The pegs on the decorated tubules of the contractile vacuole complex of Paramecium are proton pumps

Abstract

Our previous study has shown that the decorated tubules (collectively known as the decorated spongiome) of the contractile vacuole complex (CVC) in Paramecium are the site of fluid segregation, as the binding of microinjected monoclonal antibody (mAb) DS-1 to the tubules reduced the CVC's fluid output. In this study, we showed by immunogold labeling on cryosections that the antigenic sites for mAb DS-1 were located on the 15 nm ‘pegs’ protruding from the cytosolic surface of the decorated tubules. In immunofluorescence studies, both polyclonal antibodies against the subunits of the V-ATPase of Dictyostelium discoideum and against the 57 kDa B-subunit of the V-ATPase of chromaffin granules gave identical labeling patterns to that produced by mAb DS-1. On cryosections, all three antigens were located most consistently near or on the pegs of the decorated tubules. These data support the notion that the pegs on the membrane of the decorated tubules represent the V1 complex of a proton pump. Concanamycin B, a potent inhibitor of V-ATPase activity and of acidification of lysosomes and endosomes, strongly and reversibly inhibited fluid output from the CVC but had minimal effect on the integrity of the decorated spongiome as observed by immunofluorescence. Such inhibition suggests that a V-ATPase is intimately involved in fluid segregation. Exposing Paramecium to 12 degrees C or 1 degrees C for 30 minutes resulted in the dissociation of the decorated tubules from the smooth spongiome that borders the collecting canals; thus the DS-1-reactive A4 antigen, the 75 kDa and 66 kDa antigens were all found dispersed in the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)

A conserved pressure-driven mechanism for regulating cytosolic osmolarity

“…We next sought to determine how water moves from the cytosol into contractile vacuole networks. Other species, 14, 19, 20, 22, 61 including Dictyostelium , 15, 16 transport protons into their contractile vacuoles using vacuolar-type proton pumps, resulting in a contractile vacuole lumen that is hyperosmotic relative to the cytosol. 21 Aquaporins in the contractile vacuole membrane then allow water to flow passively into the contractile vacuole.…”

Structure and dynamics of the contractile vacuole complex inTetrahymena thermophila

“…Though the CV bladder and spongiome both contain Dop1p and Vps8Dp, these two compartments are likely to serve non-identical functions and therefore differ in their molecular composition. Vacuolar ATPases have been localized to a spongiome in both the amoebozoan Dictyostelium discoideum and the ciliate Paramecium tetraurelia (Fok et al, 1995, Heuser et al, 1993, Fok et al, 1993, Nolta et al, 1993). In T. thermophila , the vATPase E-subunit-encoding gene, VMA4 , is a potential CVC determinant (Cheng et al, 2016).…”

“…The most detailed molecular studies have been pursued in Dictyostelium discoideum , Paramecium (species multimicronucleatum and tetraurelia ), and the Trypanosomid T. cruzi , which represent three highly unrelated lineages (Amoebozoa, Alveolata, and Rhizaria, respectively). In all three, water transfer from the cytosol into the CVC lumen involves the activity of proton-pumping vacuolar ATPases in the organellar membrane (Fok et al, 1995, Gronlien et al, 2002, Temesvari et al, 1996). However, because the CVC lumen is not detectibly acidified (Stock et al, 2002a), it is believed that the proton gradients produced by these pumps are harnessed to produce coupled gradients of other ions, which are more directly involved in transport of water (Jimenez and Docampo, 2015) and other small molecules (Stock et al, 2001, Stock et al, 2002a, Stock et al, 2002b, Heuser et al, 1993).…”

“…In Dictyostelium, both the bladder and spongiome are rich in vacuolar ATPase, and the two structures may interconvert during contractile cycles (Gerisch et al, 2002). In Trypanosoma cruzi the V-ATPase appears concentrated in the bladder (Ulrich 2011 PLOS One), while the Paramecium V-ATPase is strictly concentrated in a sub-domain of the spongiome, called “decorated” (Fok et al, 1995). The Paramecium CVC, which altogether appears to be more highly structured than in Dictyostelium or Trypanosoma, also includes a third distinct zone comprised of undecorated tubular arms, that links the bladder and decorated spongiome (Ishida et al, 1996, McKanna, 1976).…”

Hydro-osmotic Instabilities in Active Membrane Tubes

“…environment, thereby regulating cell volume [9,14]. Water influx into the CVC is due to an osmotic gradient generated by ATP-hydrolysing proton pumps in the membrane that move protons into the CVC [12,[15][16][17]. In many organisms such as Paramecium multimicronucleatum, the CVC includes several membrane tubular arms connected to the main vesicles, which are thought to be associated with the primary sites of proton pumping and water influx activity [18].…”