Inhibitors of the V0 subunit of the vacuolar H+-ATPase prevent segregation of lysosomal- and secretory-pathway proteins

Sobota, Jacqueline A.; Back, Nathan; Eipper, Betty A.; Mains, Richard E.

doi:10.1242/jcs.034298

Cited by 66 publications

(61 citation statements)

References 70 publications

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“…The physiological significance of this blockade can be explained by the reduction of subunit B-associated ATP consumption of the V 1 domain (9,65). Similar results were obtained after knockdown of subunit a of the V 0 domain (45).…”

Section: Discussionsupporting

confidence: 67%

Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex

Serra‐Peinado¹,

Sicart²,

Llopis³

et al. 2016

Journal of Biological Chemistry

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We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and the trans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H ؉ -translocating ATPase (V-ATPase), whose V 1 domain subunits B and C bind actin. We have generated a GFPtagged subunit B2 construct (GFP-B2) that is incorporated into the V 1 domain, which in turn is coupled to the V 0 sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V 1 -V 0 domains, which entails subunit B2 translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunits B2 and C1 and actin were detected. In addition, Golgi membrane lipid order disruption by D-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V 1 -V 0 domains of V-ATPase through the binding of microfilaments to subunits B and C and preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH.The secretory pathway is characterized by progressive lumen acidification of its organelles, from almost neutral in the endoplasmic reticulum (ER) 4 (pH Ϸ7.1-7.2), along the cis-to-trans Golgi stack (pH Ϸ6.7-6.0), to more acidic in the trans-Golgi network (TGN) and secretory vesicles/granules (pH Ϸ5.0) (1-3). This pH gradient is crucial for post-translational modifications and membrane trafficking events (4, 5). The main molecular determinant of the progressive fall in pH along the secretory pathway is the vacuolar [H ϩ ]ATPase (V-ATPase) (6 -8). V-ATPase is a multisubunit complex composed of two large domains, V 0 and V 1 . The V 0 domain is a 260-kDa integral membrane complex made up of five different subunits (a, b, c, cЈ, cЉ, d, and e), which mediates proton translocation; the V 1 domain is a 600 -650-kDa peripheral complex composed of eight different subunits (A, B, C, D, E, F, G, and H), which is responsible for the ATP hydrolysis that provides the mechanical force necessary for proton (H ϩ ) translocation (7, 9 -11). Whereas they are the primary source of proton delivery to endomembranes consuming ATP, the final steady-state pH in the secretory pathway is the result of the balance between active H ϩ pumping by the V-ATPase, passive H ϩ efflux through organelle endogenous H ϩ permeability, and differences in counter-ion conductance (3, 12).How differences in the pH of individual secretory compartments are generated is not well understood. Differential VATPase density and/or local regulatory mechanisms in secretory organelles and subcompartments are possible (13). In this respect, V-ATPase-dependent proton translocation could be regulated by several mechanisms, which include the following: (a) differential V-ATPase subunit expression; (b) intracel...

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Section: Discussionsupporting

confidence: 67%

Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex

Serra‐Peinado¹,

Sicart²,

Llopis³

et al. 2016

Journal of Biological Chemistry

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“…Late endosomes/lysosomes were compromised in their morphology after NH 4 Cl or Concanamycin A treatment as previously described (16,17) (Figure 7A). Consistently, when cells were incubated with pHrodo™dextran and treated with NH 4 Cl or Concanamycin A, the fluorescence intensity of the pHrodo™dextran staining decreased significantly ( Figure 7A).…”

Section: Interfering With Host Acidic Vesicles Disrupts Parasite Growthsupporting

confidence: 74%

The Host Endocytic Pathway is Essential for Plasmodium berghei Late Liver Stage Development

Silva

Thieleke‐Matos

Cabrita‐Santos

et al. 2012

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The obligate intracellular liver stage of the Plasmodium parasite represents a bottleneck in the parasite life cycle and remains a promising target for therapeutic intervention. During this stage, parasites undergo dramatic morphological changes and achieve one of the fastest replication rates among eukaryotic species. Nevertheless, relatively little is known about the parasite interactions with the host hepatocyte. Using immunofluorescence, live cell imaging and electron microscopy, we show that Plasmodium berghei parasites are surrounded by vesicles from the host late endocytic pathway. We found that these vesicles are acidic and contain the membrane markers Rab7a, CD63 and LAMP1. When host cell vesicle acidification was disrupted using ammonium chloride or Concanamycin A during the late liver stage of infection, parasite survival was not affected, but schizont size was significantly decreased. Furthermore, when the host cell endocytic pathway was loaded with BSA-gold, gold particles were found within the parasite cytoplasm, showing the transport of material from the host endocytic pathway toward the parasite interior. These observations reveal a novel Plasmodium-host interaction and suggest that vesicles from the host endolysosomal pathway could represent an important source of nutrients exploited by the fastgrowing late liver stage parasites.

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“…Two forms of active CTSB are known: (i) the one chain form (31 kDA) and (ii) the two chain form (25/26 1 5 kDA). [31][32][33] As extracellular signaling and cell-cell communications play an important role during metastatic processes 34 and VATPase inhibitors modify endo-and exocytotic events by altering membrane trafficking 15,35 the following hypothesis arouse: archazolid alters the secretion profile of invasive tumor cells contributing to its antimetastatic potential. We analyzed the secretome of archazolid-treated migratory carcinoma cells, found an impaired cathepsin B maturation process and evaluated its functional impact in vitro as well as in vivo.…”

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confidence: 99%

V-ATPase inhibition by archazolid leads to lysosomal dysfunction resulting in impaired cathepsin B activationin vivo

et al. 2013

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The myxobacterial agent archazolid inhibits the vacuolar proton pump V-ATPase. V-ATPases are ubiquitously expressed ATPdependent proton pumps, which are known to regulate the pH in endomembrane systems and thus play a crucial role in endo-and exocytotic processes of the cell. As cancer cells depend on a highly active secretion of proteolytic proteins in order to invade tissue and form metastases, inhibition of V-ATPase is proposed to affect the secretion profile of cancer cells and thus potentially abrogate their metastatic properties. Archazolid is a novel V-ATPase inhibitor. Here, we show that the secretion pattern of archazolid treated cancer cells includes various prometastatic lysosomal proteins like cathepsin A, B, C, D and Z. In particular, archazolid induced the secretion of the proforms of cathepsin B and D. Archazolid treatment abrogates the cathepsin B maturation process leading to reduced intracellular mature cathepsin B protein abundance and finally decreased cathepsin B activity, by inhibiting mannose-6-phoshate receptor-dependent trafficking. Importantly, in vivo reduced cathepsin B protein as well as a decreased proteolytic cathepsin B activity was detected in tumor tissue of archazolid-treated mice. Our results show that inhibition of V-ATPase by archazolid reduces the activity of prometastatic proteases like cathepsin B in vitro and in vivo.Vacuolar (H1)-ATPases (V-ATPases) are found on various membranes, including lysosomes, endosomes, vesicles and the plasma membrane. As V-ATPases are ATP-dependent proton pumps they are crucial for maintaining the pH of these compartments. Thus, they play a vital role in various cellular processes, like intracellular targeting of lysosomal enzymes, protein processing and degradation as well as receptor-mediated endocytosis.

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Inhibitors of the V0 subunit of the vacuolar H+-ATPase prevent segregation of lysosomal- and secretory-pathway proteins

Cited by 66 publications

References 70 publications

Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex

Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex

The Host Endocytic Pathway is Essential for Plasmodium berghei Late Liver Stage Development

V-ATPase inhibition by archazolid leads to lysosomal dysfunction resulting in impaired cathepsin B activationin vivo

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