V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway

The recruitment of the small GTPase Arf6 and ARNO from cytosol to endosomal membranes is driven by V-ATPase-dependent intra-endosomal acidification. The molecular mechanism that mediates this pH-sensitive recruitment and its role are unknown. Here, we demonstrate that Arf6 interacts with the c-subunit, and ARNO with the a2-isoform of V-ATPase. The a2-isoform is targeted to early endosomes, interacts with ARNO in an intra-endosomal acidification-dependent manner, and disruption of this interaction results in reversible inhibition of endocytosis. Inhibition of endosomal acidification abrogates protein trafficking between early and late endosomal compartments. These data demonstrate the crucial role of early endosomal acidification and V-ATPase/ARNO/Arf6 interactions in the regulation of the endocytic degradative pathway. They also indicate that V-ATPase could modulate membrane trafficking by recruiting and interacting with ARNO and Arf6; characteristics that are consistent with the role of V-ATPase as an essential component of the endosomal pH-sensing machinery.     

In situ measurement of the electrical potential across the lysosomal membrane using FRET

The progressive acidification of the endocytic pathway is generated by H(+) pumping of electrogenic vacuolar-type ATPases (V-ATPases) on the endosomal/lysosomal membrane. The determinants of pH during endosome maturation are not completely understood, but the permeability to ions that neutralize the electrogenic effect of the V-ATPase has been proposed to play a central role. If counter-ion conductance becomes limiting, the generation of a large membrane potential would dominate the proton-motive force (pmf), diminishing the pH gradient proportionally. Validation of this notion requires direct measurement of the electrical potential that develops across the endosomal/lysosomal membrane. To date, the measurement of lysosomal membrane potential (ψ(φ) ) in situ has been hampered by the inability to access endosomes by electrophysiological means and the fact that individual organelles cannot be discerned when using potentiometric fluorescent dyes. Here, we describe a noninvasive procedure to estimate ψ(φ) in intact cells, based on fluorescence resonance energy transfer (FRET). At steady state, ψ(φ) averaged 19 mV (lumen positive) and was only partially dissipated by inhibition of the V-ATPase with concanamycin A (CcA). ψ(φ) was considerably increased by alkalinization of the lysosome lumen by NH(4) Cl, implying that at steady state the V-ATPase operates at submaximal rates and that the contribution of ψ(φ) to pmf is relatively small. Our method should enable systematic studies of endosomal/lysosomal potential.     

Caveolin-1 is ubiquitinated and targeted to intralumenal vesicles in endolysosomes for degradation

Caveolae are long-lived plasma membrane microdomains composed of caveolins, cavins, and a cholesterol-rich membrane. Little is known about how caveolae disassemble and how their coat components are degraded. We studied the degradation of caveolin-1 (CAV1), a major caveolar protein, in CV1 cells. CAV1 was degraded very slowly, but turnover could be accelerated by compromising caveolae assembly. Now, CAV1 became detectable in late endosomes (LE) and lysosomes where it was degraded. Targeting to the degradative pathway required ubiquitination and the endosomal sorting complex required for transport (ESCRT) machinery for inclusion into intralumenal vesicles in endosomes. A dual-tag strategy allowed us to monitor exposure of CAV1 to the acidic lumen of individual, maturing LE in living cells. Importantly, we found that "caveosomes," previously described by our group as independent organelles distinct from endosomes, actually correspond to late endosomal compartments modified by the accumulation of overexpressed CAV1 awaiting degradation. The findings led us to a revised model for endocytic trafficking of CAV1.     

MLN64 is involved in actin-mediated dynamics of late endocytic organelles

MLN64 is a late endosomal cholesterol-binding membrane protein of an unknown function. Here, we show that MLN64 depletion results in the dispersion of late endocytic organelles to the cell periphery similarly as upon pharmacological actin disruption. The dispersed organelles in MLN64 knockdown cells exhibited decreased association with actin and the Arp2/3 complex subunit p34-Arc. MLN64 depletion was accompanied by impaired fusion of late endocytic organelles and delayed cargo degradation. MLN64 overexpression increased the number of actin and p34-Arc-positive patches on late endosomes, enhanced the fusion of late endocytic organelles in an actin-dependent manner, and stimulated the deposition of sterol in late endosomes harboring the protein. Overexpression of wild-type MLN64 was capable of rescuing the endosome dispersion in MLN64-depleted cells, whereas mutants of MLN64 defective in cholesterol binding were not, suggesting a functional connection between MLN64-mediated sterol transfer and actin-dependent late endosome dynamics. We propose that local sterol enrichment by MLN64 in the late endosomal membranes facilitates their association with actin, thereby governing actin-dependent fusion and degradative activity of late endocytic organelles.     

Retrieval of the Vacuolar H+-ATPase from Phagosomes Revealed by Live Cell Imaging

Background

The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.

Methodology

To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.

Principal Findings

We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.

Conclusions/Signficance

Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.     

ARF1 regulates pH-dependent COP functions in the early endocytic pathway

Coat proteins of the COP family were recently shown by us and others to be involved in membrane transport in the endocytic pathway, in addition to their known functions in the biosynthetic pathway. We have also shown that membrane association of endosomal COPs depends on the acidic endosomal pH, in contrast to biosynthetic COPs. In this paper, we report that both membrane recruitment of endosomal COPs and in vitro biogenesis of transport intermediates destined for late endosomes, depend on a cytosolic factor, which we identified as the small GTP-binding protein ARF1. Our data indicate that ARF1 does not act via activation of an endosomal phospholipase D. We also find that ARF1 membrane association is regulated by the endosomal pH, and that this controls the pH-dependent association of endosomal COPs. These studies thus show that ARF1 regulates COP functions in the endocytic pathway, and indicate that ARF1 acts as the cytosolic component of a transmembrane pH-sensing mechanism.     

Endosomal Maturation,Rab7 GTPase and Phosphoinositides in African Swine Fever Virus Entry

Here we analyzed the dependence of African swine fever virus (ASFV) infection on the integrity of the endosomal pathway. Using confocal immunofluorescence with antibodies against viral capsid proteins, we found colocalization of incoming viral particles with early endosomes (EE) during the first minutes of infection. Conversely, viral capsid protein was not detected in acidic late endosomal compartments, multivesicular bodies (MVBs), late endosomes (LEs) or lysosomes (LY). Using an antibody against a viral inner core protein, we found colocalization of viral cores with late compartments from 30 to 60 minutes postinfection. The absence of capsid protein staining in LEs and LYs suggested that virus desencapsidation would take place at the acid pH of these organelles. In fact, inhibitors of intraluminal acidification of endosomes caused retention of viral capsid staining virions in Rab7 expressing endosomes and more importantly, severely impaired subsequent viral protein production. Endosomal acidification in the first hour after virus entry was essential for successful infection but not thereafter. In addition, altering the balance of phosphoinositides (PIs) which are responsible of the maintenance of the endocytic pathway impaired ASFV infection. Early infection steps were dependent on the production of phosphatidylinositol 3-phosphate (PtdIns3P) which is involved in EE maturation and multivesicular body (MVB) biogenesis and on the interconversion of PtdIns3P to phosphatidylinositol 3, 5-biphosphate (PtdIns(3,5)P₂). Likewise, GTPase Rab7 activity should remain intact, as well as processes related to LE compartment physiology, which are crucial during early infection. Our data demonstrate that the EE and LE compartments and the integrity of the endosomal maturation pathway orchestrated by Rab proteins and PIs play a central role during early stages of ASFV infection.     

A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast

V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.     

Annexin A8 regulates late endosome organization and function

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. Late endosomes, organelles of the degradative lysosomal route, seem to require associated actin filaments for proper localization and function. We show here that the F-actin and phospholipid binding protein annexin A8 is associated specifically with late endosomes. Altering intracellular annexin A8 levels drastically affected the morphology and intracellular distribution of late endosomes. Trafficking through the degradative pathway was delayed in the absence of annexin A8, resulting in attenuated ligand-induced degradation of the epidermal growth factor receptor and prolonged epidermal growth factor-induced activation of mitogen-activated protein kinase. Depletion of annexin A8 reduced the association of late endosomal membranes with actin filaments. These results indicate that the defective cargo transport through the late endocytic pathway and the imbalanced signaling of activated receptors observed in the absence of annexin A8 results from the disturbed association of late endosomal membranes with the actin network, resulting in impaired actin-based late endosome motility.     

Polar transmembrane domains target proteins to the interior of the yeast vacuole

Membrane proteins transported to the yeast vacuole can have two fates. Some reach the outer vacuolar membrane, whereas others enter internal vesicles, which form in late endosomes, and are ultimately degraded. The vacuolar SNAREs Nyv1p and Vam3p avoid this fate by using the AP-3-dependent pathway, which bypasses late endosomes, but the endosomal SNARE Pep12p must avoid it more directly. Deletion analysis revealed no cytoplasmic sequences necessary to prevent the internalization of Pep12p in endosomes. However, introduction of acidic residues into the cytoplasmic half of the transmembrane domain created a dominant internalization signal. In other contexts, this same feature diverted proteins from the Golgi to endosomes and slowed their exit from the endoplasmic reticulum. The more modestly polar transmembrane domains of Sec12p and Ufe1p, which normally serve to hold these proteins in the endoplasmic reticulum, also cause Pep12p to be internalized, as does that of the vacuolar protein Cps1p. It seems that quality control mechanisms recognize polar transmembrane domains at multiple points in the secretory and endocytic pathways and in endosomes sort proteins for subsequent destruction in the vacuole. These mechanisms may minimize the damaging effects of abnormally exposed polar residues while being exploited for the localization of some normal proteins.     

The big brain aquaporin is required for endosome maturation and notch receptor trafficking

Activity of the big brain (bib) gene influences Notch signaling during Drosophila nervous system development. We demonstrate that Bib, which belongs to the aquaporin family of channel proteins, is required for endosome maturation in Drosophila epithelial cells. In the absence of Bib, early endosomes arrest and form abnormal clusters, and cells exhibit reduced acidification of endocytic trafficking organelles. Bib acts downstream of Hrs in early endosome morphogenesis and regulates biogenesis of endocytic compartments prior to the formation of Rab7-containing late endosomes. Abnormal endosome morphology caused by loss of Bib is accompanied by overaccumulation of Notch, Delta, and other signaling molecules as well as reduced intracellular trafficking of Notch to nuclei. Analysis of several endosomal trafficking mutants reveals a correlation between endosomal acidification and levels of Notch signaling. Our findings reveal an unprecedented role for an aquaporin in endosome maturation, trafficking, and acidification.     

Characterization and intracellular trafficking pattern of vacuoles containing Chlamydia pneumoniae in human epithelial cells

Chlamydiae are obligate intracellular pathogens that reside within a membrane-bound vacuole throughout their developmental cycle. In this study, the intraphagosomal pH of Chlamydia pneumoniae ( Cpn ) was qualitatively assessed, and the intracellular fate of the pathogen-containing vacuole and its interaction with endocytic organelles in human epithelial cells were analysed using conventional immunofluorescence and confocal microscopy. The pH-sensitive probes acridine orange (AO), LysoTracker (LyT) and DAMP did not accumulate in the bacterial inclusion. In addition, exposure of cells to bafilomycin A1 (BafA1), a potent acidification inhibitor, did not inhibit or delay chlamydial growth. The chlamydial compartment was not accessible to the fluid-phase tracer Texas Red (TR)-dextran and did not exhibit any level of staining for the late endosomal marker cation-independent mannose-6-phosphate receptor (Ci-M6PR) or for the lysosomal-associated membrane proteins (LAMP-1 and -2) and CD63. In addition, transferrin receptor (TfR)-enriched vesicles were observed close to Cpn vacuoles, potentially indicating a specific translocation of these organelles through the cytoplasm to the vicinity of the vacuole. We conclude that Cpn , like other chlamydial spp., circumvents the host endocytic pathway and inhabits a non-acidic vacuole, which is dissociated from late endosomes and lysosomes, but selectively accumulates early endosomes.     

Dynamic movements of organelles containing Niemann-Pick C1 protein: NPC1 involvement in late endocytic events

People homozygous for mutations in the Niemann-Pick type C1 (NPC1) gene have physiological defects, including excess accumulation of intracellular cholesterol and other lipids, that lead to drastic neural and liver degeneration. The NPC1 multipass transmembrane protein is resident in late endosomes and lysosomes, but its functions are unknown. We find that organelles containing functional NPC1-fluorescent protein fusions undergo dramatic movements, some in association with extending strands of endoplasmic reticulum. In NPC1 mutant cells the NPC1-bearing organelles that normally move at high speed between perinuclear regions and the periphery of the cell are largely absent. Pulse-chase experiments with dialkylindocarbocyanine low-density lipoprotein showed that NPC1 organelles function late in the endocytic pathway; NPC1 protein may aid the partitioning of endocytic and lysosomal compartments. The close connection between NPC1 and the drug U18666A, which causes NPC1-like organelle defects, was established by rescuing drug-treated cells with overproduced NPC1. U18666A inhibits outward movements of NPC1 organelles, trapping membranes and cholesterol in perinuclear organelles similar to those in NPC1 mutant cells, even when cells are grown in lipoprotein-depleted serum. We conclude that NPC1 protein promotes the creation and/or movement of particular late endosomes, which rapidly transport materials to and from the cell periphery.     

A pH-sensitive luminal His-cluster promotes interaction of PAM with V-ATPase along the secretory and endocytic pathways of peptidergic cells

The biosynthetic and endocytic pathways of secretory cells are characterized by progressive luminal acidification, a process which is crucial for posttranslational modifications and membrane trafficking. This progressive fall in luminal pH is mainly achieved by the vacuolar-type-H⁺ ATPase (V-ATPase). V-ATPases are large, evolutionarily ancient rotary proton pumps that consist of a peripheral V1 complex, which hydrolyzes ATP, and an integral membrane V0 complex, which transports protons from the cytosol into the lumen. Upon sensing the desired luminal pH, V-ATPase activity is regulated by reversible dissociation of the complex into its V1 and V0 components. Molecular details of how intraluminal pH is sensed and transmitted to the cytosol are not fully understood. Peptidylglycine α-amidating mono-oxygenase (PAM; EC 1.14.17.3), a secretory pathway membrane enzyme which shares similar topology with two V-ATPase accessory proteins (Ac45 and prorenin receptor), has a pH-sensitive luminal linker region. Immunofluorescence and sucrose gradient analysis of peptidergic cells (AtT-20) identified distinct subcellular compartments exhibiting spatial co-occurrence of PAM and V-ATPase. In vitro binding assays demonstrated direct binding of the cytosolic domain of PAM to V1H. Blue native PAGE identified heterogeneous high-molecular weight complexes of PAM and V-ATPase. A PAM-1 mutant (PAM-1/H3A) with altered pH sensitivity had diminished ability to form high-molecular weight complexes. In addition, V-ATPase assembly status was altered in PAM-1/H3A expressing cells. Our analysis of the secretory and endocytic pathways of peptidergic cells supports the hypothesis that PAM serves as a luminal pH-sensor, regulating V-ATPase action by altering its assembly status.     

Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis

Ubiquitin functions as a signal for sorting cargo at multiple steps of the endocytic pathway and controls the activity of trans-acting components of the endocytic machinery (reviewed in refs 1, and 2). By contrast to proteasome degradation, which generally requires a polyubiquitin chain that is at least four subunits long, internalization and sorting of endocytic cargo at the late endosome are mediated by mono-ubiquitination. Here, we demonstrate that ubiquitin-interacting motifs (UIMs) found in epsins and Vps27p (ref. 9) from Saccharomyces cerevisiae are required for ubiquitin binding and protein transport. Epsin UIMs are important for the internalization of receptors into vesicles at the plasma membrane. Vps27p UIMs are necessary to sort biosynthetic and endocytic cargo into vesicles that bud into the lumen of a late endosomal compartment, the multivesicular body. We propose that mono-ubiquitin regulates internalization and endosomal sorting by interacting with modular ubiquitin-binding domains in core components of the protein transport machinery. UIM domains are found in a broad spectrum of proteins, consistent with the idea that mono-ubiquitin can function as a regulatory signal to control diverse biological activities.     

Analyses of the Recycling Receptor, FcRn, in Live Cells Reveal Novel Pathways for Lysosomal Delivery

Lysosomes play a central role in the degradation of proteins and other macromolecules. The mechanisms by which receptors are transferred to lysosomes for constitutive degradation are poorly understood. We have analyzed the processes that lead to the lysosomal delivery of the Fc receptor, FcRn. These studies provide support for a novel pathway for receptor delivery. Specifically, unlike other receptors that enter intraluminal vesicles in late endosomes, FcRn is transferred from the limiting membrane of such endosomes to lysosomes, and is rapidly internalized into the lysosomal lumen. By contrast, LAMP-1 persists on the limiting membrane. Receptor transfer is mediated by tubular extensions from late endosomes to lysosomes, or by interactions of the two participating organelles in kiss-and-linger-like processes, whereas full fusion is rarely observed. The persistence of FcRn on the late endosomal limiting membrane, together with selective transfer to lysosomes, allows this receptor to undergo recycling or degradation. Consequently, late endosomes have functional plasticity, consistent with the presence of the Rab5 GTPase in discrete domains on these compartments.     

Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery

After reaching early endosomes by receptor-mediated endocytosis, diphtheria toxin (DT) molecules have two possible fates. A large pool enters the degradative pathway whereas a few molecules become cytotoxic by translocating their catalytic fragment A (DTA) into the cytosol. Impairment of DT degradation by microtubule depolymerization does not block DT cytotoxicity. Therefore, DTA membrane translocation into the cytosol occurs from an endocytic compartment located upstream of late endosomes. Comparisons between early endosomes and endocytic carrier vesicles in a cell-free translocation assay have demonstrated that early endosomes are the earliest endocytic compartment from which DTA translocates. DTA translocation is ATP-dependent, requires early endosomal acidification, and is increased by the addition of cytosol. Cytosol-dependent DTA translocation is GTPγS-insensitive but is blocked by anti-βCOP antibodies.     

Endocytosed cation-independent mannose 6-phosphate receptor traffics via the endocytic recycling compartment en route to the trans-Golgi network and a subpopulation of late endosomes

Although the distribution of the cation-independent mannose 6-phosphate receptor (CI-MPR) has been well studied, its intracellular itinerary and trafficking kinetics remain uncertain. In this report, we describe the endocytic trafficking and steady-state localization of a chimeric form of the CI-MPR containing the ecto-domain of the bovine CI-MPR and the murine transmembrane and cytoplasmic domains expressed in a CHO cell line. Detailed confocal microscopy analysis revealed that internalized chimeric CI-MPR overlaps almost completely with the endogenous CI-MPR but only partially with individual markers for the trans-Golgi network or other endosomal compartments. After endocytosis, the chimeric receptor first enters sorting endosomes, and it then accumulates in the endocytic recycling compartment. A large fraction of the receptors return to the plasma membrane, but some are delivered to the trans-Golgi network and/or late endosomes. Over the course of an hour, the endocytosed receptors achieve their steady-state distribution. Importantly, the receptor does not start to colocalize with late endosomal markers until after it has passed through the endocytic recycling compartment. In CHO cells, only a small fraction of the receptor is ever detected in endosomes bearing substrates destined for lysosomes (kinetically defined late endosomes). These data demonstrate that CI-MPR takes a complex route that involves multiple sorting steps in both early and late endosomes.