Dynamics of the vacuolar H(+)-ATPase in the contractile vacuole complex and the endosomal pathway of Dictyostelium cells.

The vacuolar H(+)-ATPase (V-ATPase) is a multi-subunit enzyme that plays important roles in eukaryotic cells. In Dictyostelium, it is found primarily in membranes of the contractile vacuole complex, where it energizes fluid accumulation by this osmoregulatory organelle and also in membranes of endolysosomes, where it serves to acidify the endosomal lumen. In the present study, a fusion was created between vatM, the gene encoding the 100 kDa transmembrane subunit of the V-ATPase, and the gene encoding Green Fluorescent Protein (GFP). When expressed in Dictyostelium cells, this fusion protein, VatM-GFP, was correctly targeted to contractile vacuole and endolysosomal membranes and was competent to direct assembly of the V-ATPase enzyme complex. Protease treatment of isolated endosomes indicated that the GFP moiety, located on the C-terminus of VatM, was exposed to the cytoplasmic side of the endosomal membrane rather than to the lumenal side. VatM-GFP labeling of the contractile vacuole complex revealed clearly the dynamics of this pleiomorphic vesiculotubular organelle. VatM-GFP labeling of endosomes allowed direct visualization of the trafficking of vacuolar proton pumps in this pathway, which appeared to be entirely independent from the contractile vacuole membrane system. In cells whose endosomes were pre-labeled with TRITC-dextran and then fed yeast particles, VatM-GFP was delivered to newly formed yeast phagosomes with the same time course as TRITC-dextran, consistent with transfer via a direct fusion of endosomes with phagosomes. Several minutes were required before the intensity of the VatM-GFP labeling of new phagosomes reached the level observed in older phagosomes, suggesting that this fusion process was progressive and continuous. VatM-GFP was retrieved from the phagosome membrane prior to exocytosis of the indigestible remnants of the yeast particle. These data suggest that vacuolar proton pumps are recycled by fusion of advanced with newly formed endosomes.

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

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.

Mitochondrial membrane dynamics are altered in cluA- mutants of Dictyostelium.

In cluA- mutants of Dictyostelium, mitochondria are clustered near the cell center rather than being dispersed throughout the cytoplasm. We have examined two possible mechanisms that could account for this phenotype. First, we sought evidence that the cytoskeleton or a presumptive mitochondrion-cytoskeleton linkage was altered in mutant cells. We found that cytoskeletal structures in cluA- cells appeared normal by immunostaining, and that the distribution of peroxisomes in mutant cells was indistinguishable from that in wild type cells. Treatment of wild type cells with drugs that disrupted microtubules or actin filaments did not mimic the cluA- phenotype. Thus, cytoskeletal defects seemed unlikely to account for the mitochondrial clustering in cluA- cells. Observation of the movement of GFP-tagged mitochondria in wild type cells suggested that mitochondria are transported along microtubules, as in mammalian cells, rather than along actin filaments, as in budding yeast. Therefore, the similar phenotypes of cluA- Dictyostelium cells and clu1delta yeast cells argued against CluA/Clu1p acting as a mitochondrion-cytoskeleton linker. We next examined the ultrastructure of mitochondria in freeze-substituted, thin-sectioned cells. We found that the clustered mitochondria in cluA- cells are interconnected. Often, adjacent mitochondria are linked by narrow membranous strands, although sometimes the mitochondria are partially merged. The presence of narrow constrictions at presumptive division sites argues that the constriction step of division proceeds normally. Our data suggest that cluA- cells may be blocked at a very late step in fission of the outer mitochondrial membrane.