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Neurons are highly polarized cells with long neurites. Vesicular transport is required for neurite extension. We recently identified protrudin as a key regulator of vesicular transport during neurite extension. Expression of protrudin in nonneuronal cells thus induces formation of neurite-like membrane protrusions. We adopted a proteomics approach to identify proteins that associate with protrudin. Among the protrudin-associated proteins, including many with a function related to intracellular trafficking, we focused on KIF5, a motor protein that mediates anterograde vesicular transport in neurons. A coimmunoprecipitation assay confirmed that endogenous protrudin and KIF5 interact in mouse brain. Overexpression of KIF5 induced the formation of membrane protrusions in HeLa cells, reminiscent of the effect of protrudin overexpression. Forced expression of both protrudin and KIF5 promoted protrusion extension in a synergistic manner, whereas depletion of either protein attenuated protrusion formation. Protrudin facilitated the interaction of KIF5 with Rab11, VAP-A and -B, Surf4, and RTN3, suggesting that protrudin serves as an adaptor protein and that the protrudin-KIF5 complex contributes to the transport of these proteins in neurons. Given that mutation of protrudin or KIF5 is a cause of human hereditary spastic paraplegia, the protrudin-KIF5 axis appears to be integral to neuronal function.
The AP (adaptor protein) complexes are heterotetrameric protein complexes that mediate intracellular membrane trafficking along endocytic and secretory transport pathways. There are five different AP complexes: AP-1, AP-2 and AP-3 are clathrin-associated complexes; whereas AP-4 and AP-5 are not. These five AP complexes localize to different intracellular compartments and mediate membrane trafficking in distinct pathways. They recognize and concentrate cargo proteins into vesicular carriers that mediate transport from a donor membrane to a target organellar membrane. AP complexes play important roles in maintaining the normal physiological function of eukaryotic cells. Dysfunction of AP complexes has been implicated in a variety of inherited disorders, including: MEDNIK (mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis and keratodermia) syndrome, Fried syndrome, HPS (Hermansky-Pudlak syndrome) and HSP (hereditary spastic paraplegia).
Binding of motor proteins to cellular cargoes is regulated by adaptor proteins. HAP1 and GRIP1 are kinesin-1 adaptors that have been implicated individually in the transport of vesicular cargoes in the dendrites of neurons. We find that HAP1a and GRIP1 form a protein complex in the brain, and co-operate to activate the kinesin-1 subunit KIF5C in vitro Based upon this co-operative activation of kinesin-1, we propose a modification to the kinesin activation model that incorporates stabilisation of the central hinge region known to be critical to autoinhibition of kinesin-1.
In eukaryotes, EPSINs are Epsin N-terminal Homology (ENTH) domain-containing proteins that serve as monomeric clathrin adaptors at the plasma membrane (PM) or the trans-Golgi Network (TGN)/early endosomes (EE). The model plant Arabidopsis thaliana encodes for seven ENTH proteins, of which so far, only AtEPSIN1 (AtEPS1) and MODIFIED TRANSPORT TO THE VACUOLE1 (AtMTV1) localize to the TGN/EE and contribute to cargo trafficking to both the cell surface and the vacuole. However, relatively little is known about role(s) of any plant EPSIN in governing physiological responses. We have recently shown that AtEPS1 is a positive modulator of plant immune signaling and pattern-triggered immunity against flagellated Pseudomonas syringae pv. tomato (Pto) DC3000 bacteria. In eps1 mutants, impaired immune responses correlate with reduced accumulation of the receptor FLAGELLIN SENSING2 (AtFLS2) and the convergent immune co-receptor BRASSINOSTEROID INSENTIVE1-ASSOCIATED RECEPTOR KINASE1 (AtBAK1) in the PM. Here, we report that in contrast to AtEPS1, the TGN/EE-localized AtMTV1 did not contribute significantly to immunity against pathogenic Pto DC3000 bacteria. We also compared the amino acid sequences, peptide motif structures and in silico tertiary structures of the ENTH domains of AtEPS1 and AtMTV1 in more detail. We conclude that despite sharing the classical tertiary alpha helical ENTH-domain structure and clathrin-binding motifs, the overall low amino acid identity and differences in peptide motifs may explain their role(s) in trafficking of some of the same as well as distinct cargo components to their site of function, with the latter potentially contributing to differences in physiological responses.
Biosynthetic sorting of newly synthesized transmembrane cargos to endosomes and lysosomes is thought to occur at the TGN through recognition of sorting signals in the cytosolic tails of the cargos by adaptor proteins, leading to cargo packaging into coated vesicles destined for the endolysosomal system. Here we present evidence for a different mechanism in which two sets of endolysosomal proteins undergo early segregation to distinct domains of the Golgi complex by virtue of the proteins' luminal and transmembrane domains. Proteins in one Golgi domain exit into predominantly vesicular carriers by interaction of sorting signals with adaptor proteins, but proteins in the other domain exit into predominantly tubular carriers shared with plasma membrane proteins, independently of signal-adaptor interactions. These findings demonstrate that sorting of endolysosomal proteins begins at an earlier stage and involves mechanisms that partly differ from those described by classical models.
Coat proteins have a central role in vesicular transport by binding to cargoes for their sorting into intracellular pathways. Cargo recognition is mediated by components of the coat complex known as adaptor proteins1-3. We previously showed that Arf-GAP with coil-coil, ANK repeat and PH domain-containing protein 1 (ACAP1) functions as an adaptor for a clathrin coat complex that has a function in endocytic recycling4-6. Here, we show that the protein kinase Akt acts as a co-adaptor in this complex, and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling. In addition to advancing the understanding of endocytic recycling, we uncover a fundamentally different function in which a kinase acts, as Akt in this case is an effector rather than a regulator in a cellular event.
AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B-/- mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B-/- mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.
The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes.
Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37 degreesC, ultrastructural studies on cryosections failed to detect B-fragment-specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.
The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B-dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B-dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.
Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.
Intracellular trafficking is essential for cell structure and function. In order to perform key tasks such as phagocytosis, secretion or migration, cells must coordinate their intracellular trafficking, and cytoskeleton dynamics. This relies on certain classes of proteins endowed with specialized and conserved domains that bridge membranes with effector proteins. Of particular interest are proteins capable of interacting with membrane subdomains enriched in specific phosphatidylinositol lipids, tightly regulated by various kinases and phosphatases. Here, we focus on the poorly studied RUFY family of adaptor proteins, characterized by a RUN domain, which interacts with small GTP-binding proteins, and a FYVE domain, involved in the recognition of phosphatidylinositol 3-phosphate. We report recent findings on this protein family that regulates endosomal trafficking, cell migration and upon dysfunction, can lead to severe pathology at the organismal level.
Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication and contributes to immune evasion in vivo, but the underlying molecular mechanisms remain incompletely defined. Nef interferes with host cell actin dynamics to restrict T lymphocyte responses to chemokine stimulation and T cell receptor engagement. This relies on the assembly of a labile multiprotein complex including the host kinase PAK2 that Nef usurps to phosphorylate and inactivate the actin-severing factor cofilin. Components of the exocyst complex (EXOC), an octameric protein complex involved in vesicular transport and actin remodeling, were recently reported to interact with Nef via the same molecular surface that mediates PAK2 association. Exploring the functional relevance of EXOC in Nef-PAK2 complex assembly/function, we found Nef-EXOC interactions to be specifically mediated by the PAK2 interface of Nef, to occur in infected human T lymphocytes, and to be conserved among lentiviral Nef proteins. In turn, EXOC was dispensable for direct downstream effector functions of Nef-associated PAK2. Surprisingly, PAK2 was essential for Nef-EXOC association, which required a functional Rac1/Cdc42 binding site but not the catalytic activity of PAK2. EXOC was dispensable for Nef functions in vesicular transport but critical for inhibition of actin remodeling and proximal signaling upon T cell receptor engagement. Thus, Nef exploits PAK2 in a stepwise mechanism in which its kinase activity cooperates with an adaptor function for EXOC to inhibit host cell actin dynamics.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive loss of motor neurons (MNs) in the brain and spinal cord. It is caused by multiple factors, including mutations in any one of several specific genes. Optineurin (OPTN) mutation is an essential cause of some familial and sporadic ALS. Besides, as a multifunctional protein, OPTN is highly expressed and conserved in the central nervous system. OPTN exerts its functions by interacting with various proteins, often acting as an adaptor to provide a link between two or more core proteins related to autophagy and inflammation, etc. OPTN mutation mainly results in its function deficiency, which alters these interactions, leading to functional impairment in many processes. Meanwhile, OPTN immunopositive inclusions are also confirmed in the cases of ALS due to C9ORF72, FUS, TARDBP, and SOD1 mutations. Therefore, OPTN gene may play fundamental roles in the molecular pathology of ALS in addition to OPTN mutation. In this review, we summarize the recent advances in the ALS pathology of OPTN defect, such as mitophagy disorder, neuroinflammation, neuronal axonal degeneration, vesicular transport dysfunction, etc., which will provide a reference for research on the pathogenesis and treatment of ALS.
The synaptic removal of AMPA-type glutamate receptors (AMPARs) is a core mechanism for hippocampal long-term depression (LTD). In this study, we address the role of microtubule-dependent transport of AMPARs as a driver for vesicular trafficking and sorting during LTD. Here, we show that the kinesin-1 motor KIF5A/C is strictly required for LTD expression in CA3-to-CA1 hippocampal synapses. Specifically, we find that KIF5 is required for an efficient internalization of AMPARs after NMDA receptor activation. We show that the KIF5/AMPAR complex is assembled in an activity-dependent manner and associates with microsomal membranes upon LTD induction. This interaction is facilitated by the vesicular adaptor protrudin, which is also required for LTD expression. We propose that protrudin links KIF5-dependent transport to endosomal sorting, preventing AMPAR recycling to synapses after LTD induction. Therefore, this work identifies an activity-dependent molecular motor and the vesicular adaptor protein that executes AMPAR synaptic removal during LTD.
Epithelial-to-mesenchymal transition (EMT) is a transcriptionally governed process by which cancer cells establish a front-rear polarity axis that facilitates motility and invasion. Dynamic assembly of focal adhesions and other actin-based cytoskeletal structures on the leading edge of motile cells requires precise spatial and temporal control of protein trafficking. Yet, the way in which EMT-activating transcriptional programs interface with vesicular trafficking networks that effect cell polarity change remains unclear. Here, by utilizing multiple approaches to assess vesicular transport dynamics through endocytic recycling and retrograde trafficking pathways in lung adenocarcinoma cells at distinct positions on the EMT spectrum, we find that the EMT-activating transcription factor ZEB1 accelerates endocytosis and intracellular trafficking of plasma membrane-bound proteins. ZEB1 drives turnover of the MET receptor tyrosine kinase by hastening receptor endocytosis and transport to the lysosomal compartment for degradation. ZEB1 relieves a plus-end-directed microtubule-dependent kinesin motor protein (KIF13A) and a clathrin-associated adaptor protein complex subunit (AP1S2) from microRNA-dependent silencing, thereby accelerating cargo transport through the endocytic recycling and retrograde vesicular pathways, respectively. Depletion of KIF13A or AP1S2 mitigates ZEB1-dependent focal adhesion dynamics, front-rear axis polarization, and cancer cell motility. Thus, ZEB1-dependent transcriptional networks govern vesicular trafficking dynamics to effect cell polarity change.
The time course of signaling by peptide hormones, neural peptides, and other neuromodulators depends on their storage inside dense core vesicles (DCVs). Adaptor protein 3 (AP-3) assembles the membrane proteins that confer regulated release of DCVs and is thought to promote their trafficking from endosomes directly to maturing DCVs. We now find that regulated monoamine release from DCVs requires sorting nexin 5 (SNX5). Loss of SNX5 disrupts trafficking of the vesicular monoamine transporter (VMAT) to DCVs. The mechanism involves a role for SNX5 in retrograde transport of VMAT from endosomes to the TGN. However, this role for SNX5 conflicts with the proposed function of AP-3 in trafficking from endosomes directly to DCVs. We now identify a transient role for AP-3 at the TGN, where it associates with DCV cargo. Thus, retrograde transport from endosomes by SNX5 enables DCV assembly at the TGN by AP-3, resolving the apparent antagonism. A novel role for AP-3 at the TGN has implications for other organelles that also depend on this adaptor.
We describe unrelated individuals with ichthyosis, failure to thrive, thrombocytopenia, photophobia, and progressive hearing loss. Each have bi-allelic mutations in AP1B1, the gene encoding the β subunit of heterotetrameric adaptor protein 1 (AP-1) complexes, which mediate endomembrane polarization, sorting, and transport. In affected keratinocytes the AP-1 β subunit is lost, and the γ subunit is greatly reduced, demonstrating destabilization of the AP-1 complex. Affected cells and tissue contain an abundance of abnormal vesicles and show hyperproliferation, abnormal epidermal differentiation, and derangement of intercellular junction proteins. Transduction of affected cells with wild-type AP1B1 rescues the vesicular phenotype, conclusively establishing that loss of AP1B1 function causes this disorder.
Among the various coats involved in vesicular transport, the clathrin associated coats that contain the adaptor complexes AP-1 and AP-2 are the most extensively characterized. The function of the recently described adaptor complex AP-3, which is similar to AP-1 and AP-2 in protein composition but does not associate with clathrin, is not known. By monitoring surface plasmon resonance we observed that AP-3 is able to interact with the tail of the lysosomal integral membrane protein LIMP-II and that this binding depends on a DEXXXLI sequence in the LIMP-II tail. Furthermore, AP-3 bound to the cytoplasmic tail of the melanosome-associated protein tyrosinase which contains a related EEXXXLL sequence. The tails of LIMP-II and tyrosinase either did not interact, or only interacted poorly, with AP-1 or AP-2. In contrast, the cytoplasmic tails of other membrane proteins containing di-leucine and/or tyrosine-based sorting signals did not bind AP-3, but AP-1 and/or AP-2. This points to a function of AP-3 in intracellular sorting to lysosomes and melanosomes of a subset of cargo proteins via di-leucine-based sorting motifs.
Local endocytic events involving receptors for axon guidance cues play a central role in controlling growth cone behaviour. Yet, little is known about the fate of internalized receptors, and whether the sorting events directing them to distinct endosomal pathways control guidance decisions. Here, we show that the receptor Plexin-D1 contains a sorting motif that interacts with the adaptor protein GIPC1 to facilitate transport to recycling endosomes. This sorting process promotes colocalization of Plexin-D1 with vesicular pools of active R-ras, leading to its inactivation. In the absence of interaction with GIPC1, missorting of Plexin-D1 results in loss of signalling activity. Consequently, Gipc1 mutant mice show specific defects in axonal projections, as well as vascular structures, that rely on Plexin-D1 signalling for their development. Thus, intracellular sorting steps that occur after receptor internalization by endocytosis provide a critical level of control of cellular responses to guidance signals.
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