Altered expression of the 100 kDa subunit of theDictyosteliumvacuolar proton pump impairs enzyme assembly, endocytic function and cytosolic pH regulation

Abstract

The vacuolar proton pump (V-ATPase) appears to be essential for viability of Dictyostelium cells. To investigate the function of VatM, the 100 kDa transmembrane V-ATPase subunit, we altered its level. By means of homologous recombination, the promoter for the chromosomal vatM gene was replaced with the promoter for the act6 gene, yielding the mutant strain VatMpr. The act6 promoter is much more active in cells growing axenically than on bacteria. Thus, transformants were selected under axenic growth conditions, then shifted to bacteria to determine the consequences of reduced vatM expression. When VatMpr cells were grown on bacteria,the level of the 100 kDa V-ATPase subunit dropped, cell growth slowed, and the A subunit, a component of the peripheral catalytic domain of the V-ATPase,became mislocalized. These defects were complemented by transformation of the mutant cells with a plasmid expressing vatM under the control of its own promoter. Although the principal locus of vacuolar proton pumps in Dictyostelium is membranes of the contractile vacuole system, mutant cells did not manifest osmoregulatory defects. However, bacterially grown VatMpr cells did exhibit substantially reduced rates of phagocytosis and a prolonged endosomal transit time. In addition, mutant cells manifested alterations in the dynamic regulation of cytosolic pH that are characteristic of normal cells grown in acid media, which suggested that the V-ATPase also plays a role in cytosolic pH regulation.

Ankyrin‐repeat proteins from sponge symbionts modulate amoebal phagocytosis

“…7) within the autofluorescent sponge tissue (dark yellowgreen). Diatoms were also present (large blue cells), consistent with previous observation for C. concentrica (Liu et al 2011). The gamma-proteobacterial cells detected with the HER16 probe were predominately and directly associated with sponge cells (bright yellow-white in Fig.…”

“…The formation of large food vacuoles observed here is similar to the morphological changes found for Dictyostelium when exposed to concanamycin A (CMA), an inhibitor of H + ATPase function (Temesvari et al 1996). Acidification of the phagosome by H + ATPase is crucial for the fusion of new phagosomes with late lysosomes containing pH-sensitive, digestive enzymes, and disruptions in H + ATPase activity drastically reduces endocytosis in Dictyostelium, thus leading to abnormal or homotypic fusions (Temesvari et al 1996;Liu et al 2002). Similar morphological changes occur also with other H + ATPase inhibitory compounds (Henomatsu et al 1993), and we propose that SSA1 and SSA3 may also potentially affect H + ATPase activity or associated interactions to interfer with phagosome development.…”

“…The samples were rinsed with calcium-and magnesium-free artificial seawater (CMFSW; 25 g NaCl, 0.8 g KCl, 1 g Na 2 SO 4 , 0.04 g NaHCO 3 per 1 L) and immediately transferred to 15% sucrose solution. The specimens were then transferred back to the laboratory on ice within 1 h. Sample preparation and FISH were performed as described in (Liu et al 2011) with minor modifications. Briefly, hybridization was performed at 46 °C for 2 h in hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl (pH 7.4), 0.01% SDS and 30% formamide) with a probe concentration of 5 ng/lL.…”

“…Recent metagenomic studies have discovered the surprising presence and abundance of genes encoding for eukaryotic-like proteins (ELPs) in sponge-associated bacteria (Thomas et al 2010;Liu et al 2011;Siegl et al 2011;Fan et al 2012). Specifically, many proteins with ankyrin repeats (ANK) are encoded in the genomes of dominant bacteria found in the sponge Cymbastela concentrica and five other sponge species (Thomas et al 2010;Fan et al 2012).…”

Quantification of GPCR internalization by single-molecule microscopy in living cells

“…Celllines were maintained in 6-well plates in axenic medium with addition of penicillin and streptomycin at 22 1C. Cells were maintained overnight in low fluorescence medium 50 and starved for 4-6 h in phosphate buffer prior to single-molecule measurement. All measurements were performed at 22 1C or 10 1C to slow down dephosphorylation kinetics.…”