Cytohesins are highly homologous proteins composed of four distinct structural domains: (i) an N-terminal coiled-coil domain (ii) a central Sec7-domain (iii) a pleckstrin homology (PH) domain and (iv) a C-terminal polybasic domain. Importantly, our recent study revealed that the other three a-subunit isoforms (a1, a3 and a4) also bind to CTH2, suggesting its ubiquitous nature and cell biological significance of signaling between V-ATPase and CTH2 ( Merkulova et al., 2011).Ĭytohesin-2 (CTH2, also known as ARNO) together with cytohesins-1, -3 and -4, belongs to cytohesin subfamily of Arf-GEFs, activators of Arf small GTPases ( Casanova, 2007). Since CTH2 and Arf small GTPases are the key regulators of receptors signaling, vesicular trafficking and actin cytoskeleton rearrangement ( Hurtado-Lorenzo et al., 2006 Merkulova, Bakulina, Thaker, Grüber, & Marshansky, et al., 2010), these findings provided new insights into the functional link between V-ATPase dependent organellar acidification and vesicular trafficking.
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Initially we found, that the cytosolic N-terminal domain of a2-subunit (a2N 1–402) directly interacts with cytohesin-2 (CTH2), which acts as Arf-GEF (GDP/GTP exchange factor) and activator of Arf-family small GTPases. While a-subunit isoforms are highly homologous to one another, nevertheless they perform non-redundant functions. In mammals there are four a-subunit isoforms, (a1, a2, a3 and a4) that contain two major domains: a cytosolic N-terminal domain (aN ~ 400 aa) and a membrane-integrated C-terminal domain (aC ~ 400 aa), containing eight transmembrane spanning helices ( Marshansky, 2007 Toei, Toei, & Forgac, 2011). It is noteworthy that during the disassembly of V 1-sector from V O-sector, the protein-protein interactions between a-subunit and G/E-stalks as well as C-subunit and G/E-stalk are destabilized through a yet unknown molecular mechanism ( Oot et al., 2017). In particular, while two of three G/ E-stalks are directly bound to a-subunit, the third G/E-stalk is bound to subunit C, which itself interacts simultaneously with a-subunit and the second G/E-peripheral stalk ( Oot, Couoh-Cardel, Sharma, & Wilkens, 2017 Oot & Wilkens, 2012 Zhang et al., 2008). Each of these stalks is composed by a G/E heterodimer, which anchors the A 3B 3 headpiece to the membrane through either: (i) direct binding to the N-terminal tail of a-subunit or (ii) indirectly through subunit C.
In order to accomplish an efficient transfer of ATP hydrolysis energy to c-ring rotation, the rotation of A 3B 3 headpiece is prevented by three peripheral stalks ( Zhang et al., 2008). The rotation of the c-ring is driven by ATP hydrolysis catalyzed by the stationary A 3B 3 headpiece of the V 1-sector. Proton pumping across the membrane occurs via coupling of ATP-hydrolysis with rotary-mechanism of protons translocation in the interface between the rotating c-ring and the stationary a-subunit of V-ATPase.
In mammalian cells eight different proteins are combined in the following stoichiometry A 3B 3C 1D 1E 3F 1G 3H 1 to form a V 1-sector while at least six different proteins a 1c 5c″ 1d 1e 1Ac45 1 form the transmembrane V O-sector. Each of the two sectors are composed of multiple different subunits. This dissociation results in reduced ATPase activity and a shutting down of proton translocation of V-ATPase, and is one of the main mechanisms of down-regulation of V-ATPase function ( Marshansky et al., 2014 Marshansky & Futai, 2008).
Functional V-ATPases are composed of two parts: a cytoplasmic V 1- and a transmembrane V O-sector, which may dissociate from each other in response to some stimuli. V-ATPases are very complex multi-subunit enzymes that function as proton-pumping rotary nano-motors ( Marshansky et al., 2014 Marshansky & Futai, 2008). The direct and indirect roles of V-ATPase were previously reviewed by us ( Marshansky & Futai, 2008 Marshansky, Rubinstein, & Grüber, 2014) and others ( Forgac, 2007 Hinton, Bond, & Forgac, 2009). V-ATPases maintain pH homeostasis at the cellular and the whole organism level, and also play a critical role in cellular function via direct interaction with a variety of proteins, whose functions are unrelated to pH homeostasis. The V-ATPases are ubiquitous proton pumps, that use the energy of ATP to translocate protons from cytosol to intracellular compartments or extracellular space. Gerhard Grüber, in Current Topics in Membranes, 2019 1 Introduction
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