SLC3A2 N-glycosylation and Golgi remodeling regulate SLC7A amino acid exchangers and stress mitigation.

Proteostasis requires oxidative metabolism (ATP) and mitigation of the associated damage by glutathione, in an increasingly dysfunctional relationship with aging. SLC3A2 (4F2hc, CD98) plays a role as a disulfide-linked adaptor to the SLC7A5 and SLC7A11 exchangers which import essential amino acids and cystine while exporting Gln and Glu, respectively. The positions of N-glycosylation sites on SLC3A2 have evolved with the emergence of primates, presumably in synchrony with metabolism. Herein, we report that each of the four sites in SLC3A2 has distinct profiles of Golgi-modified N-glycans. N-glycans at the primate-derived site N381 stabilized SLC3A2 in the galectin-3 lattice against coated-pit endocytosis, while N365, the site nearest the membrane promoted glycolipid-galectin-3 (GL-Lect)-driven endocytosis. Our results indicate that surface retention and endocytosis are precisely balanced by the number, position, and remodeling of N-glycans on SLC3A2. Furthermore, proteomics and functional assays revealed an N-glycan-dependent clustering of the SLC3A2∗SLC7A5 heterodimer with amino-acid/Na+ symporters (SLC1A4, SLC1A5) that balances branched-chain amino acids and Gln levels, at the expense of ATP to maintain the Na+/K+ gradient. In replete conditions, SLC3A2 interactions require Golgi-modified N-glycans at N365D and N381D, whereas reducing N-glycosylation in the endoplasmic reticulum by fluvastatin treatment promoted the recruitment of CD44 and transporters needed to mitigate stress. Thus, SLC3A2 N-glycosylation and Golgi remodeling of the N-glycans have distinct roles in amino acids import for growth, maintenance, and metabolic stresses.

Site-specific N-glycan profiles of α5 ß1 integrin from rat liver.

BACKGROUND INFORMATION: Like most other cell surface proteins, α5 ß1 integrin is glycosylated, which is required for its various activities in ways that mostly remain to be determined. RESULTS: Here, we have established the first comprehensive site-specific glycan map of α5 ß1 integrin that was purified from a natural source, that is, rat liver. This analysis revealed striking site selective variations in glycan composition. Complex bi, tri, or tetraantennary N-glycans were predominant at various proportions at most potential N-glycosylation sites. A few of these sites were nonglycosylated or contained high mannose or hybrid glycans, indicating that early N-glycan processing was hindered. Almost all complex N-glycans had fully galactosylated and sialylated antennae. Moderate levels of core fucosylation and high levels of O-acetylation of NeuAc residues were observed at certain sites. An O-linked HexNAc was found in an EGF-like domain of ß1 integrin. The extensive glycan information that results from our study was projected onto a map of α5 ß1 integrin that was obtained by homology modeling. We have used this model for the discussion of how glycosylation might be used in the functional cycle of α5 ß1 integrin. A striking example concerns the involvement of glycan-binding galectins in the regulation of the molecular homeostasis of glycoproteins at the cell surface through the formation of lattices or endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis. CONCLUSION: We expect that the glycoproteomics data of the current study will serve as a resource for the exploration of structural mechanisms by which glycans control α5 ß1 integrin activity and endocytic trafficking. SIGNIFICANCE: Glycosylation of α5 ß1 integrin has been implicated in multiple aspects of integrin function and structure. Yet, detailed knowledge of its glycosylation, notably the specific sites of glycosylation, is lacking. Furthermore, the α5 ß1 integrin preparation that was analyzed here is from a natural source, which is of importance as there is not a lot of literature in the field about the glycosylation of "native" glycoproteins.

Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells.

E-cadherin (E-cad) is the main component of epithelial junctions in multicellular organisms, where it is essential for cell-cell adhesion. The localisation of E-cad is often strongly polarised in the apico-basal axis. However, the mechanisms required for its polarised distribution are still largely unknown. We performed a systematic RNAi screen in vivo to identify genes required for the strict E-cad apical localisation in C. elegans epithelial epidermal cells. We found that the loss of clathrin, its adaptor AP-1 and the AP-1 interactor SOAP-1 induced a basolateral localisation of E-cad without affecting the apico-basal diffusion barrier. We further found that SOAP-1 controls AP-1 localisation, and that AP-1 is required for clathrin recruitment. Finally, we also show that AP-1 controls E-cad apical delivery and actin organisation during embryonic elongation, the final morphogenetic step of embryogenesis. We therefore propose that a molecular pathway, containing SOAP-1, AP-1 and clathrin, controls the apical delivery of E-cad and morphogenesis.

AP-1 is required for the maintenance of apico-basal polarity in the C. elegans intestine.

Epithelial tubes perform functions that are essential for the survival of multicellular organisms. Understanding how their polarised features are maintained is therefore crucial. By analysing the function of the clathrin adaptor AP-1 in the C. elegans intestine, we found that AP-1 is required for epithelial polarity maintenance. Depletion of AP-1 subunits does not affect epithelial polarity establishment or the formation of the intestinal lumen. However, the loss of AP-1 affects the polarised distribution of both apical and basolateral transmembrane proteins. Moreover, it triggers de novo formation of ectopic apical lumens between intestinal cells along the lateral membranes later during embryogenesis. We also found that AP-1 is specifically required for the apical localisation of the small GTPase CDC-42 and the polarity determinant PAR-6. Our results demonstrate that AP-1 controls an apical trafficking pathway required for the maintenance of epithelial polarity in vivo in a tubular epithelium.

Endocytic Roles of Glycans on Proteins and Lipids.

Most cell surface proteins are decorated by glycans, and the plasma membrane is rich in glycosylated lipids. The mechanisms by which the enormous complexity of these glycan structures on proteins and lipids is exploited to control glycoprotein activity by setting their cell surface residence time and the ways by which they are taken up into cells are still under active investigation. Here, two mechanisms are presented, termed galectin lattices and glycolipid-lectin (GL-Lect)-driven endocytosis, which are among the most prominent to establish a link between glycan information and endocytosis. Types of glycans on glycoproteins and glycolipids are reviewed from the angle of their interaction with glycan-binding proteins that are at the heart of galectin lattices and GL-Lect-driven endocytosis. Examples are given to show how these mechanisms affect cellular functions ranging from cell migration and signaling to vascularization and immune modulation. Finally, outstanding challenges on the link between glycosylation and endocytosis are discussed.

Transcytosis of Galectin-3 in Mouse Intestine.

The GlycoLipid-Lectin (GL-Lect) hypothesis provides a conceptual framework to explain how endocytic pits are built in processes of clathrin-independent endocytosis. According to this hypothesis, oligomeric cellular or pathogenic lectins interact with glycosylated plasma membrane lipids in a way such as to drive the formation of tubular endocytic pits that then detach to generate clathrin-independent endocytic carriers for the cellular uptake of cellular or pathogenic products. This process operates in a complementary manner to the conventional clathrin pathway for biological function linked to cell polarity. Up to date, the premises of the GL-Lect hypothesis have been based on model membrane and cell culture experiments. It has therefore become urgent to extend its exploration to complex organisms. In the current protocol, we describe methods to study the endocytosis and transcytosis of a key driver of the GL-Lect mechanism, the cellular galectin-3, and of one of its cargoes, lactotransferrin, in enterocytes of the intact jejunum of mice. In a step-by-step manner, we present the generation of fluorescent endocytic ligands, tissue preparation for cellular uptake measurements, binding and internalization assays, tissue fixation and preparation for sectioning, light and electron microscopical observations, and quantification of data by image processing. Pitfalls are discussed to optimize the chances of success with the described methods.

Solubilization and Purification of α5ß1 Integrin from Rat Liver for Reconstitution into Nanodiscs.

Transmembrane proteins (or integral membrane proteins) are synthesized in the endoplasmic reticulum where most of them are core glycosylated prior to folding and in some cases assembly into quaternary structures. Correctly glycosylated, folded, and assembled transmembrane proteins are then shuttled to the Golgi apparatus for additional posttranslational modifications such as complex-type glycosylations, sulfation or proteolytic clipping. At the plasma membrane, the glycosylated extracellular domains are key to communicate with the cellular environment for a variety of functions, such as binding to the extracellular matrix for cell adhesion and migration, to neighboring cells for cell-cell interaction, or to extracellular components for nutrient uptake and cell signaling. Intracellular domains are essential to mediate signaling cascades, or to connect to cytosolic adaptors for internalization and intracellular compartmentalization. Despite its importance for the understanding of molecular mechanisms of transmembrane protein function, the determination of their structures has remained a challenging task. In recent years, their reconstitution in lipid nanodiscs in combination with high resolution cryo-electron microscopy has provided novel avenues to render this process more accessible. Here, we describe detailed protocols for the solubilization of heavily glycosylated α5ß1 integrin from rat livers, its purification and reconstitution into nanodiscs. At the plasma membrane of many cells, including tumor metastases, this essential heterodimeric transmembrane protein mediates the communication between extracellular matrix and cytosolic cytoskeleton in processes of cell adhesion and migration. We expect that the protocols that are described here will provide new opportunities for the determination of the full structure of α5ß1 integrin, as well as for the understanding of how interacting partners can regulate function and activity of this transmembrane protein.

Galectin-3 Binding to α5ß1 Integrin in Pore Suspended Biomembranes.

Galectin-3 (Gal3) is a ß-galactoside binding lectin that mediates many physiological functions, including the binding of cells to the extracellular matrix for which the glycoprotein α5ß1 integrin is of critical importance. The mechanisms by which Gal3 interacts with membranes have not been widely explored to date due to the complexity of cell membranes and the difficulty of integrin reconstitution within model membranes. Herein, to study their interaction, Gal3 and α5ß1 were purified, and the latter reconstituted into pore-suspended lipid bilayers comprised eggPC:eggPA. Using electrochemical impedance and fluorescence lifetime correlation spectroscopy, we found that on incubation with low nanomolar concentrations of wild-type Gal3, the membrane's admittance and fluidity, as well as integrin's lateral diffusivity, were enhanced. These effects were diminished in the following conditions: (i) absence of integrin, (ii) presence of lactose as a competitive inhibitor of glycan-Gal3 interaction, and (iii) use of a Gal3 mutant that lacked the N-terminal oligomerization domain (Gal3ΔNter). These findings indicated that WTGal3 oligomerized on α5ß1 integrin in a glycan-dependent manner and that the N-terminal domain interacted directly with membranes in a way that is yet to be fully understood. At concentrations above 10 nM of WTGal3, membrane capacitance started to decrease and very slowly diffusing molecular species appeared, which indicated the formation of protein clusters made from WTGal3-α5ß1 integrin assemblies. Overall, our study demonstrates the capacity of WTGal3 to oligomerize in a cargo protein-dependent manner at low nanomolar concentrations. Of note, these WTGal3 oligomers appeared to have membrane active properties that could only be revealed using our sensitive methods. At slightly higher WTGal3 concentrations, the capacity to generate lateral assemblies between cargo proteins was observed. In cells, this could lead to the construction of tubular endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis or to the formation of galectin lattices, depending on cargo glycoprotein stability at the membrane, the local Gal3 concentration, or plasma membrane intrinsic parameters. The study also demonstrates the utility of microcavity array-suspended lipid bilayers to address the biophysics of transmembrane proteins.

Quantitative Methods to Study Endocytosis and Retrograde Transport of Cargo Proteins.

Endocytosis and intracellular retrograde trafficking from endosomes to the Golgi apparatus are key cellular processes. Endocytosis is directly or indirectly involved in many if not all cellular functions ranging from nutrient uptake and receptor signaling to mitosis, cell division, and migration (Scita, Di Fiore. Nature 463(7280):464-473, 2010; McMahon, Boucrot. Nat Rev Mol Cell Biol 12(8):517-533, 2011). Retrograde trafficking is emerging as a key driver for cell polarity. Robust methods are needed to quantify these processes. At the example of the bacterial Shiga toxin and the endogenous α5ß1 integrin, we here describe generic methods to differentiate (1) internalized from cell surface-accessible cargo proteins and (2) endocytic cargo proteins that have reached the Golgi apparatus via the retrograde route from those that have not. The choice of antibodies or natural ligands allows to adjust these methods to virtually any chosen biological system.

Retrograde and Anterograde Transport of Lat-Vesicles during the Immunological Synapse Formation: Defining the Finely-Tuned Mechanism.

LAT is an important player of the signaling cascade induced by TCR activation. This adapter molecule is present at the plasma membrane of T lymphocytes and more abundantly in intracellular compartments. Upon T cell activation the intracellular pool of LAT is recruited to the immune synapse (IS). We previously described two pathways controlling LAT trafficking: retrograde transport from endosomes to the TGN, and anterograde traffic from the Golgi to the IS. We address the specific role of four proteins, the GTPase Rab6, the t-SNARE syntaxin-16, the v-SNARE VAMP7 and the golgin GMAP210, in each pathway. Using different methods (endocytosis and Golgi trap assays, confocal and TIRF microscopy, TCR-signalosome pull down) we show that syntaxin-16 is regulating the retrograde transport of LAT whereas VAMP7 is regulating the anterograde transport. Moreover, GMAP210 and Rab6, known to contribute to both pathways, are in our cellular context, specifically and respectively, involved in anterograde and retrograde transport of LAT. Altogether, our data describe how retrograde and anterograde pathways coordinate LAT enrichment at the IS and point to the Golgi as a central hub for the polarized recruitment of LAT to the IS. The role that this finely-tuned transport of signaling molecules plays in T-cell activation is discussed.

Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes.

Glycoproteins and glycolipids at the plasma membrane contribute to a range of functions from growth factor signaling to cell adhesion and migration. Glycoconjugates undergo endocytic trafficking. According to the glycolipid-lectin (GL-Lect) hypothesis, the construction of tubular endocytic pits is driven in a glycosphingolipid-dependent manner by sugar-binding proteins of the galectin family. Here, we provide evidence for a function of the GL-Lect mechanism in transcytosis across enterocytes in the mouse intestine. We show that galectin-3 (Gal3) and its newly identified binding partner lactotransferrin are transported in a glycosphingolipid-dependent manner from the apical to the basolateral membrane. Transcytosis of lactotransferrin is perturbed in Gal3 knockout mice and can be rescued by exogenous Gal3. Inside enterocytes, Gal3 is localized to hallmark structures of the GL-Lect mechanism, termed clathrin-independent carriers. These data pioneer the existence of GL-Lect endocytosis in vivo and strongly suggest that polarized trafficking across the intestinal barrier relies on this mechanism.

Clathrin-independent endocytosis, retrograde trafficking, and cell polarity.

Several mechanisms allow for cargo internalization into cells within membrane-bound endocytic carriers. How these internalization processes couple to specific pathways of intracellular distribution remains poorly explored. Here, we review uptake reactions that are independent of the conventional clathrin machinery. We discuss how these link to retrograde trafficking from endosomes to the Golgi apparatus and exemplify biological situations in which the polarized secretion capacity of the Golgi apparatus allows for retrograde cargoes to be delivered to specialized areas of the plasma membrane, such as the leading edge of migratory cells or the immunological synapse of immune cells. We also address the evidence that allows to position apicobasal polarity of epithelial cells in this context. The underlying theme is thereby the functional coupling between specific types of endocytosis to intracellular retrograde trafficking for protein cargoes that need to be localized in a highly polarized and dynamic manner to plasmalemmal subdomains.

Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166.

While several clathrin-independent endocytic processes have been described so far, their biological relevance often remains elusive, especially in pathophysiological contexts such as cancer. In this study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a clathrin-independent cargo. We show that endophilin-A3-but neither A1 nor A2 isoforms-functionally associates with CD166-containing early endocytic carriers and physically interacts with the cargo. Our data further demonstrates that the three endophilin-A isoforms control the uptake of distinct subsets of cargoes. In addition, we provide strong evidence that the construction of endocytic sites from which CD166 is taken up in an endophilin-A3-dependent manner is driven by extracellular galectin-8. Taken together, our data reveal the existence of a previously uncharacterized clathrin-independent endocytic modality, that modulates the abundance of CD166 at the cell surface, and regulates adhesive and migratory properties of cancer cells.

Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation.

The adapter molecule linker for activation of T cells (LAT) orchestrates the formation of signalosomes upon T cell receptor (TCR) stimulation. LAT is present in different intracellular pools and is dynamically recruited to the immune synapse upon stimulation. However, the intracellular traffic of LAT and its function in T lymphocyte activation are ill defined. We show herein that LAT, once internalized, transits through the Golgi-trans-Golgi network (TGN), where it is repolarized to the immune synapse. This retrograde transport of LAT depends on the small GTPase Rab6 and the target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) Syntaxin-16, two regulators of the endosome-to-Golgi/TGN retrograde transport. We also show in vitro in Syntaxin-16- or Rab6-silenced human cells and in vivo in CD4+ T lymphocytes of the Rab6 knockout mouse that this retrograde traffic controls TCR stimulation. These results establish that the retrograde traffic of LAT from the plasma membrane to the Golgi-TGN controls the polarized delivery of LAT at the immune synapse and T lymphocyte activation.

Glycolipids and Lectins in Endocytic Uptake Processes.

A host of endocytic processes has been described at the plasma membrane of eukaryotic cells. Their categorization has most commonly referenced cytosolic machinery, of which the clathrin coat has occupied a preponderant position. In what concerns intra-membrane constituents, the focus of interest has been on phosphatidylinositol lipids and their capacity to orchestrate endocytic events on the cytosolic leaflet of the membrane. The contribution of extracellular determinants to the construction of endocytic pits has received much less attention, depite the fact that (glyco)sphingolipids are exoplasmic leaflet fabric of membrane domains, termed rafts, whose contributions to predominantly clathrin-independent internalization processes is well recognized. Furthermore, sugar modifications on extracellular domains of proteins, and sugar-binding proteins, termed lectins, have also been linked to the uptake of endocytic cargoes at the plasma membrane. In this review, we first summarize these contributions by extracellular determinants to the endocytic process. We thus propose a molecular hypothesis - termed the GL-Lect hypothesis - on how GlycoLipids and Lectins drive the formation of compositional nanoenvrionments from which the endocytic uptake of glycosylated cargo proteins is operated via clathrin-independent carriers. Finally, we position this hypothesis within the global context of endocytic pathway proposals that have emerged in recent years.

Persistent cell migration and adhesion rely on retrograde transport of ß(1) integrin.

Integrins have key functions in cell adhesion and migration. How integrins are dynamically relocalized to the leading edge in highly polarized migratory cells has remained unexplored. Here, we demonstrate that ß1 integrin (known as PAT-3 in Caenorhabditis elegans), but not ß3, is transported from the plasma membrane to the trans-Golgi network, to be resecreted in a polarized manner. This retrograde trafficking is restricted to the non-ligand-bound conformation of ß1 integrin. Retrograde trafficking inhibition abrogates several ß1-integrin-specific functions such as cell adhesion in early embryonic development of mice, and persistent cell migration in the developing posterior gonad arm of C. elegans. Our results establish a paradigm according to which retrograde trafficking, and not endosomal recycling, is the key driver for ß1 integrin function in highly polarized cells. These data more generally suggest that the retrograde route is used to relocalize plasma membrane machinery from previous sites of function to the leading edge of migratory cells.

Spatiotemporal control of interferon-induced JAK/STAT signalling and gene transcription by the retromer complex.

Type-I interferons (IFNs) play a key role in the immune defences against viral and bacterial infections, and in cancer immunosurveillance. We have established that clathrin-dependent endocytosis of the type-I interferon (IFN-α/ß) receptor (IFNAR) is required for JAK/STAT signalling. Here we show that the internalized IFNAR1 and IFNAR2 subunits of the IFNAR complex are differentially sorted by the retromer at the early endosome. Binding of the retromer VPS35 subunit to IFNAR2 results in IFNAR2 recycling to the plasma membrane, whereas IFNAR1 is sorted to the lysosome for degradation. Depletion of VPS35 leads to abnormally prolonged residency and association of the IFNAR subunits at the early endosome, resulting in increased activation of STAT1- and IFN-dependent gene transcription. These experimental data establish the retromer complex as a key spatiotemporal regulator of IFNAR endosomal sorting and a new factor in type-I IFN-induced JAK/STAT signalling and gene transcription.

SNAP-tagging the retrograde route.

We have developed a chemical biology strategy to identify proteins that follow the retrograde transport route from the plasma membrane to the Golgi apparatus, via endosomes. The general principle is the following: plasma membrane proteins are covalently tagged with a first probe. Only the ones that are then transported to trans-Golgi/TGN membranes are covalently bound to a capture reagent that has been engineered into this compartment. Specifically, the first probe is benzylguanine (BG) that is conjugated onto primary amino groups of plasma-membrane proteins. The capture reagent includes an O(6)-alkylguanine-DNA alkyltransferase-derived fragment, the SNAP-tag, which forms a covalent linkage with BG. The SNAP-tag is fused to the GFP-tagged Golgi membrane anchor from galactosyl transferase for proper targeting to trans-Golgi/TGN membranes. Cell-surface BG-tagged proteins that are transported to trans-Golgi/TGN membranes (i.e., that are retrograde cargoes) are thereby covalently captured by the SNAP-tag fusion protein. For identification, the latter is immunopurified using GFP-Trap, and associated retrograde cargo proteins are identified by mass spectrometry. We here provide a step-by-step protocol of this method.