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APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting.

ABSTRACT: RAB GTPases are central modulators of membrane trafficking. They are under the dynamic regulation of activating guanine exchange factors (GEFs) and inactivating GTPase-activating proteins (GAPs). Once activated, RABs recruit a large spectrum of effectors to control trafficking functions of eukaryotic cells. Multiple proteomic studies, using pull-down or yeast two-hybrid approaches, have identified a number of RAB interactors. However, due to the in vitro nature of these approaches and inherent limitations of each technique, a comprehensive definition of RAB interactors is still lacking. By comparing quantitative affinity purifications of GFP:RAB21 with APEX2-mediated proximity labeling of RAB4a, RAB5a, RAB7a, and RAB21, we find that APEX2 proximity labeling allows for the comprehensive identification of RAB regulators and interactors. Importantly, through biochemical and genetic approaches, we establish a novel link between RAB21 and the WASH and retromer complexes, with functional consequences on cargo sorting. Hence, APEX2-mediated proximity labeling of RAB neighboring proteins represents a new and efficient tool to define RAB functions.

SUBMITTER: Del Olmo T

PROVIDER: S-EPMC6362359 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting

Project description:RAB GTPases are central modulators of membrane trafficking. They are under the dynamic regulation of activating guanine exchange factors (GEFs) and inactivating GTPase activating proteins (GAPs). Once activated, RABs recruit a large spectrum of effectors to control trafficking functions of eukaryotic cells. Multiple proteomic studies, using pull-down or yeast two-hybrid approaches, have identified a number of RAB interactors. However, due to the in vitro nature of these and to inherent limitations of each technique, a comprehensive definition of RAB interactors is still lacking. By comparing quantitative affinity purifications of GFP:RAB21 with APEX2-mediated proximity labeling of RAB4a, RAB5a, RAB7a and RAB21, we find that APEX2 proximity labeling allows for the comprehensive identification of RAB regulators and interactors. Importantly, through biochemical and genetic approaches, we establish a novel link between RAB21 and the WASH and retromer complexes, with functional consequences on cargo sorting. Hence, APEX2-mediated proximity labeling of RAB neighboring proteins represents a new and efficient tool to define RAB functions.

| S-SCDT-91748_2_1551690886_jats | biostudies-other

4 RABs proxymity labelling - APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting

Project description:APEX2-mediated proximity labeling was performed with 4 endosomal RABs allowed an efficient and comprehensive identification of their protein neighbors. APEX2-mediated proximity labeling was performed in triplicate. Protein enrichments obtained from all RABs were compared to APEX2-nude proximity labeling results. Finally, bioinformatic and biochemical approaches were used to confirm functional validity of RAB protein neighbor’s identification.

2019-01-07 | PXD010950 | Pride

Microtubule-dependent endosomal sorting of clathrin-independent cargo by Hook1.

Project description:Many plasma membrane (PM) proteins enter cells nonselectively through clathrin-independent endocytosis (CIE). Here, we present evidence that cytoplasmic sequences in three CIE cargo proteins-CD44, CD98, and CD147-were responsible for the rapid sorting of these proteins into endosomal tubules away from endosomes associated with early endosomal antigen 1 (EEA1). We found that Hook1, a microtubule- and cargo-tethering protein, recognized the cytoplasmic tail of CD147 to help sort it and CD98 into Rab22a-dependent tubules associated with recycling. Depletion of Hook1 from cells altered trafficking of CD44, CD98, and CD147 toward EEA1 compartments and impaired the recycling of CD98 back to the PM. In contrast, another CIE cargo protein, major histocompatibility complex class I, which normally traffics to EEA1 compartments, was not affected by depletion of Hook1. Loss of Hook1 also led to an inhibition of cell spreading, implicating a role for Hook1 sorting of specific CIE cargo proteins away from bulk membrane and back to the PM.

| S-EPMC3628520 | biostudies-literature

Sorting of Clathrin-Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin-Mediated Endocytosis.

Project description:Clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) co-exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6-associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6-GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6-GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin-coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6-GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.

| S-EPMC4543536 | biostudies-literature

Directed evolution of APEX2 for electron microscopy and proximity labeling.

Project description:APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.

| S-EPMC4296904 | biostudies-literature

Syp1 regulates the clathrin-mediated and clathrin-independent endocytosis of multiple cargo proteins through a novel sorting motif.

Project description:Internalization of proteins from the plasma membrane (PM) allows for cell-surface composition regulation, signaling of network modulation, and nutrient uptake. Clathrin-mediated endocytosis (CME) is a major internalization route for PM proteins. During CME, endocytic adaptor proteins bind cargoes at the cell surface and link them to the PM and clathrin coat. Muniscins are a conserved family of endocytic adaptors, including Syp1 in budding yeast and its mammalian orthologue, FCHo1. These adaptors bind cargo via a C-terminal μ-homology domain (μHD); however, few cargoes exhibiting muniscin-dependent endocytosis have been identified, and the sorting sequence recognized by the µHD is unknown. To reveal Syp1 cargo-sorting motifs, we performed a phage display screen and used biochemical methods to demonstrate that the Syp1 µHD binds DxY motifs in the previously identified Syp1 cargo Mid2 and the v-SNARE Snc1. We also executed an unbiased visual screen, which identified the peptide transporter Ptr2 and the ammonium permease Mep3 as Syp1 cargoes containing DxY motifs. Finally, we determined that, in addition to regulating cargo entry through CME, Syp1 can promote internalization of Ptr2 through a recently identified clathrin-independent endocytic pathway that requires the Rho1 GTPase. These findings elucidate the mechanism of Syp1 cargo recognition and its role in trafficking.

| S-EPMC5576906 | biostudies-literature

Identification of Lipid Droplet Proteomes by Proximity Labeling Proteomics Using APEX2.

Project description:Lipid droplets (LDs) are ubiquitous lipid storage organelles composed of a neutral lipid core surrounded by a phospholipid monolayer that is decorated with integral and peripheral proteins. Accurate identification of LD proteins using biochemical fractionation methods has been challenging due to the presence of contaminant proteins from co-fractionating organelles. Here, we describe a method to identify high-confidence LD proteomes that employs an engineered ascorbate peroxidase (APEX2) to induce spatially and temporally restricted biotinylation of LD proteins. This proximity labeling method can be broadly applied to define the composition of the LD proteome in any cultured cell line and can be utilized to examine LD proteome dynamics.

| S-EPMC6609093 | biostudies-literature

APEX2-Mediated Proximity Labeling of Wnt Receptor Interactors Upon Pathway Activation.

Project description:The Wnt signaling pathway regulates metazoan development, tissue homeostasis, and regeneration. Many outstanding questions in Wnt signal transduction revolve around the molecular events immediately following Wnt-receptor interactions. To identify binding partners of the Wnt receptor Frizzled 7 (Fzd7) upon pathway activation, we tagged Fzd7 with APEX2, an enzyme that allows biotinylation of proximal interactors with high temporal and spatial resolution. Upon confirming proper localization and signaling activity of APEX2-tagged Fzd7, we labeled proximal interactors of Fzd7 with or without Wnt3a stimulation. Mass spectrometry analysis of biotinylated interactors identified several known Wnt pathway proteins. Top interactors enriched upon Wnt treatment were involved in actin cytoskeleton regulation, vesicle trafficking, or phospholipid modification. Proteins enriched in the Wnt-activated Fzd7 interactome that are without established roles in Wnt signaling warrant further examination.

| S-EPMC10227642 | biostudies-literature

SEC-10 and RAB-10 coordinate basolateral recycling of clathrin-independent cargo through endosomal tubules in Caenorhabditis elegans.

Project description:Despite the increasing number of regulatory proteins identified in clathrin-independent endocytic (CIE) pathways, our understanding of the exact functions of these proteins and the sequential manner in which they function remains limited. In this study, using the Caenorhabditis elegans intestine as a model, we observed a unique structure of interconnected endosomal tubules, which is required for the basolateral recycling of several CIE cargoes including hTAC, GLUT1, and DAF-4. SEC-10 is a subunit of the octameric protein complex exocyst. Depleting SEC-10 and several other exocyst components disrupted the endosomal tubules into various ring-like structures. An epistasis analysis further suggested that SEC-10 operates at the intermediate step between early endosomes and recycling endosomes. The endosomal tubules were also sensitive to inactivation of the Rab GTPase RAB-10 and disruption of microtubules. Taken together, our data suggest that SEC-10 coordinates with RAB-10 and microtubules to form the endosomal tubular network for efficient recycling of particular CIE cargoes.

| S-EPMC4217428 | biostudies-literature

Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling.

Project description:Emerin is one of the best characterized proteins of the inner nuclear membrane, but can also occur at the level of the endoplasmic reticulum. We now use enhanced ascorbate peroxidase 2 (APEX2) to probe the environment of emerin. APEX2 can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. As an alternative to the standard approach with a genetic fusion of APEX2 to emerin, we also used RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC), a method with improved specificity, where the peroxidase interacts with the protein of interest (i.e., emerin) only upon addition of rapamycin to the cells. We compare these different approaches, which, together, identify well-known interaction partners of emerin like lamin A and the lamina associated polypeptide 1 (LAP1), as well as novel proximity partners.

| S-EPMC7140434 | biostudies-literature

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