A Functional Aqp1 Gene Product Localizes on The Contractile Vacuole Complex in Parameciummultimicronucleatum

Abstract

In a ciliate Paramecium, the presence of water channels on the membrane of contractile vacuole has long been predicted by both morphological and physiological data, however, to date either the biochemical or the molecular biological data have not been provided. In the present study, to examine the presence of aquaporin in Paramecium, we carried out RT-PCR with degenerated primers designed based on the ParameciumDB, and an aquaporin cDNA (aquaporin 1, aqp1) with a full-length ORF encoding 251 amino acids was obtained from Paramecium multimicronucleatum by using RACE. The deduced amino acid sequence of AQP1 had NPA-NPG motifs, and the prediction of protein secondary structure by CNR5000 and hydropathy plot showed the presence of six putative transmembrane domains and five connecting loops. Phylogenetic analysis results showed that the amino acid sequence of AQP1 was close to that of the Super-aquaporin group. The AQP1-GFP fusion protein clearly demonstrated the subcellular localization of AQP1 on the contractile vacuole complex, except for the decorated spongiome membrane. The functional analyses of aqp1 were done by RNA interference-based gene silencing, using an established feeding method. The aqp1 was found to be crucial for the total fluid output of the cell, the function of contractile vacuole membranes.

A conserved pressure-driven mechanism for regulating cytosolic osmolarity

“…21 Aquaporins in the contractile vacuole membrane then allow water to flow passively into the contractile vacuole. 3, 17, 18 In Dictyostelium , vacuolar-type proton pumps are readily visualized using deep etch electron microscopy, where they appear as 10 nm pegs protruding from the contractile vacuole membrane. 16 Similar pegs have been reported in Naegleria as well, 16 consistent with our identification of a full complement of vacuolar-type H+ATPase subunits along with aquaporins in the Naegleria genome ( Table 1, Fig.…”

“…The collection step relies on active transport of solutes and passive flow of water: vacuolar-type H + -ATPases (vacuolar-type proton pumps) embedded in contractile vacuole membranes raise the osmotic strength of the lumen, facilitating passive water flow from the cytosol through aquaporins. 3,[14][15][16][17][18][19][20][21][22] Contractile vacuole network morphology is dynamic and varies among species. Paramecium, for example, has two star-shaped contractile vacuole networks, with "canals" that extend outward into the cytosol to collect water that then flows into a central "bladder."…”

Membrane traffic and Ca2+ signals in ciliates

“…The occurrence of H + -ATPase suggests formation of an osmotic H + -gradient which serves for the exchange of Ca 2+ and for its extrusion, together with other cations and of an excess of water. This is facilitated by the presence of aquaporin, as known for a long time for other protists and documented only recently for Paramecium (Ishida et al, 2021).…”

“…It is a kind of marching on the spot. Moreover, the CVC is also endowed with organelle‐specific proteins such as aquaporin (Ishida et al, 2021) to facilitate the expulsion of an excess of water which continuously permeates into the cell, together with an excess of ions, such as Ca 2+ (Stock et al, 2002). The organelle also contains a large number of H + ‐ATPase/pump proteins (Fok et al, 2002; Wassmer et al, 2009) which, in the EM, are seen to protrude from that part of the organelle which is named the “decorated spongiome.” It is connected to the “smooth spongiome,” which forms a network around radial canals that collect the fluid to guide it, via “ampullae,” into the contractile vacuole.…”

Ciliate research: From myth to trendsetting science

“…In principle, the osmoregulatory “contractile vacuole complex” is a secretory system with isoforms of the molecules just mentioned (Plattner, 2015b) and with aquaporin for facilitating sequestration and expulsion of H 2 O, together with ions, in consequence of osmotic pressure (Ishida et al, 2021). Accordingly, it had to be expected that the system would also contain microdomain‐forming proteins of the type Reggie/Flotillin, which usually are prerequisite for the positioning of pressure‐ (mechano‐)sensitive channels (Reuter et al, 2013), just like in mammalian systems.…”