Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation.

During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.

Molecular Mechanisms of Macroautophagy, Microautophagy, and Chaperone-Mediated Autophagy.

Autophagy is a self-digestive process that is conserved in eukaryotic cells and responsible for maintaining cellular homeostasis through proteolysis. By this process, cells break down their own components in lysosomes. Autophagy can be classified into three categories: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Macroautophagy involves membrane elongation and microautophagy involves membrane internalization, and both pathways undergo selective or non-selective processes that transport cytoplasmic components into lysosomes to be degraded. CMA, however, involves selective incorporation of cytosolic materials into lysosomes without membrane deformation. All three categories of autophagy have attracted much attention due to their involvement in various biological phenomena and their relevance to human diseases, such as neurodegenerative diseases and cancer. Clarification of the molecular mechanisms behind these processes is key to understanding autophagy and recent studies have made major progress in this regard, especially for the mechanisms of initiation and membrane elongation in macroautophagy and substrate recognition in microautophagy and CMA. Furthermore, it is becoming evident that the three categories of autophagy are related to each other despite their implementation by different sets of proteins and the involvement of completely different membrane dynamics. In this review, recent progress in macroautophagy, microautophagy, and CMA are summarized.

VAMP5 and distinct sets of cognate Q-SNAREs mediate exosome release.

Small extracellular vesicles (sEVs) are largely classified into two types, plasma-membrane derived sEVs and endomembrane-derived sEVs. The latter type (referred to as exosomes herein) is originated from late endosomes or multivesicular bodies (MVBs). In order to release exosomes extracellularly, MVBs must fuse with the plasma membrane, not with lysosomes. In contrast to the mechanism responsible for MVB-lysosome fusion, the mechanism underlying the MVB-plasma membrane fusion is poorly understood. Here, we systematically analyze soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family proteins and identify VAMP5 as an MVB-localized SNARE protein required for exosome release. Depletion of VAMP5 in HeLa cells impairs exosome release. Mechanistically, VAMP5 mediates exosome release by interacting with SNAP47 and plasma membrane SNARE Syntaxin 1 (STX1) or STX4 to release exosomes. VAMP5 is also found to mediate asymmetric exosome release from polarized Madin-Darby canine kidney (MDCK) epithelial cells through interaction with the distinct sets of Q-SNAREs, suggesting that VAMP5 is a general exosome regulator in both polarized cells and non-polarized cells.Key words: exosome, small extracellular vesicle (sEV), multivesicular body, SNARE, VAMP5.

Vps9d1 regulates tubular endosome formation through specific activation of Rab22A.

The small GTPase Rab22A is an important regulator of the formation of tubular endosomes, which are one of the types of recycling endosome compartments of the clathrin-independent endocytosis pathway. In order to regulate tubular endosome formation, Rab22A must be activated by a specific guanine-nucleotide-exchange factor (GEF); however, all of the GEFs that have been reported to exhibit Rab22A-GEF activity in vitro also activate Rab5A, an essential regulator of the clathrin-mediated endocytosis pathway, and no Rab22A-specific GEF has ever been identified. Here, we identified Vps9d1, a previously uncharacterized vacuolar protein sorting 9 (VPS9) domain-containing protein, as a novel Rab22A-GEF. The formation of tubular endosome structures was found to be severely impaired in Vps9d1-depleted HeLa cells, but Rab5A localization was unaffected. Expression of a constitutively active Rab22A mutant in Vps9d1-depleted HeLa cells restored tubular endosomes, but expression of a GEF-activity-deficient Vps9d1 mutant did not. Moreover, Vps9d1 depletion altered the distribution of clathrin-independent endocytosed cargos and impaired their recycling. Our findings indicate that Vps9d1 promotes tubular endosome formation by specifically activating Rab22A.

TBC1D18 is a Rab5-GAP that coordinates endosome maturation together with Mon1.

Rab5 and Rab7 are known to regulate endosome maturation, and a Rab5-to-Rab7 conversion mediated by a Rab7 activator, Mon1-Ccz1, is essential for progression of the maturation process. However, the importance and mechanism of Rab5 inactivation during endosome maturation are poorly understood. Here, we report a novel Rab5-GAP, TBC1D18, which is associated with Mon1 and mediates endosome maturation. We found that increased active Rab5 (Rab5 hyperactivation) in addition to reduced active Rab7 (Rab7 inactivation) occurs in the absence of Mon1. We present evidence showing that the severe defects in endosome maturation in Mon1-KO cells are attributable to Rab5 hyperactivation rather than to Rab7 inactivation. We then identified TBC1D18 as a Rab5-GAP by comprehensive screening of TBC-domain-containing Rab-GAPs. Expression of TBC1D18 in Mon1-KO cells rescued the defects in endosome maturation, whereas its depletion attenuated endosome formation and degradation of endocytosed cargos. Moreover, TBC1D18 was found to be associated with Mon1, and it localized in close proximity to lysosomes in a Mon1-dependent manner.

Rab39 and its effector UACA regulate basolateral exosome release from polarized epithelial cells.

Exosomes are small extracellular vesicles that originate from the intraluminal vesicles of multivesicular bodies (MVBs). We previously reported that polarized Madin-Darby canine kidney (MDCK) epithelial cells secrete two types of exosomes, apical and basolateral exosomes, from different MVBs. However, how these MVBs are selectively targeted to the apical or basolateral membrane remained unknown. Here, we analyze members of the Rab family small GTPases and show that different sets of Rabs mediate asymmetrical exosome release. Rab27, the best-known regulator of MVB transport for exosome release, is specifically but partially involved in apical exosome release, and Rab37, a close homolog of Rab27, is an additional apical exosome regulator. By contrast, Rab39 functions as a specific regulator of basolateral exosome release. Mechanistically, Rab39 interacts with its effector UACA, and UACA then recruits Lyspersin, a component of BLOC-1-related complex (BORC). Our findings suggest that the Rab39-UACA-BORC complex specifically mediates basolateral exosome release.

ALIX and ceramide differentially control polarized small extracellular vesicle release from epithelial cells.

Exosomes, important players in cell-cell communication, are small extracellular vesicles of endocytic origin. Although single cells are known to release various kinds of exosomes (referred to as exosomal heterogeneity), very little is known about the mechanisms by which they are produced and released. Here, we established methods of studying exosomal heterogeneity by using polarized epithelial cells and showed that distinct types of small extracellular vesicles (more specifically CD9- and CD63-positive, Annexin I-negative small extracellular vesicles, which we refer to as exosomes herein) are differentially secreted from the apical and basolateral sides of polarized epithelial cells. We also identify GPRC5C (G protein-coupled receptor class C group 5 member C) as an apical exosome-specific protein. We further demonstrate that basolateral exosome release depends on ceramide, whereas ALIX, an ESCRT (endosomal sorting complexes required for transport)-related protein, not the ESCRT machinery itself, is required for apical exosome release. Thus, two independent machineries, the ALIX-Syntenin1-Syndecan1 machinery (apical side) and the sphingomyelinase-dependent ceramide production machinery (basolateral side), are likely to be responsible for the polarized exosome release from epithelial cells.

RBD11, a bioengineered Rab11-binding module for visualizing and analyzing endogenous Rab11.

The small GTPase Rab11 (herein referring to the Rab11A and Rab11B isoforms) plays pivotal roles in diverse physiological phenomena, including the recycling of membrane proteins, cytokinesis, neurite outgrowth and epithelial morphogenesis. One effective method of analyzing the function of endogenous Rab11 is to overexpress a Rab11-binding domain from one of its effectors, for example, the C-terminal domain of Rab11-FIP2 (Rab11-FIP2-C), as a dominant-negative construct. However, the drawback of this method is the broader Rab-binding specificity of the effector domain, because Rab11-FIP2-C binds to Rabs other than Rab11, for example, to Rab14 and Rab25. In this study, we bioengineered an artificial Rab11-specific binding domain, named RBD11. Expression of RBD11 allowed visualization of endogenous Rab11 without affecting its localization or function, whereas expression of a tandem RBD11, named 2×RBD11, inhibited epithelial morphogenesis and induced a multi-lumen phenotype characteristic of Rab11-deficient cysts. We also developed two tools for temporally and reversibly analyzing Rab11-dependent membrane trafficking - tetracycline-inducible 2×RBD11 and an artificially oligomerized domain (FM)-tagged RBD11.

YKT6 as a second SNARE protein of mammalian autophagosomes.

Mammalian autophagosomes possess the Qa-SNARE STX17 (syntaxin 17) for fusion with lysosomes. However, STX17 is not absolutely required for fusion because STX17 knockout cells partially retain autophagosome-lysosome fusion activity. We recently identified YKT6, an R-SNARE, as another autophagosomal SNARE protein that acts independently of STX17 in mammals. Here, we discuss the features and functions of autophagosomal SNARE proteins by comparing STX17 and YKT6. Abbreviations: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; STX17, syntaxin 17.

Autophagosomal YKT6 is required for fusion with lysosomes independently of syntaxin 17.

Macroautophagy is an evolutionarily conserved catabolic mechanism that delivers intracellular constituents to lysosomes using autophagosomes. To achieve degradation, lysosomes must fuse with closed autophagosomes. We previously reported that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin (STX) 17 translocates to autophagosomes to mediate fusion with lysosomes. In this study, we report an additional mechanism. We found that autophagosome-lysosome fusion is retained to some extent even in STX17 knockout (KO) HeLa cells. By screening other human SNAREs, we identified YKT6 as a novel autophagosomal SNARE protein. Depletion of YKT6 inhibited autophagosome-lysosome fusion partially in wild-type and completely in STX17 KO cells, suggesting that YKT6 and STX17 are independently required for fusion. YKT6 formed a SNARE complex with SNAP29 and lysosomal STX7, both of which are required for autophagosomal fusion. Recruitment of YKT6 to autophagosomes depends on its N-terminal longin domain but not on the C-terminal palmitoylation and farnesylation that are essential for its Golgi localization. These findings suggest that two independent SNARE complexes mediate autophagosome-lysosome fusion.

An Autophagic Flux Probe that Releases an Internal Control.

Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is critically important but remains complicated and challenging. Here, we have developed GFP-LC3-RFP-LC3ΔG, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. GFP-LC3 is degraded by autophagy, while RFP-LC3ΔG remains in the cytosol, serving as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Using this probe, we re-evaluated previously reported autophagy-modulating compounds, performed a high-throughput screen of an approved drug library, and identified autophagy modulators. Furthermore, we succeeded in measuring both induced and basal autophagic flux in embryos and tissues of zebrafish and mice. The GFP-LC3-RFP-LC3ΔG probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms.

Dennd3 functions as a guanine nucleotide exchange factor for small GTPase Rab12 in mouse embryonic fibroblasts.

Small GTPase Rab12 regulates mTORC1 (mammalian target of rapamycin complex 1) activity and autophagy through controlling PAT4 (proton/amino acid transporter 4) trafficking from recycling endosomes to lysosomes, where PAT4 is degraded. However, the precise regulatory mechanism of the Rab12-mediated membrane trafficking pathway remained to be determined because a physiological Rab12-GEF (guanine nucleotide exchange factor) had yet to be identified. In this study we performed functional analyses of Dennd3, which has recently been shown to possess a GEF activity toward Rab12 in vitro. The results showed that knockdown of Dennd3 in mouse embryonic fibroblast cells caused an increase in the amount of PAT4 protein, the same as Rab12 knockdown did, and knockdown of Dennd3 and overexpression of Dennd3 were found to result in an increase and a decrease, respectively, in the intracellular amino acid concentration. Dennd3 overexpression was also found to reduce mTORC1 activity and promoted autophagy in a Rab12-dependent manner. Unexpectedly, however, Dennd3 knockdown had no effect on mTORC1 activity or autophagy despite increasing the intracellular amino acid concentration. Further study showed that Dennd3 knockdown reduced Akt activity, and the reduction in Akt activity is likely to have canceled out amino acid-induced mTORC1 activation through PAT4. These findings indicated that Dennd3 not only functions as a Rab12-GEF but also modulates Akt signaling in mouse embryonic fibroblast cells.

Methods of analysis of the membrane trafficking pathway from recycling endosomes to lysosomes.

The transferrin receptor (TfR) is responsible for iron uptake through its trafficking between the plasma membrane and recycling endosomes, and as a result it has become a well-known marker for recycling endosomes. Although the molecular basis of the TfR recycling pathway has been thoroughly investigated, the TfR degradation mechanism has been poorly understood. Exposure of cultured cells to two drugs, the protein synthesis inhibitor cycloheximide and the V-ATPase inhibitor bafilomycin A1, recently showed that TfR is not only recycled back to the plasma membrane after endocytosis but is constitutively transported to lysosomes for degradation. The results of genome-wide screening of mouse Rab small GTPases (common regulators of membrane trafficking in all eukaryotes) have indicated that Rab12 regulates TfR trafficking to lysosomes independently of the known membrane trafficking pathways, for example, the conventional endocytic pathway and recycling pathway. This chapter summarizes the methods that the authors used to analyze the membrane trafficking pathway from recycling endosomes to lysosomes that is specifically regulated by Rab12.

Small GTPase Rab39A interacts with UACA and regulates the retinoic acid-induced neurite morphology of Neuro2A cells.

We screened for a Rab39-specific effector by performing a yeast two-hybrid assay with GTP-locked Rab39A/B as the bait and identified UACA (uveal autoantigen with coiled-coil domains and ankyrin repeats) as a specific Rab39A/B-binding protein. Deletion analysis revealed that a C-terminal coiled-coil domain of UACA functions as a GTP-dependent Rab39-binding domain. shRNA-mediated knockdown of endogenous Rab39A or UACA in mouse neuroblastoma Neuro2A cells resulted in a change in retinoic acid-induced neurite morphology from a multipolar morphology to a bipolar morphology. Taken together, these findings indicate that UACA functions as a Rab39A effector in the retinoic acid-induced differentiation of Neuro2A cells.

Rab12 regulates mTORC1 activity and autophagy through controlling the degradation of amino-acid transporter PAT4.

Autophagy is an evolutionarily conserved catabolic mechanism that targets intracellular molecules and damaged organelles to lysosomes. Autophagy is achieved by a series of membrane trafficking events, but their regulatory mechanisms are poorly understood. Here, we report small GTPase Rab12 as a new type of autophagic regulator that controls the degradation of an amino-acid transporter. Knockdown of Rab12 results in inhibition of autophagy and in increased activity of mTORC1 (mammalian/mechanistic target of rapamycin complex 1), an upstream regulator of autophagy. We also found that Rab12 promotes constitutive degradation of PAT4 (proton-coupled amino-acid transporter 4), whose accumulation in Rab12-knockdown cells modulates mTORC1 activity and autophagy. Our findings reveal a new mechanism of regulation of mTORC1 signalling and autophagy, that is, quality control of PAT4 by Rab12.

Rabex-5 protein regulates dendritic localization of small GTPase Rab17 and neurite morphogenesis in hippocampal neurons.

Small GTPase Rab17 has recently been shown to regulate dendritic morphogenesis of mouse hippocampal neurons; however, the exact molecular mechanism of Rab17-mediated dendritogenesis remained to be determined, because no guanine nucleotide exchange factor (GEF) for Rab17 had been identified. In this study we screened for the Rab17-GEF by performing yeast two-hybrid assays with a GDP-locked Rab17 mutant as bait and found that Rabex-5 and ALS2, both of which were originally described as Rab5-GEFs, interact with Rab17. We also found that expression of Rabex-5, but not of ALS2, promotes translocation of Rab17 from the cell body to the dendrites of developing mouse hippocampal neurons. The shRNA-mediated knockdown of Rabex-5 or its known downstream target Rab5 in hippocampal neurons inhibited morphogenesis of both axons and dendrites, whereas knockdown of Rab17 affected dendrite morphogenesis alone. Based on these findings, we propose that Rabex-5 regulates neurite morphogenesis of hippocampal neurons by activating at least two downstream targets, Rab5, which is localized in both axons and dendrites, and Rab17, which is localized in dendrites alone.

Decoding the regulation of mast cell exocytosis by networks of Rab GTPases.

Exocytosis is a key event in mast cell functions. By this process, mast cells release inflammatory mediators, contained in secretory granules (SGs), which play important roles in immunity and wound healing but also provoke allergic and inflammatory responses. The mechanisms underlying mast cell exocytosis remained poorly understood. An essential step toward deciphering the mechanisms behind exocytosis is the identification of the cellular components that regulate this process. Because Rab GTPases regulate specific trafficking pathways, we screened 44 Rabs for their functional impacts on exocytosis triggered by the FcεRI or combination of Ca ²âº ionophore and phorbol ester. Because exocytosis involves the continuous reorganization of the actin cytoskeleton, we also repeated our screen in the presence of cytochalasin D that inhibits actin polymerization. In this paper, we report on the identification of 30 Rabs as regulators of mast cell exocytosis, the involvement of 26 of which has heretofore not been recognized. Unexpectedly, these Rabs regulated exocytosis in a stimulus-dependent fashion, unless the actin skeleton was disrupted. Functional clustering of the identified Rabs suggested their classification as Rabs involved in SGs biogenesis or Rabs that control late steps of exocytosis. The latter could be further divided into Rabs that localize to the SGs and Rabs that regulate transport from the endocytic recycling compartment. Taken together, these findings unveil the Rab networks that control mast cell exocytosis and provide novel insights into their mechanisms of action.

The Rab interacting lysosomal protein (RILP) homology domain functions as a novel effector domain for small GTPase Rab36: Rab36 regulates retrograde melanosome transport in melanocytes.

Small GTPase Rab functions as a molecular switch that drives membrane trafficking through specific interaction with its effector molecule. Thus, identification of its specific effector domain is crucial to revealing the molecular mechanism that underlies Rab-mediated membrane trafficking. Because of the large numbers of Rab isoforms in higher eukaryotes, however, the effector domains of most of the vertebrate- or mammalian-specific Rabs have yet to be determined. In this study we screened for effector molecules of Rab36, a previously uncharacterized Rab isoform that is largely conserved in vertebrates, and we succeeded in identifying nine Rab36-binding proteins, including RILP (Rab interacting lysosomal protein) family members. Sequence comparison revealed that five of nine Rab36-binding proteins, i.e. RILP, RILP-L1, RILP-L2, and JIP3/4, contain a conserved coiled-coil domain. We identified the coiled-coil domain as a RILP homology domain (RHD) and characterized it as a common Rab36-binding site. Site-directed mutagenesis of the RHD of RILP revealed the different contributions by amino acids in the RHD to binding activity toward Rab7 and Rab36. Expression of RILP in melanocytes, but not expression of its Rab36 binding-deficient mutants, induced perinuclear aggregation of melanosomes, and this effect was clearly attenuated by knockdown of endogenous Rab36 protein. Moreover, knockdown of Rab36 in Rab27A-deficient melanocytes, which normally exhibit perinuclear melanosome aggregation because of increased retrograde melanosome transport activity, caused dispersion of melanosomes from the perinucleus to the cell periphery, but knockdown of Rab7 did not. Our findings indicated that Rab36 mediates retrograde melanosome transport in melanocytes through interaction with RILP.

Small GTPase Rab17 regulates dendritic morphogenesis and postsynaptic development of hippocampal neurons.

Neurons are compartmentalized into two morphologically, molecularly, and functionally distinct domains: axons and dendrites, and precise targeting and localization of proteins within these domains are critical for proper neuronal functions. It has been reported that several members of the Rab family small GTPases that are key mediators of membrane trafficking, regulate axon-specific trafficking events, but little has been elucidated regarding the molecular mechanisms that underlie dendrite-specific membrane trafficking. Here we show that Rab17 regulates dendritic morphogenesis and postsynaptic development in mouse hippocampal neurons. Rab17 is localized at dendritic growth cones, shafts, filopodia, and mature spines, but it is mostly absent in axons. We also found that Rab17 mediates dendrite growth and branching and that it does not regulate axon growth or branching. Moreover, shRNA-mediated knockdown of Rab17 expression resulted in a dramatically reduced number of dendritic spines, probably because of impaired filopodia formation. These findings have revealed the first molecular link between membrane trafficking and dendritogenesis.

Small GTPase Rab12 regulates constitutive degradation of transferrin receptor.

Transferrin receptor (TfR) is a well-characterized plasma membrane protein that travels between the plasma membrane and intracellular membrane compartments. Although TfR itself should undergo degradation, the same as other intracellular proteins, whether a specific TfR degradation pathway exists has never been investigated. In this study, we screened small GTPase Rab proteins, common regulators of membrane traffic in all eukaryotes, for proteins that are specifically involved in TfR degradation. We performed the screening by three sequential methods, i.e. colocalization of Rab with TfR, colocalization with lysosomes, and knockdown of Rab by specific small interfering RNA (siRNA), and succeeded in identifying Rab12, a previously uncharacterized Rab isoform, as a prime candidate among the 60 human or mouse Rabs screened. We showed that expression of a constitutive active mutant of Rab12 reduced the amount of TfR protein, whereas functional ablation of Rab12 by knockdown of either Rab12 itself or its upstream activator Dennd3 increased the amount of TfR protein. Interestingly, however, knockdown of Rab12 had no effect on the degradation of epidermal growth factor receptor (EGFR) protein, i.e. on a conventional degradation pathway. Our findings indicated that TfR is constitutively degraded by a Rab12-dependent pathway (presumably from recycling endosomes to lysosomes), which is independent of the conventional degradation pathway.