Swedish Alzheimer's disease variant perturbs activity of retrograde molecular motors and causes widespread derangement of axonal transport pathways.

Experimental studies in flies, mice, and humans suggest a significant role of impaired axonal transport in the pathogenesis of Alzheimer's disease (AD). The mechanisms underlying these impairments in axonal transport, however, remain poorly understood. Here we report that the Swedish familial AD mutation causes a standstill of the amyloid precursor protein (APP) in the axons at the expense of its reduced anterograde transport. The standstill reflects the perturbed directionality of the axonal transport of APP, which spends significantly more time traveling in the retrograde direction. This ineffective movement is accompanied by an enhanced association of dynactin-1 with APP, which suggests that reduced anterograde transport of APP is the result of enhanced activation of the retrograde molecular motor dynein by dynactin-1. The impact of the Swedish mutation on axonal transport is not limited to the APP vesicles since it also reverses the directionality of a subset of early endosomes, which become enlarged and aberrantly accumulate in distal locations. In addition, it also reduces the trafficking of lysosomes due to their less effective retrograde movement. Altogether, our experiments suggest a pivotal involvement of retrograde molecular motors and transport in the mechanisms underlying impaired axonal transport in AD and reveal significantly more widespread derangement of axonal transport pathways in the pathogenesis of AD.

Rab21 regulates caveolin-1-mediated endocytic trafficking to promote immature neurite pruning.

Transmembrane proteins are internalized by clathrin- and caveolin-dependent endocytosis. Both pathways converge on early endosomes and are thought to share the small GTPase Rab5 as common regulator. In contrast to this notion, we show here that the clathrin- and caveolin-mediated endocytic pathways are differentially regulated. Rab5 and Rab21 localize to distinct populations of early endosomes in cortical neurons and preferentially regulate clathrin- and caveolin-mediated pathways, respectively, suggesting heterogeneity in the early endosomes, rather than a converging point. Suppression of Rab21, but not Rab5, results in decreased plasma membrane localization and total protein levels of caveolin-1, which perturbs immature neurite pruning of cortical neurons, an in vivo-specific step of neuronal maturation. Taken together, our data indicate that clathrin- and caveolin-mediated endocytic pathways run in parallel in early endosomes, which show different molecular regulation and physiological function.

The temporal association of CapZ with early endosomes regulates endosomal trafficking and viral entry into host cells.

BACKGROUND: Many viruses enter host cells by hijacking endosomal trafficking. CapZ, a canonical actin capping protein, participates in endosomal trafficking, yet its precise role in endocytosis and virus infection remains elusive. RESULTS: Here, we showed that CapZ was transiently associated with early endosomes (EEs) and was subsequently released from the matured EEs after the fusion of two EEs, which was facilitated by PI(3)P to PI(3,5)P2 conversion. Vacuolin-1 (a triazine compound) stabilized CapZ at EEs and thus blocked the transition of EEs to late endosomes (LEs). Likewise, artificially tethering CapZ to EEs via a rapamycin-induced protein-protein interaction system blocked the early-to-late endosome transition. Remarkably, CapZ knockout or artificially tethering CapZ to EEs via rapamycin significantly inhibited flaviviruses, e.g., Zika virus (ZIKV) and dengue virus (DENV), or beta-coronavirus, e.g., murine hepatitis virus (MHV), infection by preventing the escape of RNA genome from endocytic vesicles. CONCLUSIONS: These results indicate that the temporal association of CapZ with EEs facilitates early-to-late endosome transition (physiologically) and the release of the viral genome from endocytic vesicles (pathologically).

Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1.

Selective autophagy of damaged organelles is important to maintain cellular homeostasis. The mechanisms how autophagy selects specific targets is often poorly understood. Rabaptin5 was previously known as a major regulator of early endosome identity and maturation. Here, we identify two novel Rabaptin5 interactors: FIP200, a subunit of the ULK1 autophagy initiator complex, and ATG16L1, a central component of the E3-like enzyme in LC3 lipidation. Autophagy of early endosomes damaged by chloroquine or monensin treatment requires Rabaptin5 and particularly a short sequence motif that binds to the WD domain of ATG16L1. Rabaptin5 and its interaction with ATG16L1 further contributes to the autophagic elimination of Salmonella enterica early after infection, when it resides in phagosomes with early endosomal characteristics. Our results demonstrate a novel function of Rabaptin5 in quality control of early endosomes in the selective targeting of autophagy to damaged early endosomes and phagosomes.

circEIF3I facilitates the recruitment of SMAD3 to early endosomes to promote TGF-ß signalling pathway-mediated activation of MMPs in pancreatic cancer.

BACKGROUND: Among digestive tract tumours, pancreatic ductal adenocarcinoma (PDAC) shows the highest mortality trend. Moreover, although PDAC metastasis remains a leading cause of cancer-related deaths, the biological mechanism is poorly understood. Recent evidence demonstrates that circular RNAs (circRNAs) play important roles in PDAC progression. METHODS: Differentially expressed circRNAs in normal and PDAC tissues were screened via bioinformatics analysis. Sanger sequencing, RNase R and actinomycin D assays were performed to confirm the loop structure of circEIF3I. In vitro and in vivo functional experiments were conducted to assess the role of circEIF3I in PDAC. MS2-tagged RNA affinity purification, mass spectrometry, RNA immunoprecipitation, RNA pull-down assay, fluorescence in situ hybridization, immunofluorescence and RNA-protein interaction simulation and analysis were performed to identify circEIF3I-interacting proteins. The effects of circEIF3I on the interactions of SMAD3 with TGFßRI or AP2A1 were measured through co-immunoprecipitation and western blotting. RESULTS: A microarray data analysis showed that circEIF3I was highly expressed in PDAC cells and correlated with TNM stage and poor prognosis. Functional experiments in vitro and in vivo revealed that circEIF3I accelerated PDAC cells migration, invasion and metastasis by increasing MMPs expression and activity. Mechanistic research indicated that circEIF3I binds to the MH2 domain of SMAD3 and increases SMAD3 phosphorylation by strengthening the interactions between SMAD3 and TGFßRI on early endosomes. Moreover, AP2A1 binds with circEIF3I directly and promotes circEIF3I-bound SMAD3 recruitment to TGFßRI on early endosomes. Finally, we found that circEif3i exerts biological functions in mice similar to those of circEIF3I in humans PDAC. CONCLUSIONS: Our study reveals that circEIF3I promotes pancreatic cancer progression. circEIF3I is a molecular scaffold that interacts with SMAD3 and AP2A1 to form a ternary complex, that facilitates the recruitment of SMAD3 to early endosomes and then activates the TGF-ß signalling pathway. Hence, circEIF3I is a potential prognostic biomarker and therapeutic target in PDAC.

The Nma1 protein promotes long distance transport mediated by early endosomes in Ustilago maydis.

Early endosomes (EEs) are part of the endocytic transport pathway and resemble the earliest class of transport vesicles between the internalization of extracellular material, their cellular distribution or vacuolar degradation. In filamentous fungi, EEs fulfill important functions in long distance transport of cargoes as mRNAs, ribosomes, and peroxisomes. Formation and maturation of early endosomes is controlled by the specific membrane-bound Rab-GTPase Rab5 and tethering complexes as CORVET (class C core vacuole/endosome tethering). In the basidiomycete Ustilago maydis, Rab5a is the prominent GTPase to recruit CORVET to EEs; in rab5a deletion strains, this function is maintained by the second EE-associated GTPase Rab5b. The tethering- and core-subunits of CORVET are essential, buttressing a central role for EE transport in U. maydis. The function of EEs in long distance transport is supported by the Nma1 protein that interacts with the Vps3 subunit of CORVET. The interaction stabilizes the binding of Vps3 to the CORVET core complex that is recruited to Rab5a via Vps8. Deletion of nma1 leads to a significantly reduced number of EEs, and an increased conversion rate of EEs to late endosomes. Thus, Nma1 modulates the lifespan of EEs to ensure their availability for the various long distance transport processes.

Human Cytomegalovirus Manipulates Syntaxin 6 for Successful Trafficking and Subsequent Infection of Monocytes.

Human cytomegalovirus (HCMV) exhibits a complex host-pathogen interaction with peripheral blood monocytes. We have identified a unique, cell-type specific retrograde-like intracellular trafficking pattern that HCMV utilizes to gain access to the monocyte nucleus and for productive infection. We show that infection of primary human monocytes, epithelial cells, and fibroblasts leads to an increase in the amount of the trafficking protein Syntaxin 6 (Stx6). However, only knockdown (KD) of Stx6 in monocytes inhibited viral trafficking to the trans-Golgi network (TGN), a requisite step for nuclear translocation in monocytes. Conversely, KD of Stx6 in epithelial cells and fibroblasts did not change the kinetics of nuclear translocation and productive infection. Stx6 predominantly functions at the level of the TGN where it facilitates retrograde transport, a trafficking pathway used by only a few cellular proteins and seldom by pathogens. We also newly identify that in monocytes, Stx6 exhibits an irregular vesicular localization rather than being concentrated at the TGN as seen in other cell-types. Lastly, we implicate that viral particles that associate with both Stx6 and EEA1 early in infection are the viral population that successfully traffics to the TGN at later time points and undergo nuclear translocation. Additionally, we show for the first time that HCMV enters the TGN, and that lack of Stx6 prevents viral trafficking to this organelle. We argue that we have identified an essential cell-type specific regulator that controls early steps in efficient productive infection of a cell-type required for viral persistence and disease. IMPORTANCE Human cytomegalovirus (HCMV) infection causes severe and often fatal disease in the immunocompromised. It is one of the leading infectious causes of birth defects and causes severe complications in transplant recipients. By uncovering the unique pathways used by the virus to infect key cells, such as monocytes, responsible for dissemination and persistence, we provide new potential targets for therapeutic intervention.

APP-BACE1 Interaction and Intracellular Localization Regulate Aß Production in iPSC-Derived Cortical Neurons.

Alzheimer's disease (AD) is characterized pathologically by amyloid ß (Aß)-containing plaques. Generation of Aß from amyloid precursor protein (APP) by two enzymes, ß- and γ-secretase, has therefore been in the AD research spotlight for decades. Despite this, how the physical interaction of APP with the secretases influences APP processing is not fully understood. Herein, we compared two genetically identical human iPSC-derived neuronal cell types: low Aß-secreting neuroprogenitor cells (NPCs) and high Aß-secreting mature neurons, as models of low versus high Aß production. We investigated levels of substrate, enzymes and products of APP amyloidogenic processing and correlated them with the proximity of APP to ß- and γ-secretase in endo-lysosomal organelles. In mature neurons, increased colocalization of full-length APP with the ß-secretase BACE1 correlated with increased ß-cleavage product sAPPß. Increased flAPP/BACE1 colocalization was mainly found in early endosomes. In the same way, increased colocalization of APP-derived C-terminal fragment (CTF) with presenilin-1 (PSEN1), the catalytic subunit of γ-secretase, was seen in neurons as compared to NPCs. Furthermore, most of the interaction of APP with BACE1 in low Aß-secreting NPCs seemed to derive from CTF, the remaining APP part after BACE1 cleavage, indicating a possible novel product-enzyme inhibition. In conclusion, our results suggest that interaction of APP and APP cleavage products with their secretases can regulate Aß production both positively and negatively. ß- and γ-Secretases are difficult targets for AD treatment due to their ubiquitous nature and wide range of substrates. Therefore, targeting APP-secretase interactions could be a novel treatment strategy for AD. Colocalization of APP species with BACE1 in a novel model of low- versus high-Aß secretion-Two genetically identical human iPSC-derived neuronal cell types: low Aß-secreting neuroprogenitor cells (NPCs) and high Aß secreting mature neurons, were compared. Increased full-length APP (flAPP)/BACE1 colocalization in early endosomes was seen in neurons, while APP-CTF/BACE1 colocalization was much higher than flAPP/BACE1 colocalization in NPCs, although the cellular location was not determined.

The Rab11-regulated endocytic pathway and BDNF/TrkB signaling: Roles in plasticity changes and neurodegenerative diseases.

Neurons are highly polarized cells that rely on the intracellular transport of organelles. This process is regulated by molecular motors such as dynein and kinesins and the Rab family of monomeric GTPases that together help move cargo along microtubules in dendrites, somas, and axons. Rab5-Rab11 GTPases regulate receptor trafficking along early-recycling endosomes, which is a process that determines the intracellular signaling output of different signaling pathways, including those triggered by BDNF binding to its tyrosine kinase receptor TrkB. BDNF is a well-recognized neurotrophic factor that regulates experience-dependent plasticity in different circuits in the brain. The internalization of the BDNF/TrkB complex results in signaling endosomes that allow local signaling in dendrites and presynaptic terminals, nuclear signaling in somas and dynein-mediated long-distance signaling from axons to cell bodies. In this review, we briefly discuss the organization of the endocytic pathway and how Rab11-recycling endosomes interact with other endomembrane systems. We further expand upon the roles of the Rab11-recycling pathway in neuronal plasticity. Then, we discuss the BDNF/TrkB signaling pathways and their functional relationships with the postendocytic trafficking of BDNF, including axonal transport, emphasizing the role of BDNF signaling endosomes, particularly Rab5-Rab11 endosomes, in neuronal plasticity. Finally, we discuss the evidence indicating that the dysfunction of the early-recycling pathway impairs BDNF signaling, contributing to several neurodegenerative diseases.

The Dynamic Range of Acidity: Tracking Rules for the Unidirectional Penetration of Cellular Compartments.

Labeled ammonium cations with pKa ∼7.4 accumulate in acidic organelles because they can be neutralized transiently to cross the membrane at cytosolic pH 7.2 but not at their internal pH<5.5. Retention in early endosomes with less acidic internal pH was achieved recently using weaker acids of up to pKa 9.8. We report here that primary ammonium cations with higher pKa 10.6, label early endosomes more efficiently. This maximized early endosome tracking coincides with increasing labeling of Golgi networks with similarly weak internal acidity. Guanidinium cations with pKa 13.5 cannot cross the plasma membrane in monomeric form and label the plasma membrane with selectivity for vesicles embarking into endocytosis. Self-assembled into micelles, guanidinium cations enter cells like arginine-rich cell-penetrating peptides and, driven by their membrane potential, penetrate mitochondria unidirectionally despite their high inner pH. The resulting tracking rules with an approximated dynamic range of pKa change ∼3.5 are expected to be generally valid, thus enabling the design of chemistry tools for biology research in the broadest sense. From a practical point of view, most relevant are two complementary fluorescent flipper probes that can be used to image the mechanics at the very beginning of endocytosis.

Class IA PI3Ks regulate subcellular and functional dynamics of IDO1.

Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities-namely, the catalytic and signaling functions-are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3-kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus, IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity.

KDEL receptor is a cell surface receptor that cycles between the plasma membrane and the Golgi via clathrin-mediated transport carriers.

KDEL receptor cycles between the ER and the Golgi to retrieve ER-resident chaperones that get leaked to the secretory pathway during protein export from the ER. Recent studies have shown that a fraction of KDEL receptor may reside in the plasma membrane and function as a putative cell surface receptor. However, the trafficking itinerary and mechanism of cell surface expressed KDEL receptor remains largely unknown. In this study, we used N-terminally Halo-tagged KDEL receptor to investigate its endocytosis from the plasma membrane and trafficking itinerary of the endocytosed receptor through the endolysosomal compartments. Our results indicate that surface-expressed KDEL receptor undergoes highly complex recycling pathways via the Golgi and peri-nuclear recycling endosomes that are positive for Rab11 and Rab14, respectively. Unexpectedly, KDEL receptor appears to preferentially utilize clathrin-mediated endocytic pathway as well as clathrin-dependent transport carriers for export from the trans-Golgi network. Taken together, we suggest that KDEL receptor may be a bona fide cell surface receptor with a complex, yet well-defined trafficking itinerary through the endolysosomal compartments.

The endosomal RIN2/Rab5C machinery prevents VEGFR2 degradation to control gene expression and tip cell identity during angiogenesis.

Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic 'tip' and 'stalk' cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis.

Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling.

Despite recently uncovered connections between autophagy and the endocytic pathway, the role of autophagy in regulating endosomal function remains incompletely understood. Here, we find that the ablation of autophagy-essential players disrupts EGF-induced endocytic trafficking of EGFR. Cells lacking ATG7 or ATG16L1 exhibit increased levels of phosphatidylinositol-3-phosphate (PI(3)P), a key determinant of early endosome maturation. Increased PI(3)P levels are associated with an accumulation of EEA1-positive endosomes where EGFR trafficking is stalled. Aberrant early endosomes are recognised by the autophagy machinery in a TBK1- and Gal8-dependent manner and are delivered to LAMP2-positive lysosomes. Preventing this homeostatic regulation of early endosomes by autophagy reduces EGFR recycling to the plasma membrane and compromises downstream signalling and cell survival. Our findings uncover a novel role for the autophagy machinery in maintaining early endosome function and growth factor sensing.

Toll-Like Receptor 9-Mediated Neuronal Innate Immune Reaction Is Associated with Initiating a Pro-Regenerative State in Neurons of the Dorsal Root Ganglia Non-Associated with Sciatic Nerve Lesion.

One of the changes brought about by Wallerian degeneration distal to nerve injury is disintegration of axonal mitochondria and consequent leakage of mitochondrial DNA (mtDNA)-the natural ligand for the toll-like receptor 9 (TLR9). RT-PCR and immunohistochemical or Western blot analyses were used to detect TLR9 mRNA and protein respectively in the lumbar (L4-L5) and cervical (C7-C8) dorsal root ganglia (DRG) ipsilateral and contralateral to a sterile unilateral sciatic nerve compression or transection. The unilateral sciatic nerve lesions led to bilateral increases in levels of both TLR9 mRNA and protein not only in the lumbar but also in the remote cervical DRG compared with naive or sham-operated controls. This upregulation of TLR9 was linked to activation of the Nuclear Factor kappa B (NFκB) and nuclear translocation of the Signal Transducer and Activator of Transcription 3 (STAT3), implying innate neuronal immune reaction and a pro-regenerative state in uninjured primary sensory neurons of the cervical DRG. The relationship of TLR9 to the induction of a pro-regenerative state in the cervical DRG neurons was confirmed by the shorter lengths of regenerated axons distal to ulnar nerve crush following a previous sciatic nerve lesion and intrathecal chloroquine injection compared with control rats. The results suggest that a systemic innate immune reaction not only triggers the regenerative state of axotomized DRG neurons but also induces a pro-regenerative state further along the neural axis after unilateral nerve injury.

Fluorescent Membrane Tension Probes for Early Endosomes.

Fluorescent flipper probes have been introduced recently to image membrane tension in live cells, and strategies to target these probes to specific membranes are emerging. In this context, early endosome (EE) targeting without the use of protein engineering is especially appealing because it translates into a fascinating transport problem. Weakly basic probes, commonly used to track the inside of acidic late endosomes and lysosomes, are poorly retained in EE because they are sufficiently neutralized in weakly acidic EE, thus able to diffuse out. Here, we disclose a rational strategy to target EE using a substituted benzylamine with a higher pKa value as a head group of the flipper probe. The resulting EE flippers are validated for preserved mechanosensitivity, ready for use in biology, particularly to elucidate the mechanics of endocytosis.

Engaging myosin VI tunes motility, morphology and identity in endocytosis.

While unconventional myosins interact with different stages of the endocytic pathway, they are ascribed a transport function that is secondary to the protein complexes that control organelle identity. Endosomes are subject to a dynamic, continuous flux of proteins that control their characteristic properties, including their motility within the cell. Efforts to describe the changes in identity of this compartment have largely focused on the adaptors present on the compartment and not on the motile properties of the compartment itself. In this study, we use a combination of optogenetic and chemical-dimerization strategies to target exogenous myosin VI to early endosomes, and probe its influence on organelle motility, morphology and identity. Our analysis across timescales suggests a model wherein the artificial engagement of myosin VI motility on early endosomes restricts microtubule-based motion, followed by morphological changes characterized by the rapid condensation and disintegration of organelles, ultimately leading to the enhanced overlap of markers that demarcate endosomal compartments. Together, our findings show that synthetic engagement of myosin VI motility is sufficient to alter organelle homeostasis in the endocytic pathway.

Cholera toxin inhibits SNX27-retromer-mediated delivery of cargo proteins to the plasma membrane.

Cholera toxin (CT) causes severe diarrhea by increasing intracellular cAMP leading to a PKA-dependent increase in Cl- secretion through CFTR and decreased Na+ absorption through inhibition of Na+/H+ exchanger 3 (NHE3; also known as SLC9A3). The mechanism(s) by which CT inhibits NHE3 is partially understood, although no drug therapy has been successful at reversing this inhibition. We now describe that CT phosphorylates an amino acid in the PDZ domain of SNX27, which inhibits SNX27-mediated trafficking of NHE3 from the early endosomes to the plasma membrane (PM), and contributes to reduced basal NHE3 activity through a mechanism that involves reduced PM expression and reduced endocytic recycling. Importantly, mutagenesis studies (Ser to Asp) showed that the effect of this phosphorylation of SNX27 phenocopies the effects seen upon loss of SNX27 function, affecting PM trafficking of cargo proteins that bind SNX27-retromer. Additionally, CT destabilizes retromer function by decreasing the amount of core retromer proteins. These effects of CT can be partially rescued by enhancing retromer stability by using 'pharmacological chaperones'. Moreover, pharmacological chaperones can be used to increase basal and cholera toxin-inhibited NHE3 activity and fluid absorption by intestinal epithelial cells.This article has an associated First Person interview with the first author of the paper.

Amyloid precursor protein traffics from the Golgi directly to early endosomes in an Arl5b- and AP4-dependent pathway.

The intracellular trafficking and proteolytic processing of the membrane-bound amyloid precursor protein (APP) are coordinated events leading to the generation of pathogenic amyloid-beta (Aß) peptides. The membrane transport of newly synthesized APP from the Golgi to the endolysosomal system is not well defined, yet it is likely to be critical for regulating its processing by ß-secretase (BACE1) and γ-secretase. Here, we show that the majority of newly synthesized APP is transported from the trans-Golgi network (TGN) directly to early endosomes and then subsequently to the late endosomes/lysosomes with very little transported to the cell surface. We show that Arl5b, a small G protein localized to the TGN, and AP4 are essential for the post-Golgi transport of APP to early endosomes. Arl5b is physically associated with AP4 and is required for the recruitment of AP4, but not AP1, to the TGN. Depletion of either Arl5b or AP4 results in the accumulation of APP, but not BACE1, in the Golgi, and an increase in APP processing and Aß secretion. These findings demonstrate that APP is diverted from BACE1 at the TGN for direct transport to early endosomes and that the TGN represents a site for APP processing with the subsequent secretion of Aß.

Function of the cargo sorting dileucine motif in a cytomegalovirus immune evasion protein.

As an immune evasion mechanism, cytomegaloviruses (CMVs) have evolved proteins that interfere with cell surface trafficking of MHC class-I (MHC-I) molecules to tone down recognition by antiviral CD8 T cells. This interference can affect the trafficking of recently peptide-loaded MHC-I from the endoplasmic reticulum to the cell surface, thus modulating the presentation of viral peptides, as well as the recycling of pre-existing cell surface MHC-I, resulting in reduction of the level of overall MHC-I cell surface expression. Murine cytomegalovirus (mCMV) was paradigmatic in that it led to the discovery of this immune evasion strategy of CMVs. Members of its m02-m16 gene family code for type-I transmembrane glycoproteins, proven or predicted, most of which carry cargo sorting motifs in their cytoplasmic, C-terminal tail. For the m06 gene product m06 (gp48), the cargo has been identified as being MHC-I, which is linked by m06 to cellular adapter proteins AP-1A and AP-3A through the dileucine motif EPLARLL. Both APs are involved in trans-Golgi network (TGN) cargo sorting and, based on transfection studies, their engagement by the dileucine motif was proposed to be absolutely required to prevent MHC-I exposure at the cell surface. Here, we have tested this prediction in an infection system with the herein newly described recombinant virus mCMV-m06AA, in which the dileucine motif is destroyed by replacing EPLARLL with EPLARAA. This mutation has a phenotype in that the transition of m06-MHC-I complexes from early endosomes (EE) to late endosomes (LE)/lysosomes for degradation is blocked. Consistent with the binding of the MHC-I α-chain to the luminal domain of m06, the m06-mediated disposal of MHC-I did not require the ß2m chain of mature MHC-I. Unexpectedly, however, disconnecting MHC-I cargo from AP-1A/3A by the motif mutation in m06 had no notable rescuing impact on overall cell surface MHC-I, though it resulted in some improvement of the presentation of viral antigenic peptides by recently peptide-loaded MHC-I. Thus, the current view on the mechanism by which m06 mediates immune evasion needs to be revised. While the cargo sorting motif is critically involved in the disposal of m06-bound MHC-I in the endosomal/lysosomal pathway at the stage of EE to LE transition, this motif-mediated disposal is not the critical step by which m06 causes immune evasion. We rather propose that engagement of AP-1A/3A by the cargo sorting motif in m06 routes the m06-MHC-I complexes into the endosomal pathway and thereby detracts them from the constitutive cell surface transport.