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.

CryoEM of endogenous mammalian V-ATPase interacting with the TLDc protein mEAK-7.

V-ATPases are rotary proton pumps that serve as signaling hubs with numerous protein binding partners. CryoEM with exhaustive focused classification allowed detection of endogenous proteins associated with porcine kidney V-ATPase. An extra C subunit was found in ∼3% of complexes, whereas ∼1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 showed that mEAK-7's TLDc domain interacts with V-ATPase's stator, whereas its C-terminal α helix binds V-ATPase's rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike the yeast TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that the mEAK-7:V-ATPase interaction is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. These results show that V-ATPase binding by TLDc domain proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzyme's activity.

Endosomal LC3C-pathway selectively targets plasma membrane cargo for autophagic degradation.

Autophagy selectively targets cargo for degradation, yet mechanistic understanding remains incomplete. The ATG8-family plays key roles in autophagic cargo recruitment. Here by mapping the proximal interactome of ATG8-paralogs, LC3B and LC3C, we uncover a LC3C-Endocytic-Associated-Pathway (LEAP) that selectively recruits plasma-membrane (PM) cargo to autophagosomes. We show that LC3C localizes to peripheral endosomes and engages proteins that traffic between PM, endosomes and autophagosomes, including the SNARE-VAMP3 and ATG9, a transmembrane protein essential for autophagy. We establish that endocytic LC3C binds cargo internalized from the PM, including the Met receptor tyrosine kinase and transferrin receptor, and is necessary for their recruitment into ATG9 vesicles targeted to sites of autophagosome initiation. Structure-function analysis identified that LC3C-endocytic localization and engagement with PM-cargo requires the extended carboxy-tail unique to LC3C, the TBK1 kinase, and TBK1-phosphosites on LC3C. These findings identify LEAP as an unexpected LC3C-dependent pathway, providing new understanding of selective coupling of PM signalling with autophagic degradation.

A proximity-dependent biotinylation map of a human cell.

Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy1 and biochemical fractionation coupled with mass spectrometry2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells5-7. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.

Rag GTPases and phosphatidylinositol 3-phosphate mediate recruitment of the AP-5/SPG11/SPG15 complex.

Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both phosphatidylinositol 3-phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation.

Mitochondrial Threonyl-tRNA Synthetase TARS2 Is Required for Threonine-Sensitive mTORC1 Activation.

Mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and proliferation by sensing fluctuations in environmental cues such as nutrients, growth factors, and energy levels. The Rag GTPases (Rags) serve as a critical module that signals amino acid (AA) availability to modulate mTORC1 localization and activity. Recent studies have demonstrated how AAs regulate mTORC1 activity through Rags. Here, we uncover an unconventional pathway that activates mTORC1 in response to variations in threonine (Thr) levels via mitochondrial threonyl-tRNA synthetase TARS2. TARS2 interacts with inactive Rags, particularly GTP-RagC, leading to increased GTP loading of RagA. mTORC1 activity in cells lacking TARS2 is resistant to Thr repletion, showing that TARS2 is necessary for Thr-dependent mTORC1 activation. The requirement of TARS2, but not cytoplasmic threonyl-tRNA synthetase TARS, for this effect demonstrates an additional layer of complexity in the regulation of mTORC1 activity.

The GATOR-Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids.

The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR-Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase-independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.

The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9.

PCSK9 is a secreted protein that regulates plasma cholesterol levels and cardiovascular disease risk. Prior studies suggested the presence of an ER cargo receptor that recruits PCSK9 into the secretory pathway, but its identity has remained elusive. Here, we apply a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify SURF4, a homologue of the yeast cargo receptor Erv29p, as a primary mediator of PCSK9 secretion in HEK293T cells. The functional contribution of SURF4 to PCSK9 secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. SURF4 was found to localize to the early secretory pathway where it physically interacts with PCSK9. Deletion of SURF4 resulted in ER accumulation and decreased extracellular secretion of PCSK9. These findings support a model in which SURF4 functions as an ER cargo receptor mediating the efficient cellular secretion of PCSK9.

The Lysosome and Intracellular Signalling.

In addition to being the terminal degradative compartment of the cell's endocytic and autophagic pathways, the lysosome is a multifunctional signalling hub integrating the cell's response to nutrient status and growth factor/hormone signalling. The cytosolic surface of the limiting membrane of the lysosome is the site of activation of the multiprotein complex mammalian target of rapamycin complex 1 (mTORC1), which phosphorylates numerous cell growth-related substrates, including transcription factor EB (TFEB). Under conditions in which mTORC1 is inhibited including starvation, TFEB becomes dephosphorylated and translocates to the nucleus where it functions as a master regulator of lysosome biogenesis. The signalling role of lysosomes is not limited to this pathway. They act as an intracellular Ca2+ store, which can release Ca2+ into the cytosol for both local effects on membrane fusion and pleiotropic effects within the cell. The relationship and crosstalk between the lysosomal and endoplasmic reticulum (ER) Ca2+ stores play a role in shaping intracellular Ca2+ signalling. Lysosomes also perform other signalling functions, which are discussed. Current views of the lysosomal compartment recognize its dynamic nature. It includes endolysosomes, autolysosome and storage lysosomes that are constantly engaged in fusion/fission events and lysosome regeneration. How signalling is affected by individual lysosomal organelles being at different stages of these processes and/or at different sites within the cell is poorly understood, but is discussed.

Functions of the COPII gene paralogs SEC23A and SEC23B are interchangeable in vivo.

Approximately one-third of the mammalian proteome is transported from the endoplasmic reticulum-to-Golgi via COPII-coated vesicles. SEC23, a core component of coat protein-complex II (COPII), is encoded by two paralogous genes in vertebrates (Sec23a and Sec23b). In humans, SEC23B deficiency results in congenital dyserythropoietic anemia type-II (CDAII), while SEC23A deficiency results in a skeletal phenotype (with normal red blood cells). These distinct clinical disorders, together with previous biochemical studies, suggest unique functions for SEC23A and SEC23B. Here we show indistinguishable intracellular protein interactomes for human SEC23A and SEC23B, complementation of yeast Sec23 by both human and murine SEC23A/B, and rescue of the lethality of sec23b deficiency in zebrafish by a sec23a-expressing transgene. We next demonstrate that a Sec23a coding sequence inserted into the murine Sec23b locus completely rescues the lethal SEC23B-deficient pancreatic phenotype. We show that SEC23B is the predominantly expressed paralog in human bone marrow, but not in the mouse, with the reciprocal pattern observed in the pancreas. Taken together, these data demonstrate an equivalent function for SEC23A/B, with evolutionary shifts in the transcription program likely accounting for the distinct phenotypes of SEC23A/B deficiency within and across species, a paradigm potentially applicable to other sets of paralogous genes. These findings also suggest that enhanced erythroid expression of the normal SEC23A gene could offer an effective therapeutic approach for CDAII patients.

Proteomic and Biochemical Comparison of the Cellular Interaction Partners of Human VPS33A and VPS33B.

Multi-subunit tethering complexes control membrane fusion events in eukaryotic cells. Class C core vacuole/endosome tethering (CORVET) and homotypic fusion and vacuole protein sorting (HOPS) are two such complexes, both containing the Sec1/Munc18 protein subunit VPS33A. Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. It has been recently suggested that VPS33B and VIPAR comprise two subunits of a novel multi-subunit tethering complex (named "CHEVI"), perhaps analogous in configuration to CORVET and HOPS. We utilized the BioID proximity biotinylation assay to compare and contrast the interactomes of VPS33A and VPS33B. Overall, few proteins were identified as associating with both VPS33A and VPS33B, suggesting that these proteins have distinct sub-cellular localizations. Consistent with previous reports, we observed that VPS33A was co-localized with many components of class III phosphatidylinositol 3-kinase (PI3KC3) complexes: PIK3C3, PIK3R4, NRBF2, UVRAG and RUBICON. Although VPS33A clearly co-localized with several subunits of CORVET and HOPS in this assay, no proteins with the canonical CORVET/HOPS domain architecture were found to co-localize with VPS33B. Instead, we identified that VPS33B interacts directly with CCDC22, a member of the CCC complex. CCDC22 does not co-fractionate with VPS33B and VIPAR in gel filtration of human cell lysates, suggesting that CCDC22 interacts transiently with VPS33B/VIPAR rather than forming a stable complex with these proteins in cells. We also observed that the protein complex containing VPS33B and VIPAR is considerably smaller than CORVET/HOPS, suggesting that the CHEVI complex comprises just VPS33B and VIPAR.

Translational control of ERK signaling through miRNA/4EHP-directed silencing.

MicroRNAs (miRNAs) exert a broad influence over gene expression by directing effector activities that impinge on translation and stability of mRNAs. We recently discovered that the cap-binding protein 4EHP is a key component of the mammalian miRNA-Induced Silencing Complex (miRISC), which mediates gene silencing. However, little is known about the mRNA repertoire that is controlled by the 4EHP/miRNA mechanism or its biological importance. Here, using ribosome profiling, we identify a subset of mRNAs that are translationally controlled by 4EHP. We show that the Dusp6 mRNA, which encodes an ERK1/2 phosphatase, is translationally repressed by 4EHP and a specific miRNA, miR-145. This promotes ERK1/2 phosphorylation, resulting in augmented cell growth and reduced apoptosis. Our findings thus empirically define the integral role of translational repression in miRNA-induced gene silencing and reveal a critical function for this process in the control of the ERK signaling cascade in mammalian cells.

N-acetylglucosamine: more than a silent partner in insulin resistance.

Pedersen et al. (Pedersen HK, Gudmundsdottir V, Nielsen HB, Hyotylainen T, Nielsen T, Jensen BA, Forslund K, Hildebrand F, Prifti E, Falony G, et al. 2016. Human gut microbes impact host serum metabolome and insulin sensitivity. Nature. 535: 376-381.) report that human serum levels of branched-chain amino acids (BCAA) and N-acetylglucosamine (GlcNAc) increase in proportion to insulin resistance. They focus on the microbiome and the contributing subset of microbe species, thereby demonstrating disease causality in mice. As either oral GlcNAc or BCAA in mice are known to increase insulin resistance and weight gain, we note that recently published molecular data argues for a cooperative interaction.

Cap-binding protein 4EHP effects translation silencing by microRNAs.

MicroRNAs (miRNAs) play critical roles in a broad variety of biological processes by inhibiting translation initiation and by destabilizing target mRNAs. The CCR4-NOT complex effects miRNA-mediated silencing, at least in part through interactions with 4E-T (eIF4E transporter) protein, but the precise mechanism is unknown. Here we show that the cap-binding eIF4E-homologous protein 4EHP is an integral component of the miRNA-mediated silencing machinery. We demonstrate that the cap-binding activity of 4EHP contributes to the translational silencing by miRNAs through the CCR4-NOT complex. Our results show that 4EHP competes with eIF4E for binding to 4E-T, and this interaction increases the affinity of 4EHP for the cap. We propose a model wherein the 4E-T/4EHP interaction engenders a closed-loop mRNA conformation that blocks translational initiation of miRNA targets.

Parallel Exploration of Interaction Space by BioID and Affinity Purification Coupled to Mass Spectrometry.

Complete understanding of cellular function requires knowledge of the composition and dynamics of protein interaction networks, the importance of which spans all molecular cell biology fields. Mass spectrometry-based proteomics approaches are instrumental in this process, with affinity purification coupled to mass spectrometry (AP-MS) now widely used for defining interaction landscapes. Traditional AP-MS methods are well suited to providing information regarding the temporal aspects of soluble protein-protein interactions, but the requirement to maintain protein-protein interactions during cell lysis and AP means that both weak-affinity interactions and spatial information is lost. A more recently developed method called BioID employs the expression of bait proteins fused to a nonspecific biotin ligase, BirA*, that induces in vivo biotinylation of proximal proteins. Coupling this method to biotin affinity enrichment and mass spectrometry negates many of the solubility and interaction strength issues inherent in traditional AP-MS methods, and provides unparalleled spatial context for protein interactions. Here we describe the parallel implementation of both BioID and FLAG AP-MS allowing simultaneous exploration of both spatial and temporal aspects of protein interaction networks.

Myosin VI facilitates connexin 43 gap junction accretion.

In this study, we demonstrate myosin VI enrichment at Cx43 (also known as GJA1)-containing gap junctions (GJs) in heart tissue, primary cardiomyocytes and cell culture models. In primary cardiac tissue and in fibroblasts from the myosin VI-null mouse as well as in tissue culture cells transfected with siRNA against myosin VI, we observe reduced GJ plaque size with a concomitant reduction in intercellular communication, as shown by fluorescence recovery after photobleaching (FRAP) and a new method of selective calcein administration. Analysis of the molecular role of myosin VI in Cx43 trafficking indicates that myosin VI is dispensable for the delivery of Cx43 to the cell surface and connexon movement in the plasma membrane. Furthermore, we cannot corroborate clathrin or Dab2 localization at gap junctions and we do not observe a function for the myosin-VI-Dab2 complex in clathrin-dependent endocytosis of annular gap junctions. Instead, we found that myosin VI was localized at the edge of Cx43 plaques by using total internal reflection fluorescence (TIRF) microscopy and use FRAP to identify a plaque accretion defect as the primary manifestation of myosin VI loss in Cx43 homeostasis. A fuller understanding of this derangement may explain the cardiomyopathy or gliosis associated with the loss of myosin VI.

BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers.

Endomembrane organelle maturation requires cargo delivery via fusion with membrane transport intermediates and recycling of fusion factors to their sites of origin. Melanosomes and other lysosome-related organelles obtain cargoes from early endosomes, but the fusion machinery involved and its recycling pathway are unknown. Here, we show that the v-SNARE VAMP7 mediates fusion of melanosomes with tubular transport carriers that also carry the cargo protein TYRP1 and that require BLOC-1 for their formation. Using live-cell imaging, we identify a pathway for VAMP7 recycling from melanosomes that employs distinct tubular carriers. The recycling carriers also harbor the VAMP7-binding scaffold protein VARP and the tissue-restricted Rab GTPase RAB38. Recycling carrier formation is dependent on the RAB38 exchange factor BLOC-3. Our data suggest that VAMP7 mediates fusion of BLOC-1-dependent transport carriers with melanosomes, illuminate SNARE recycling from melanosomes as a critical BLOC-3-dependent step, and likely explain the distinct hypopigmentation phenotypes associated with BLOC-1 and BLOC-3 deficiency in Hermansky-Pudlak syndrome variants.

VARP is recruited on to endosomes by direct interaction with retromer, where together they function in export to the cell surface.

VARP is a Rab32/38 effector that also binds to the endosomal/lysosomal R-SNARE VAMP7. VARP binding regulates VAMP7 participation in SNARE complex formation and can therefore influence VAMP7-mediated membrane fusion events. Mutant versions of VARP that cannot bind Rab32:GTP, designed on the basis of the VARP ankyrin repeat/Rab32:GTP complex structure described here, unexpectedly retain endosomal localization, showing that VARP recruitment is not dependent on Rab32 binding. We show that recruitment of VARP to the endosomal membrane is mediated by its direct interaction with VPS29, a subunit of the retromer complex, which is involved in trafficking from endosomes to the TGN and the cell surface. Transport of GLUT1 from endosomes to the cell surface requires VARP, VPS29, and VAMP7 and depends on the direct interaction between VPS29 and VARP. Finally, we propose that endocytic cycling of VAMP7 depends on its interaction with VARP and, consequently, also on retromer.

A mutation causing Brugada syndrome identifies a mechanism for altered autonomic and oxidant regulation of cardiac sodium currents.

BACKGROUND: The mechanisms of the electrocardiographic changes and arrhythmias in Brugada syndrome (BrS) remain controversial. Mutations in the sodium channel gene, SCN5A, and regulatory proteins that reduce or eliminate sodium current (INa) have been linked to BrS. We studied the properties of a BrS-associated SCN5A mutation in a protein kinase A (PKA) consensus phosphorylation site, R526H. METHODS AND RESULTS: In vitro PKA phosphorylation was detected in the I-II linker peptide of wild-type (WT) channels but not R526H or S528A (phosphorylation site) mutants. Cell surface expression of R526H and S528A channels was reduced compared with WT. Whole-cell INa through all channel variants revealed no significant differences in the steady-state activation, inactivation, and recovery from inactivation. Peak current densities of the mutants were significantly reduced compared with WT. Infection of 2D cultures of neonatal rat ventricular myocytes with WT and mutant channels increased conduction velocity compared with noninfected cells. PKA stimulation significantly increased peak INa and conduction velocity of WT but not mutant channels. Oxidant stress inhibits cardiac INa; WT and mutant INa decreases with the intracellular application of reduced nicotinamide adenine dinucleotide (NADH), an effect that is reversed by PKA stimulation in WT but not in R526H or S528A channels. CONCLUSIONS: We identified a family with BrS and an SCN5A mutation in a PKA consensus phosphorylation site. The BrS mutation R526H is associated with a reduction in the basal level of INa and a failure of PKA stimulation to augment the current that may contribute to the predisposition to arrhythmias in patients with BrS, independent of the precipitants.