The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles inParamecium

Abstract

IntroductionThe vacuolar-proton-ATPase (V-ATPase) translocates protons across membranes against their electrochemical potential through ATP hydrolysis. The V-ATPase is responsible for the acidification of organelles like phagosomes, lysosomes, early endosomes, the trans-Golgi-network, dense core secretory granules and vacuoles of plants (Sun-Wada et al., 2003a;Nelson, 2003;Kluge et al., 2003). In some specialized cells of higher organisms, the enzyme is also localized in the plasma membrane and serves to secrete protons to the extracellular space. For example, the acidification of the extracellular matrix by proton secretion of osteoclasts is important for bone resorption (Toyomura et al., 2003). An important aspect of the proton translocation by the V-ATPase is the creation of an electrochemical potential that can be used for secondary active transport of ions. This mechanism is used in neuronal cells, where the neurotransmitters glutamate or γ-amino-butyric-acid are concentrated in the lumen of synaptic vesicles (Roseth et al., 1995;Fonnum et al., 1998) and in chromaffin granules (Apps, 1997).The V-ATPase is composed of two subcomplexes: the cytosolic V 1 -sector, where ATP binding and hydrolysis takes place; and a transmembranous V 0 -sector, through which protons are tunneled. The holoenzyme in Saccharomyces cerevisiae consists of 14 different subunits (Kawasaki-Nishi et al., 2004;Sambade and Kane, 2004). V 1 contains subunits A-H in the stoichiometry A 3 B 3 CDEFG 2 H 1-2 . V 0 is formed by the subunits a, c, c′, c′′, d and e in a stoichiometry of ade x c 4 c′c′′. The V-ATPase was shown to act by a rotationary mechanism similar to that of the structurally related F-ATPase (Imamura et al., 2003).The enzyme activity can be regulated by reversible dissociation of V 1 from V 0 . It was found in yeast that the ratio of assembled/disassembled enzyme is strongly influenced by the culture conditions. Most of the V 1 -domains dissociate from their V 0 counterparts at vacuoles in yeast when glucose is depleted in the medium (Kane and Smardon, 2003). Whether other physiological conditions exist that lead to a dissociation of the enzyme is unclear.Apart from its enzymatic action as a proton pump, the V 0 -subcomplex of the V-ATPase was found to be localized in the plasma membrane and to be involved in the release of neurotransmitter in mammalian cells (Falk-Vairant et al., 1996;Morel, 2003). A role of the V 0 -sector was also shown in homotypic membrane fusion of vacuoles in yeast (Peters et al., 2001). V 0 -subcomplexes were reported to build so-called 'trans-complexes' in opposing membranes and were postulated to facilitate membrane fusion. A knockout mutant of the vacuolar a-subunit of V 0 (vph1) was shown to cause fragmentation of vacuoles, also indicating an involvement of the V-ATPase in membrane dynamics (Bayer et al., 2003).Paramecium is a unicellular model organism, in which

An Ins(1,4,5)P3 receptor in Paramecium is associated with the osmoregulatory system

“…Sequences encoding the Ins(1,4,5)P 3 -binding domain of IP 3 R N (S268-L658) were amplified by PCR using primers pBD-f (5Ј-gcgctgcagatgtcaacatcttggaaaattaatctt-3Ј) and pBD-rev (5Ј-cgcctcgagaacctaatcgttcaaatagatacaatta-3Ј), and cloned in a modified pPXV-GFP vector (Wassmer et al, 2005). Paramecium cells were transformed by microinjecting DNA into the macronucleus as described by Wassmer et al (Wassmer et al, 2005).…”

“…The ORS generally consists of two identical units per cell, each composed of a contractile vacuole, with approximately six collecting canals to which a tubular membranous network is attached (reviewed in Allen and Naitoh, 2002). This network displays a part proximal to the collecting canals that has smooth membranes (smooth spongiome) and a distal part that is studded with V-type H (Allen et al, 1990;Allen, 1995;Fok et al, 1995;Tominaga et al, 1998;Wassmer et al, 2005) and which might be coupled not only to the wellestablished osmotically driven water influx (Grønlien et al, 2002) but possibly also to a hypothetical cation-exchange system (Stock et al, 2002a;Stock et al, 2002b). In the absence of a Ca 2+ -pump, Ca 2+ might, thus, be transported into the ORS.…”

Clathrin-Associated Endocytosis as a Route of Entry into Cells for Parvoviruses

“…Baf A1 blocks the V-ATPase activity causing neutralization of the acidic environment of endosomes. Baf A1 is routinely used as a suppressor of V-ATPases, and it is used to test whether specific viral entry is reliant on endosomal acidification (Clague et al, 1994;Drose & Altendorf, 1997;Jin et al, 2005;Wassmer et al, 2005). It has been widely used as a probe to study acid-mediated viral trafficking using a variety of virus models.…”

“…Chemical inhibitors are assumed to block the entry pathway at certain specific points and incomplete virus replication in the presence of inhibitor is presumed to indicate viral penetration using the pathway blocked by the drug. Commonly used drugs are chlorpromazine (prevents assembly and disassembly of clathrin lattices at the cell surface and on endosomes by inhibiting clathrin polymerization which blocks assembly of coated pits and is also a phospholipase A2 inhibitor, Blanchard et al, 2006); bafilamycin A1 (inhibits vacuolar proton ATPases inhibiting acidification of endosomes by blocking transport of protons into the vesicle, Clague et al, 1994;Drose & Altendorf, 1997;Wassmer et al, 2005); ammonium chloride (penetrates into the endosome increasing endosomal pH, Jin et al, 2002;Liebl et al, 2006); chloroquine (blocks assembly of clathrin coated pits and raises endosomal pH, Mani et al, 2006;Ros et al, 2002). Brefeldin A has no effect on the early endosome but inhibits vesicle transport and early-to-late endosome transition (Clague et al, 1994;Nebenfuhr et al, 2002).…”

Sub-Second Cellular Dynamics: Time-Resolved Electron Microscopy and Functional Correlation

“…In contrast. by expressing the different endogenous isoforms of relevant subunits as fluorescent fusion proteins in Parantecium we could largely exclude their occurrence at trichocyst exocyto sis sites (Wassmer et al, 2005) where they should have shown up in the characteristic regular spacing. Therefore.…”