Endosomal-lysosomal Trafficking Research Articles

Collapse of late endosomal pH elicits a rapid Rab7 response via the V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds and in disease states.

Ubiquitin-driven G protein-coupled receptor inflammatory signaling at the endosome.

G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-β-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes.

V-ATPase V0 subunit activation mediates maduramicin-induced methuosis through blocking endolysosomal trafficking in vitro and in vivo.

Methuosis, a novel cell death phenotype, is characterized by accumulation of cytoplasmic vacuolization upon external stimulus. Methuosis plays a critical role in maduramicin-induced cardiotoxicity despite the underlying mechanism is largely unknown. Herein, we aimed to investigate the origin and intracellular trafficking of cytoplasmic vacuoles, as well as the molecular mechanism of methuosis caused by maduramicin (1μg/mL) in myocardial cells. H9c2 cells and broiler chicken were used and were exposed to maduramicin at doses of 1μg/mL in vitro and 5ppm-30ppm in vivo. Morphological observation and dextran-Alexa Fluor 488 tracer experiment showed that endosomal compartments swelling and excessive macropinocytosis contributed to madurdamcin-induced methuosis. Cell counting kit-8 assay and morphology indicated pharmacological inhibition of macropinocytosis largely prevent H9c2 cells from maduramicin-triggered methuosis. In addition, late endosomal marker Rab7 and lysosomal associated membrane protein 1 (LAMP1) increased in a time-dependent manner after maduramicin treatment, and the recycling endosome marker Rab11 and ADP-ribosylation factor 6 (Arf6) were decreased by maduramicin. Vacuolar-H+-ATPase (V-ATPase) was activated by maduramicin, and pharmacological inhibition and genetic knockdown V0 subunit of V-ATPase restore endosomal-lysosomal trafficking and prevent H9c2 cells methuosis. Animal experiment showed that severe cardiac injury included the increase of creatine kinase (CK) and creatine kinase-MB (CK-MB), and vacuolar degeneration resembled methuosis in vivo after maduramicin treatment. Taken together, these findings demonstrate that targeting the inhibition of V-ATPase V0 subunit will prevent myocardial cells methuosis by restoring endosomal-lysosomal trafficking.

Effects of APOE4 allelic dosage on lipidomic signatures in the entorhinal cortex of aged mice

Apolipoprotein E ε4 (APOE4) is the primary genetic risk factor for the late-onset form of Alzheimer’s disease (AD). Although the reason for this association is not completely understood, researchers have uncovered numerous effects of APOE4 expression on AD-relevant brain processes, including amyloid beta (Aβ) accumulation, lipid metabolism, endosomal-lysosomal trafficking, and bioenergetics. In this study, we aimed to determine the effect of APOE4 allelic dosage on regional brain lipid composition in aged mice, as well as in cultured neurons. We performed a targeted lipidomic analysis on an AD-vulnerable brain region (entorhinal cortex; EC) and an AD-resistant brain region (primary visual cortex; PVC) from 14–15 month-old APOE3/3, APOE3/4, and APOE4/4 targeted replacement mice, as well as on neurons cultured with conditioned media from APOE3/3 or APOE4/4 astrocytes. Our results reveal that the EC possesses increased susceptibility to APOE4-associated lipid alterations compared to the PVC. In the EC, APOE4 expression showed a dominant effect in decreasing diacylglycerol (DAG) levels, and a semi-dominant, additive effect in the upregulation of multiple ceramide, glycosylated sphingolipid, and bis(monoacylglycerol)phosphate (BMP) species, lipids known to accumulate as a result of endosomal-lysosomal dysfunction. Neurons treated with conditioned media from APOE4/4 vs. APOE3/3 astrocytes showed similar alterations of DAG and BMP species to those observed in the mouse EC. Our results suggest that APOE4 expression differentially modulates regional neuronal lipid signatures, which may underlie the increased susceptibility of EC-localized neurons to AD pathology.

The Emerging and Diverse Roles of Bis(monoacylglycero) Phosphate Lipids in Cellular Physiology and Disease.

Although understudied relative to many phospholipids, accumulating evidence suggests that bis(monoacylglycero)phosphate (BMP) is an important class of regulatory lipid that plays key roles in lysosomal integrity and function. BMPs are rare in most mammalian tissues, comprising only a few percent of total cellular lipid content, but are elevated in cell types such as macrophages that rely heavily on lysosomal function. BMPs are markedly enriched in endosomal and lysosomal vesicles compared to other organelles and membranous structures, and their unique sn-1:sn-1′ stereoconfiguration may confer stability within the hydrolytic lysosomal environment. BMP-enriched vesicles serve in endosomal-lysosomal trafficking and function as docking structures for the activation of lysosomal hydrolytic enzymes, notably those involved in the catabolic breakdown of sphingolipids. BMP levels are dysregulated in lysosomal storage disorders, phospholipidosis, metabolic diseases, liver and kidney diseases and neurodegenerative disorders. However, whether BMP alteration is a mediator or simply a marker of pathological states is unclear. Likewise, although BMP acyl chain composition may be altered with disease states, the functional significance of specific BMP species remains to be resolved. Newly developed tools for untargeted lipidomic analysis, together with a deeper understanding of enzymes mediating BMP synthesis and degradation, will help shed further light on the functional significance of BMPs in cellular physiology and pathology.

Homozygosity mapping and whole exome sequencing provide exact diagnosis of Cohen syndrome in a Saudi family

BackgroundCohen syndrome (CS) is a rare multi-system autosomal recessive disorder with a high prevalence in the Finnish population. Clinical features of Finnish-type CS are homogeneous, however, in non-Finnish populations, CS diagnosis is challenging due to broad phenotypic variability. MethodsWe studied a consanguineous family having three affected individuals with clinical features of severe intellectual disability and global developmental delay. Clinical diagnosis of the phenotype could not be established based on the features. Therefore, whole genome SNP genotyping and whole exome sequencing (WES) were performed on DNA samples from affected and unaffected family members. ResultsHomozygosity mapping identified a shared loss of heterozygosity region on chromosome 8q22.1-q22.3 and WES data analysis revealed an insertion-deletion (indel) mutation (c.11519_11521delCAAinsT) in the VPS13B gene. The indel is predicted to cause a frameshift resulting in a premature termination of the VPS13B protein (NP_060360.3:p.Pro3840Leufs*2). ConclusionVPS13B encodes a giant transmembrane protein called vacuolar protein sorting 13 homolog B. VPS13B is known to play a role in the glycosylation of Golgi proteins and in endosomal-lysosomal trafficking. Moreover, it is thought to function in vesicle mediated transport and sorting of proteins within the cell. The mechanism by which abnormalities of the VPS13B protein lead to the phenotype of CS is currently unknown. Here, in this study, we successfully established a clinical diagnosis of CS cases from a family using genomic analyses. Clinical re-examination of the patients revealed characteristic ocular abnormalities.

Dietary cholesterol and apolipoprotein A-I are trafficked in endosomes and lysosomes in the live zebrafish intestine.

Difficulty in imaging the vertebrate intestine in vivo has hindered our ability to model nutrient and protein trafficking from both the lumenal and basolateral aspects of enterocytes. Our goal was to use live confocal imaging to increase understanding of intestinal trafficking of dietary cholesterol and apolipoprotein A-I (APOA-I), the main structural component of high-density lipoproteins. We developed a novel assay to visualize live dietary cholesterol trafficking in the zebrafish intestine by feeding TopFluor-cholesterol (TF-cholesterol), a fluorescent cholesterol analog, in a lipid-rich, chicken egg yolk feed. Quantitative microscopy of transgenic zebrafish expressing fluorescently tagged protein markers of early, recycling, and late endosomes/lysosomes provided the first evidence, to our knowledge, of cholesterol transport in the intestinal endosomal-lysosomal trafficking system. To study APOA-I dynamics, transgenic zebrafish expressing an APOA-I fluorescent fusion protein (APOA-I-mCherry) from tissue-specific promoters were created. These zebrafish demonstrated that APOA-I-mCherry derived from the intestine accumulated in the liver and vice versa. Additionally, intracellular APOA-I-mCherry localized to endosomes and lysosomes in the intestine and liver. Moreover, live imaging demonstrated that APOA-I-mCherry colocalized with dietary TF-cholesterol in enterocytes, and this colocalization increased with feeding time. This study provides a new set of tools for the study of cellular lipid biology and elucidates a key role for endosomal-lysosomal trafficking of intestinal cholesterol and APOA-I. NEW & NOTEWORTHY A fluorescent cholesterol analog was fed to live, translucent larval zebrafish to visualize intracellular cholesterol and apolipoprotein A-I (APOA-I) trafficking. With this model intestinal endosomal-lysosomal cholesterol trafficking was observed for the first time. A new APOA-I fusion protein (APOA-I-mCherry) expressed from tissue-specific promoters was secreted into the circulation and revealed that liver-derived APOA-I-mCherry accumulates in the intestine and vice versa. Intestinal, intracellular APOA-I-mCherry was observed in endosomes and lysosomes and colocalized with dietary cholesterol.

GCN5L1 interacts with αTAT1 and RanBP2 to regulate hepatic α-tubulin acetylation and lysosome trafficking.

Although GCN5L1 (also known as BLOC1S1) facilitates mitochondrial protein acetylation and controls endosomal-lysosomal trafficking, the mechanisms underpinning these disparate effects are unclear. As microtubule acetylation modulates endosome-lysosome trafficking, we reasoned that exploring the role of GCN5L1 in this biology may enhance our understanding of GCN5L1-mediated protein acetylation. We show that α-tubulin acetylation is reduced in GCN5L1-knockout hepatocytes and restored by GCN5L1 reconstitution. Furthermore, GCN5L1 binds to the α-tubulin acetyltransferase αTAT1, and GCN5L1-mediated α-tubulin acetylation is dependent on αTAT1. Given that cytosolic GCN5L1 has been identified as a component of numerous multiprotein complexes, we explored whether novel interacting partners contribute to this regulation. We identify RanBP2 as a novel interacting partner of GCN5L1 and αTAT1. Genetic silencing of RanBP2 phenocopies GCN5L1 depletion by reducing α-tubulin acetylation, and we find that RanBP2 possesses a tubulin-binding domain, which recruits GCN5L1 to α-tubulin. Finally, we find that genetic depletion of GCN5L1 promotes perinuclear lysosome accumulation and histone deacetylase inhibition partially restores lysosomal positioning. We conclude that the interactions of GCN5L1, RanBP2 and αTAT1 function in concert to control α-tubulin acetylation and may contribute towards the regulation of cellular lysosome positioning. This article has an associated First Person interview with the first author of the paper.

Mutations in Vps15 perturb neuronal migration in mice and are associated with neurodevelopmental disease in humans.

The formation of the vertebrate brain requires the generation, migration, differentiation and survival of neurons. Genetic mutations that perturb these critical cellular events can result in malformations of the telencephalon, providing a molecular window into brain development. Here we report the identification of an N-ethyl-N-nitrosourea-induced mouse mutant characterized by a fractured hippocampal pyramidal cell layer, attributable to defects in neuronal migration. We show that this is caused by a hypomorphic mutation in Vps15 that perturbs endosomal-lysosomal trafficking and autophagy, resulting in an upregulation of Nischarin, which inhibits Pak1 signaling. The complete ablation of Vps15 results in the accumulation of autophagic substrates, the induction of apoptosis and severe cortical atrophy. Finally, we report that mutations in VPS15 are associated with cortical atrophy and epilepsy in humans. These data highlight the importance of the Vps15-Vps34 complex and the Nischarin-Pak1 signaling hub in the development of the telencephalon.

Possible Clues for Brain Energy Translation via Endolysosomal Trafficking of APP-CTFs in Alzheimer's Disease.

Vascular dysfunctions, hypometabolism, and insulin resistance are high and early risk factors for Alzheimer's disease (AD), a leading neurological disease associated with memory decline and cognitive dysfunctions. Early defects in glucose transporters and glycolysis occur during the course of AD progression. Hypometabolism begins well before the onset of early AD symptoms; this timing implicates the vulnerability of hypometabolic brain regions to beta-secretase 1 (BACE-1) upregulation, oxidative stress, inflammation, synaptic failure, and cell death. Despite the fact that ketone bodies, astrocyte-neuron lactate shuttle, pentose phosphate pathway (PPP), and glycogenolysis compensate to provide energy to the starving AD brain, a considerable energy crisis still persists and increases during disease progression. Studies that track brain energy metabolism in humans, animal models of AD, and in vitro studies reveal striking upregulation of beta-amyloid precursor protein (β-APP) and carboxy-terminal fragments (CTFs). Currently, the precise role of CTFs is unclear, but evidence supports increased endosomal-lysosomal trafficking of β-APP and CTFs through autophagy through a vague mechanism. While intracellular accumulation of Aβ is attributed as both the cause and consequence of a defective endolysosomal-autophagic system, much remains to be explored about the other β-APP cleavage products. Many recent works report altered amino acid catabolism and expression of several urea cycle enzymes in AD brains, but the precise cause for this dysregulation is not fully explained. In this paper, we try to connect the role of CTFs in the energy translation process in AD brain based on recent findings.

Identification of Novel Vacuolin-1 Analogues as Autophagy Inhibitors by Virtual Drug Screening and Chemical Synthesis.

Autophagy is a fundamental cellular degradation process which is essential for cell homeostasis, and dysfunctional autophagy has been associated with a variety of human diseases, such as cancer. Several autophagy chemical modulators have been applied in a number of preclinical or clinical trials against these autophagy related diseases, especially cancer. Small molecule vacuolin-1 potently and reversibly inhibits both endosomal-lysosomal trafficking and autophagosome-lysosome fusion, yet the molecular mechanisms underlying vacuolin-1 mediated autophagy inhibition remain unknown. Here, we first performed the virtual drug screening and identified 14 vacuolin-1 analogues as autophagy inhibitors. Based on these virtual screening results, we further designed and synthesized 17 vacuolin-1 analogues, and found that 13 of them are autophagy inhibitors and a couple of them are as potent as vacuolin-1. In summary, these studies expanded the pool of useful autophagy inhibitors and reveal the structural-activity relationship of vacuolin-1 analogues, which is useful for future development of vacuolin-1 analogues with high potency and for identification of the molecular targets of vacuolin-1.

A Short Synthetic Peptide Mimetic of Apolipoprotein A1 Mediates Cholesterol and Globotriaosylceramide Efflux from Fabry Fibroblasts.

Fabry disease is an X-linked sphingolipid storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (AGA, EC 3.2.1.22) resulting in the intracellular accumulation of globotriaosylceramide (Gb3). We found that Gb3 storage also correlates with accumulation of endosomal-lysosomal cholesterol in Fabry fibroblasts. This cholesterol accumulation may contribute to the phenotypic pathology of Fabry disease by slowing endosomal-lysosomal trafficking. We found that LDL receptor expression is not downregulated in Fabry fibroblasts resulting in accumulation of both cholesterol and Gb3. 5A-Palmitoyl oleoyl-phosphatidylcholine (5AP) is a phospholipid complex containing a short synthetic peptide that mimics apolipoprotein A1, the main protein component of high-density lipoprotein (HDL) that mediates the efflux of cholesterol from cells via the ATP-binding cassette transporter. We used 5AP and HDL to remove cholesterol from Fabry fibroblasts to examine the fate of accumulated cellular Gb3. Using immunostaining techniques, we found that 5AP is highly effective for depleting cholesterol and Gb3 in these cells. 5AP restores the ApoA-1-mediated cholesterol efflux leading to mobilization of cholesterol and reduction of Gb3 in Fabry fibroblasts.

Cohen syndrome is associated with major glycosylation defects

Cohen syndrome (CS) is a rare autosomal recessive disorder with multisytemic clinical features due to mutations in the VPS13B gene, which has recently been described encoding a mandatory membrane protein involved in Golgi integrity. As the Golgi complex is the place where glycosylation of newly synthesized proteins occurs, we hypothesized that VPS13B deficiency, responsible of Golgi apparatus disturbance, could lead to glycosylation defects and/or mysfunction of this organelle, and thus be a cause of the main clinical manifestations of CS. The glycosylation status of CS serum proteins showed a very unusual pattern of glycosylation characterized by a significant accumulation of agalactosylated fucosylated structures as well as asialylated fucosylated structures demonstrating a major defect of glycan maturation in CS. However, CS transferrin and α1-AT profiles, two liver-derived proteins, were normal. We also showed that intercellular cell adhesion molecule 1 and LAMP-2, two highly glycosylated cellular proteins, presented an altered migration profile on SDS-PAGE in peripheral blood mononuclear cells from CS patients. RNA interference against VPS13B confirmed these glycosylation defects. Experiments with Brefeldin A demonstrated that intracellular retrograde cell trafficking was normal in CS fibroblasts. Furthermore, early endosomes were almost absent in these cells and lysosomes were abnormally enlarged, suggesting a crucial role of VPS13B in endosomal-lysosomal trafficking. Our work provides evidence that CS is associated to a tissue-specific major defect of glycosylation and endosomal-lysosomal trafficking defect, suggesting that this could be a new key element to decipher the mechanisms of CS physiopathology.

Snapin-Regulated Late Endosomal Transport Is Critical for Efficient Autophagy-Lysosomal Function in Neurons

Neuron maintenance and survival require late endocytic transport from distal processes to the soma where lysosomes are predominantly localized. Here, we report a role for Snapin in attaching dynein to late endosomes through its intermediate chain (DIC). snapin(-/-) neurons exhibit aberrant accumulation of immature lysosomes, clustering and impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis due to impaired delivery of internalized proteins and hydrolase precursors from late endosomes to lysosomes, and impaired clearance of autolysosomes, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene, but not the DIC-binding-defective Snapin-L99K mutant. Snapin overexpression in wild-type neurons enhances late endocytic transport and lysosomal function, whereas expressing the mutant defective in Snapin-DIC coupling shows a dominant-negative effect. Altogether, our study highlights new mechanistic insights into how Snapin-DIC coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function, and thus neuronal homeostasis.

The nuclear protein Waharan is required for endosomal-lysosomal trafficking in Drosophila.

Here we report Drosophila Waharan (Wah), a 170-kD predominantly nuclear protein with two potential human homologues, as a newly identified regulator of endosomal trafficking. Wah is required for neuromuscular-junction development and muscle integrity. In muscles, knockdown of Wah caused novel accumulations of tightly packed electron-dense tubules, which we termed 'sausage bodies'. Our data suggest that sausage bodies coincide with sites at which ubiquitylated proteins and a number of endosomal and lysosomal markers co-accumulate. Furthermore, loss of Wah function generated loss of the acidic LysoTracker compartment. Together with data demonstrating that Wah acts earlier in the trafficking pathway than the Escrt-III component Drosophila Shrb (snf7 in Schizosaccharomyces pombe), our results indicate that Wah is essential for endocytic trafficking at the late endosome. Highly unexpected phenotypes result from Wah knockdown, in that the distribution of ubiquitylated cargos and endolysosomal morphologies are affected despite Wah being a predominant nuclear protein. This finding suggests the existence of a relationship between nuclear functions and endolysosomal trafficking. Future studies of Wah function will give us insights into this interesting phenomenon.

EBAG9 tempers lymphocyte killing activity

The cytotoxic activity of lymphocytes is crucial for immune surveillance and homeostasis. Several independent, naturally occurring genetic models characterized by defects in granule trafficking or exocytosis have helped to decipher the multiple steps and molecules that regulate the cytotoxic process. The study by Rüder and colleagues in this issue of the JCI shows that an engineered absence of EBAG9, previously reported as a tumor-associated antigen, enhances cytotoxic activity of CTLs but not NK cells, likely acting on the endosomal-lysosomal trafficking of the cytotoxic effectors (see the related article beginning on page 2184). This finding adds a new piece to the puzzle of complex mechanisms that tightly regulate the capacity of the cytotoxic response and suggests a new target to negatively modulate CTL responsiveness.

NPC1/NPC2 function as a tag team duo to mobilize cholesterol

Cholesterol homeostasis is essential for the functional integrity of the cell (1). Both the cellular content and distribution of cholesterol within the cell is highly dynamic and tightly regulated through de novo synthesis of cholesterol by the endoplasmic reticulum (ER) (2), and by uptake of cholesterol ester-rich lipoprotein particles circulating in the serum by the low-density lipoprotein (LDL) receptor pathway (3). The main sorting station for cholesterol within the cell is the late endosome (LE), an intermediate stage in the endosomal-lysosomal trafficking pathway (Fig. 1). LEs are also referred to as “multivesicular” compartments in that they have numerous internalized membrane-enveloped structures generated by the ESCRT pathway (4) (Fig. 1). Within the lumen of the LE, cholesterol esters are hydrolyzed to free cholesterol by acid lipase. Released cholesterol joins the endogenous cholesterol pool for recycling to the cell surface, or transferred to the ER where they are re-esterified and, as necessary, placed in storage particles for later use (1).

The lipodystrophy protein seipin is found at endoplasmic reticulum lipid droplet junctions and is important for droplet morphology

Lipodystrophy is a disorder characterized by a loss of adipose tissue often accompanied by severe hypertriglyceridemia, insulin resistance, diabetes, and fatty liver. It can be inherited or acquired. The most severe inherited form is Berardinelli-Seip Congenital Lipodystrophy Type 2, associated with mutations in the BSCL2 gene. BSCL2 encodes seipin, the function of which has been entirely unknown. We now report the identification of yeast BSCL2/seipin through a screen to detect genes important for lipid droplet morphology. The absence of yeast seipin results in irregular lipid droplets often clustered alongside proliferated endoplasmic reticulum (ER); giant lipid droplets are also seen. Many small irregular lipid droplets are also apparent in fibroblasts from a BSCL2 patient. Human seipin can functionally replace yeast seipin, but a missense mutation in human seipin that causes lipodystrophy, or corresponding mutations in the yeast gene, render them unable to complement. Yeast seipin is localized in the ER, where it forms puncta. Almost all lipid droplets appear to be on the ER, and seipin is found at these junctions. Therefore, we hypothesize that seipin is important for droplet maintenance and perhaps assembly. In addition to detecting seipin, the screen identified 58 other genes whose deletions cause aberrant lipid droplets, including 2 genes encoding proteins known to activate lipin, a lipodystrophy locus in mice, and 16 other genes that are involved in endosomal-lysosomal trafficking. The genes identified in our screen should be of value in understanding the pathway of lipid droplet biogenesis and maintenance and the cause of some lipodystrophies.

Presenilin 1 Regulates Epidermal Growth Factor Receptor Turnover and Signaling in the Endosomal-Lysosomal Pathway

Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Abeta production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/beta-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS(-/-)) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS(+/+) cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS(-/-) cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.

Structural basis for viral late-domain binding to Alix

The modular protein Alix is a central node in endosomal-lysosomal trafficking and the budding of human immunodeficiency virus (HIV)-1. The Gag p6 protein of HIV-1 contains a LYPx(n)LxxL motif that is required for Alix-mediated budding and binds a region of Alix spanning residues 360-702. The structure of this fragment of Alix has the shape of the letter 'V' and is termed the V domain. The V domain has a topologically complex arrangement of 11 alpha-helices, with connecting loops that cross three times between the two arms of the V. The conserved residue Phe676 is at the center of a large hydrophobic pocket and is crucial for binding to a peptide model of HIV-1 p6. Overexpression of the V domain inhibits HIV-1 release from cells. This inhibition of release is reversed by mutations that block binding of the Alix V domain to p6.