv14, but was sent back from the Golgi to the ER in a COPI- and Rer1-dependent manner. A subset of the oligomerization-deficient Chs3 proteins evaded Golgi quality control and reached the plasma membrane, where they were enzymatically active but poorly endocytosed. This resulted in high CSIII levels, but calcofluor white resistance, explained by the reduced intercalation of calcofluor white between nascent chitin fibres. Our data show that the oligomerization of Chs3 through its N-terminus is essential for proper protein trafficking and chitin synthesis and is therefore monitored intracellularly. [ABSTRACT FROM AUTHOR]