The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function

Abstract

Vacuolar-type H+-ATPase (V-ATPase)-driven proton pumping and organellar acidification is essential for vesicular trafficking along both the exocytotic and endocytotic pathways of eukaryotic cells. Deficient function of V-ATPase and defects of vesicular acidification have been recently recognized as important mechanisms in a variety of human diseases and are emerging as potential therapeutic targets. In the past few years, significant progress has been made in our understanding of function, regulation, and the cell biological role of V-ATPase. Here, we will review these studies with emphasis on novel direct roles of V-ATPase in the regulation of vesicular trafficking events.

Figure 1

Comparative structural models and functional roles of V-ATPase and ATP-synthase (F-ATPase) expressed in endomembrane organelles and mitochondria, respectively. The comparative subunit composition of transmembrane (V_O and F_O) and peripheral (V₁ and F₁) sectors are indicated on the left, while the catalytic hexamers, stalks and proton pathways are indicated on the right. Homologous subunits, such as the A-subunit of V-ATPase and the β-subunit of F-ATPase are shown in the same colors. (a) V-ATPase is shown as a primary proton pumping nano-motor. ATP hydrolysis drives clockwise rotation of the central stalk and ring of proteolipid subunits indicated in red. This rotation leads to the translocation of protons from the cytosol to form an acidic lumen of endomembrane organelles, and generates an electrochemical proton gradient or proton-motive force (pmf) across the membrane. In endosomes the V-ATPase promotes the neutralizing current mediated by electrogenic CLC-5 (nCl⁻/H⁺-exchanger with unknown stoichiometry, which might be n = 2 as in its bacterial homologue [45]) and drives further acidification. The values of pmf components ΔΨ and ΔpH were recently determined for the early phagosomal compartment [46^••]. (b) Mitochondrial ATP-synthase (also called F-ATPase) is shown as a secondary pump. F-ATPase function (proton translocation, counter clockwise rotation, and coupled ATP synthesis) is driven by pmf that is primarily generated during the function of respiratory chain enzymes. The function and rotation of both V-ATPase and F-ATPase are reversible under certain experimental conditions. The rotation of yeast V-ATPase (see Supplementary Information, Figure S1 and Movie S1) [11^•] and bacterial F-ATPase (see Supplementary Information, Figure S2 and Movie S2) [10^•] nano-motors. Movies are adapted from Refs. [10•, 11•].

Figure 2

Differential targeting of the a-isoforms and vesicular trafficking of V-ATPase in eukaryotic cells. The scheme depicts the compartments of endocytotic (yellow/red) and exocytotic (gray) pathways. Vesicular trafficking steps are indicated for endocytosis in red arrows and for exocytosis in blue arrows. Differential targeting of V-ATPase is cell-specific and compartment-specific. Localization of V-ATPase a-isoforms is shown as demonstrated in Figure 4 and described in text. (i) In particular, V-ATPase with a1-isoform is targeted to Golgi and involved in synaptic vesicles fusion and secretion. It is also found on presynaptic plasma membrane. (ii) V-ATPase with a2-isoform is targeted either to early endosomes or to Golgi. In early endosomes a2-isoform functions as pH-sensor by recruiting small GTPases in acidification dependent manner and involved in the formation of endosomal carrier vesicles also known as multivesicular bodies (ECV/MVB). These vesicular intermediates are involved in the trafficking between early and late endosomes or in exosomes formation and secretion. (iii) V-ATPase with a3-isoform is targeted to lysosomes and in some cells is involved in lysosomal secretion and is also localized to plasma membrane. (iv) V-ATPase with a4-isoform is specifically targeted to plasma membrane of some cells.

Figure 3

Differential targeting of the a3-isoform of V-ATPase in osteoclasts. (a) Raw 264.7 cells were cultured for 7 days in medium containing sRANKL and M-CSF. Osteoclast-like cells were identified as multinuclear cells exhibiting positive staining for tartrate-resistant acid phosphatase (TRAP). (b) Targeting and colocalization of a3 and Lamp2 in lysosomes of RAW 264.7 cells and their targeting to the plasma membrane during differentiation into osteoclast-like cells. Double immunochemical staining with anti-a3 and anti-Lamp2 antibodies was performed after different days of osteoclast differentiation followed by confocal microscopy analysis. Merged images are shown both in horizontal view (x–y sections, upper panels) and lateral view (z–x sections, lower panels). Adapted from Ref. [28^••].

Figure 4

Differential targeting of the a2-isoform, a3-isoform and a4-isoform of V-ATPase in eukaryotic cells. (a–d) Specific targeting and localization of the a3-isoform to insulin containing secretory granules in pancreatic β-cells. βTC6 cells were double stained with antibodies against the a3-isoform (green) and against either (a) insulin, (b) Lamp2, (c) GM130 or (d) synaptophysin (red) as indicated. Adapted from Ref. [29^•]. (e) Differential targeting of a2-isoform to endosomes and a4-isoform to plasma membrane in kidney proximal tubule epithelial cells. Mouse kidney proximal tubules were double stained with antibodies against the a2-isoform (green) and against the a4-isoform (red). (f, g) Specific targeting of the a2-isoform to early endosomes in mouse proximal tubule cells (MTC). Cells were double stained with antibodies against the a2-isoform (red) and against either (f) EEA1 or (g) GM130 (green) as indicated. Adapted from reference [30^••]. (h) Localization of the a2-isoform to Golgi complex in osteoclasts. Raw 264.7 cells were double stained with antibodies against the a2-isoform (green) and against the GM130 (red). Adapted from Ref. [28^••].