Tonoplast targeting of VHA‐a3 relies on a Rab5‐mediated but Rab7‐independent vacuolar trafficking route

Vacuolar trafficking routes and their regulators have recently drawn lots of attention in plant cell biology.A recent study reported the discovery of a plant-specific vacuolar trafficking route,i.e.,a direct ERto-vacuole route,through analysis of VHA-a3 subcellular targeting,a key component for the tonoplast VATPases.Our recent findings showed that VHA-a3 targets to the tonoplast through a Rab5-mediated but Rab7-independent pathway,shedding new lights on the unconventional vacuolar trafficking route in plant cells.     

Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes

Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles.     

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 (Aβ) 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 β‐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 Aβ 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 Aβ.      

Rab5-dependent trafficking of the m4 muscarinic acetylcholine receptor to the plasma membrane, early endosomes, and multivesicular bodies

The m4 subtype of muscarinic acetylcholine receptor regulates many physiological processes and is a novel therapeutic target for neurologic and psychiatric disorders. However, little is known about m4 regulation because of the lack of pharmacologically selective ligands. A crucial component of G protein-coupled receptor regulation is intracellular trafficking. We thus used subtype-specific antibodies and quantitative immunocytochemistry to characterize the intracellular trafficking of m4. We show that following carbachol stimulation, m4 co-localizes with transferrin, and the selective marker of early endosomes, EEA1. In addition, m4 intracellular localization depends on Rab5 activity. The dominant negative Rab5S34N inhibits m4 endocytosis initially following carbachol stimulation, and reduces the size of m4 containing vesicles. The constitutively active Rab5Q79L enhances m4 intracellular distribution, even in unstimulated cells. Rab5Q79L also produces strikingly enlarged vacuoles, which by electron microscopy contain internal vesicles, suggesting that they are multivesicular bodies. m4 localizes both to the perimeter and interior of these vacuoles. In contrast, transferrin localizes only to the vacuole perimeter, demonstrating divergence of m4 trafficking from the pathway followed by constitutively endocytosed transferrin. We thus suggest a novel model by which multivesicular bodies sort G protein-coupled receptors from a transferrin-positive recycling pathway to a nonrecycling, possibly degradative pathway.     

Rab5b localization to early endosomes in the protozoan human pathogen Leishmania donovani

Leishmania donovani is a primitive trypanosomatid pathogen of humans. This protozoan is apically polarized such that the flagellar reservoir, the exclusive site of endocytosis and exocytosis, is situated at the anterior end. Recent evidence for the existence of an endocytic pathway in Leishmania has prompted us to investigate candidate temporal markers for endocytosis. In this study we identify the L. donovani Rab5b gene, and demonstrate the localization of a Rab5b chimera to early endosomes. A full-length Rab5b protein was fused to green fluorescent protein (GFP) to generate a chimeric protein GFP::Rab5b. Transfected L. donovani promastigotes carrying this chimeric construct displayed GFP::Rab5b localization. Additionally, incubation of transfected promastigotes with the fluid-phase marker Texas Red dextran demonstrated anterior co-localization of GFP::Rab5b and dye. This suggests Rab5b may act as a marker for early endosomes in L. donovani. Note. Nucleotide sequence data reported in this paper are available in the GenBank^TM, EMBL and DDBJ databases under the accession numbers AY357217, AL359774, AF007547, BC032740.     

Dysregulation of Rab5‐mediated endocytic pathways in Alzheimer's disease

Increasing evidence has pointed to that dysregulation of the endo‐lysosomal system is an early cellular phenotype of pathogenesis for Alzheimer's disease (AD). Rab5, a small GTPase, plays a critical role in mediating these processes. Abnormal overactivation of Rab5 has been observed in post‐mortem brain samples of Alzheimer's patients as well as brain samples of mouse models of AD. Recent genome‐wide association studies of AD have identified RIN3 (Ras and Rab interactor 3) as a novel risk factor for the disease. RIN3 that functions as a guanine nucleotide exchange factor for Rab5 may serve as an important activator for Rab5 in AD pathogenesis. In this review, we present recent research highlights on the possible roles of dysregulation of Rab5‐mediated endocytic pathways in contributing to early pathogenesis of AD.      

Rab17‐mediated recycling endosomes contribute to autophagosome formation in response to Group A Streptococcus invasion

Autophagy plays a crucial role in host defence by facilitating the degradation of invading bacteria such as Group A Streptococcus (GAS). GAS‐containing autophagosome‐like vacuoles (GcAVs) form when GAS‐targeting autophagic membranes entrap invading bacteria. However, the membrane origin and the precise molecular mechanism that underlies GcAV formation remain unclear. In this study, we found that Rab17 mediates the supply of membrane from recycling endosomes (REs) to GcAVs. We showed that GcAVs contain the RE marker transferrin receptor (TfR). Colocalization analyses demonstrated that Rab17 colocalized effectively with GcAV. Rab17 and TfR were visible as punctate structures attached to GcAVs and the Rab17‐positive dots were recruited to the GAS‐capturing membrane. Overexpression of Rab17 increased the TfR‐positive GcAV content, whereas expression of the dominant‐negative Rab17 form (Rab17 N132I) caused a decrease, thereby suggesting the involvement of Rab17 in RE–GcAV fusion. The efficiency of GcAV formation was lower in Rab17 N132I‐overexpressing cells. Furthermore, knockdown of Rabex‐5, the upstream activator of Rab17, reduced the GcAV formation efficiency. These results suggest that Rab17 and Rab17‐mediated REs are involved in GcAV formation. This newly identified function of Rab17 in supplying membrane from REs to GcAVs demonstrates that RE functions as a primary membrane source during antibacterial autophagy.     

Recycling and LFA‐1‐dependent trafficking of ICAM‐1 to the immunological synapse

Little is known about how adhesion molecules on APCs accumulate at immunological synapses. We show here that ICAM‐1 on APCs is continuously internalized and rapidly recycled back to the interface after antigen‐priming T‐cell contact. The internalization rate is high in APCs, including Raji B cells and dendritic cells, but low in endothelial cells. Internalization is significantly reduced by inhibitors of Na⁺/H⁺ exchangers (NHEs), suggesting that members of the NHE‐family regulate this process. Once internalized, ICAM‐1 is co‐localized with MHC class II in the polarized recycling compartment. Surprisingly, not only ICAM‐1, but also MHC class II, is targeted to the immunological synapse through LFA‐1‐dependent adhesion. Cytosolic ICAM‐1 is highly mobile and forms a tubular structure. Inhibitors of microtubule or actin polymerization can reduce ICAM‐1 mobility, and thereby block accumulation at immunological synapses. Membrane ICAM‐1 also moves to the T‐cell contact zone, presumably through an active, cytoskeleton‐dependent mechanism. Collectively, these results demonstrate that ICAM‐1 can be transported to the immunological synapse through the recycling compartment. Furthermore, the high‐affinity state of LFA‐1 on T cells is critical to induce targeted movements of both ICAM‐1 and MHC class II to the immunological synapse on APCs. J. Cell. Biochem. 111: 1125–1137, 2010. © 2010 Wiley‐Liss, Inc.     

SopE acts as an Rab5-specific nucleotide exchange factor and recruits non-prenylated Rab5 on Salmonella-containing phagosomes to promote fusion with early endosomes

Rab-GTPase regulates the fusion between two specific vesicles. It is well documented that, for their biological function, Rab proteins need to be prenylated for attachment to the vesicle membrane. In contrast, we showed in the present investigation that SopE, a type III secretory protein of Salmonella, translocates onto Salmonella-containing phagosomes (LSP) and mediates the recruitment of non-prenylated Rab5 (Rab5:DeltaC4) on LSP in GTP form. Simultaneously, SopE present in infected cell cytosol acts as an Rab5-specific exchange factor and converts the inactive Rab-GDP to the GTP form. The non-prenylated Rab5 subsequently promoted efficient fusion of LSP with early endosomes. This is the first demonstration that a prenylation-deficient Rab protein retains biological activity and can promote vesicle fusion, if it is recruited on the membrane by some other method.     

Abnormal Rab11‐Rab8‐vesicles cluster in enterocytes of patients with microvillus inclusion disease

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo‐tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed “secretory granules.” However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome‐edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra‐/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo‐tubular organelles as Rab11‐Rab8‐positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium‐hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.      

H-Ras dynamically interacts with recycling endosomes in CHO-K1 cells: involvement of Rab5 and Rab11 in the trafficking of H-Ras to this pericentriolar endocytic compartment

H-, N-, and K-Ras are isoforms of Ras proteins, which undergo different lipid modifications at the C terminus. These post-translational events make possible the association of Ras proteins both with the inner plasma membrane and to the cytosolic surface of endoplasmic reticulum and Golgi complex, which is also required for the proper function of these proteins. To better characterize the intracellular distribution and sorting of Ras proteins, constructs were engineered to express the C-terminal domain of H- and K-Ras fused to variants of green fluorescent protein. Using confocal microscopy, we found in CHO-K1 cells that H-Ras, which is palmitoylated and farnesylated, localized at the recycling endosome in addition to the inner leaflet of the plasma membrane. In contrast, K-Ras, which is farnesylated and nonpalmitoylated, mainly localized at the plasma membrane. Moreover, we demonstrate that sorting signals of H- and K-Ras are contained within the C-terminal domain of these proteins and that palmitoylation on this region of H-Ras might operate as a dominant sorting signal for proper subcellular localization of this protein in CHO-K1 cells. Using selective photobleaching techniques, we demonstrate the dynamic nature of H-Ras trafficking to the recycling endosome from plasma membrane. We also provide evidence that Rab5 and Rab11 activities are required for proper delivery of H-Ras to the endocytic recycling compartment. Using a chimera containing the Ras binding domain of c-Raf-1 fused to a fluorescent protein, we found that a pool of GTP-bound H-Ras localized on membranes from Rab11-positive recycling endosome after serum stimulation. These results suggest that H-Ras present in membranes of the recycling endosome might be activating signal cascades essential for the dynamic and function of the organelle.     

Foam cell‐derived 4‐hydroxynonenal induces endothelial cell senescence in a TXNIP‐dependent manner

Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co‐culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP‐1 monocyte‐derived foam cells, were analysed for the induction of senescence. Senescence associated β‐galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4‐hydroxnonenal (4‐HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4‐HNE in the co‐culture medium blunted this effect. Furthermore, both foam cells and 4‐HNE increased the expression of the pro‐oxidant thioredoxin‐interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell‐induced senescence. Previous studies showed that peroxisome proliferator‐activated receptor (PPAR)δ was activated by 4‐hydroalkenals, such as 4‐HNE. Pharmacological interventions supported the involvement of the 4‐HNE‐PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell‐released 4‐HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence.     

Rab4 function in membrane recycling from early endosomes depends on a membrane to cytoplasm cycle

The monomeric GTPase rab4 is associated with early endosomes and regulates recycling vesicle formation. Because the function of rab proteins in the biosynthetic pathway does not appear to depend on cycling between membranes and cytosol, we were interested to investigate whether or not this holds true for rab function in the endocytic pathway. We created a chimeric rab4 protein (NHrab4cbvn) in which the carboxyl-terminal prenylation motif was replaced by the transmembrane domain of cellubrevin. The chimeric protein was permanently attached to membranes, properly targeted to early endosomes, and bound guanine nucleotide to the same extent as wild type rab4. However, in transport assays we found that basolaterally endocytosed transferrin was less efficiently transported to the apical cell surface in Madin-Darby canine kidney cells transfected with NHrab4cbvn than in cells expressing wild type rab4. Hence, rab4 function requires ongoing cycles of association and dissociation from early endosomes. This cycle is altered during mitosis when rab4 accumulates in the cytoplasm through phosphorylation by a mitotic kinase. We show here, using a rab4 construct that is permanently hooked onto membranes, that the membrane-bound pool of rab4 is targeted by a mitotic kinase.     

Dietary restriction involves NAD+‐dependent mechanisms and a shift toward oxidative metabolism

Interventions that slow aging and prevent chronic disease may come from an understanding of how dietary restriction (DR) increases lifespan. Mechanisms proposed to mediate DR longevity include reduced mTOR signaling, activation of the NAD⁺‐dependent deacylases known as sirtuins, and increases in NAD⁺ that derive from higher levels of respiration. Here, we explored these hypotheses in Caenorhabditis elegans using a new liquid feeding protocol. DR lifespan extension depended upon a group of regulators that are involved in stress responses and mTOR signaling, and have been implicated in DR by some other regimens [DAF‐16 (FOXO), SKN‐1 (Nrf1/2/3), PHA‐4 (FOXA), AAK‐2 (AMPK)]. Complete DR lifespan extension required the sirtuin SIR‐2.1 (SIRT1), the involvement of which in DR has been debated. The nicotinamidase PNC‐1, a key NAD⁺ salvage pathway component, was largely required for DR to increase lifespan but not two healthspan indicators: movement and stress resistance. Independently of pnc‐1, DR increased the proportion of respiration that is coupled to ATP production but, surprisingly, reduced overall oxygen consumption. We conclude that stress response and NAD⁺‐dependent mechanisms are each critical for DR lifespan extension, although some healthspan benefits do not require NAD⁺ salvage. Under DR conditions, NAD⁺‐dependent processes may be supported by a DR‐induced shift toward oxidative metabolism rather than an increase in total respiration.     

Serum withdrawal up‐regulates human SIRT1 gene expression in a p53‐dependent manner

SIRT1, a nicotinamide adenine dinucleotide (NAD⁺)‐dependent histone/protein deacetylase, has been extensively studied recently for its critical role in the regulation of physiology, calorie restriction and aging. Studies on laboratory mice showed that expression of SIRT1 can be induced by starvation in a p53‐dependent manner and requires the p53‐binding sites present in the Sirt1 promoter. However, it remains to be determined whether these findings based on rodents apply to human beings. In this paper, we characterized a putative p53‐binding element in the human SIRT1 promoter that might be required for the up‐regulation of SIRT1 in response to nutritional stress. The p53‐binding site in the promoter of human SIRT1 is more deviant from the consensus sequence than the corresponding sequence in the mouse Sirt1. There is a C to A change at the second half site in human SIRT1, thus disrupting the core‐binding element CWWG in the canonical RRRCWWGYYY. To test whether such sequence change would affect its binding with p53 and the SIRT1 expression under stress, we studied various human cell lines with different p53 status and cells with ectopic expression of functionally distinct p53. We found that serum withdrawal also up‐regulates human SIRT1 gene expression in a p53‐dependent manner and that the p53‐binding element in SIRT1 is required for the up‐regulation. Thus, the mechanism responsible for the regulation of SIRT1 expression by p53 is conserved between mice and human beings.