No abstract

No abstract

The proton-translocating inorganic pyrophosphatases (H(+)-PPases) are primary electrogenic H(+) pumps that derive energy from the hydrolysis of inorganic pyrophosphate (PPi). They are widely distributed among most land plants and have also been found in several species of protozoan parasites. Here we describe, for the first time, the molecular cloning and functional characterization of a gene encoding an H(+)-pyrophosphatase in the protozoan scuticociliate parasite Philasterides dicentrarchi, which infects turbot. The predicted P. dicentrarchi PPase (PdPPase) consists of 587 amino acids of molecular mass 61.7 kDa and an isoelectric point of 5.0. Several motifs characteristic of plant vacuolar H(+)-PPases (V-H(+)-PPases) were also found in the PdPPase, which contains all the sequence motifs of the prototypical type I V-H(+)-PPase from Arabidopsis thaliana vacuolar pyrophosphatase type I (AVP1) plant. The PdPPase has a characteristic residue that determines strict K(+)-dependence, but unlike AVP1, PdPPase contains an N-terminal signal peptide (SP) sequence. Antibodies generated by vaccination of mice with a genetic or recombinant protein containing a partial sequence of the PdPPase and a common motif with the polyclonal antibody PABHK specific to AVP1 recognized a single band of about 62 kDa in western blots. These antibodies specifically stained both vacuole and the alveolar membranes of trophozoites of P. dicentrarchi. H+ transport was partially inhibited by the bisphosphonate pamidronate (PAM) and completely inhibited by NaF. The bisphosphonate PAM inhibited both H+-translocation and gene expression. PdPPase and PAM also inhibited in vitro growth of the ciliates. The apparent lack of V-H(+)-PPases in vertebrates and the parasite sensitivity to PPI analogues may provide a molecular target for developing new drugs to control scuticociliatosis.

No abstract

The Voltage Dependent Anion channel (VDAC) is a ubiquitous channel in the outer membrane of the mitochondrion with multiple roles in protein, metabolite and small molecule transport. In mammalian cells, VDAC, as part of a larger complex including the inositol triphosphate receptor, has been shown to have a role in mediating contacts between the mitochondria and endoplasmic reticulum (ER). We identify VDAC of the pathogenic apicomplexan Toxoplasma gondii and demonstrate its importance for parasite growth. We show that VDAC is involved in protein import and metabolite transfer to mitochondria. Further, depletion of VDAC resulted in significant morphological changes of the mitochondrion and ER, suggesting a role in mediating contacts between these organelles in T. gondii.