Cloning and sequencing of a protein involved in phagosomal membrane fusionin Paramecium

An mAb was raised to the C5 phagosomal antigen in Paramecium multimicronucl eatum. To determine its function, the cDNA and genomic DNA encoding C5 were cloned. This antigen consisted of 315 amino acid residues with a predicted molecular weight of 36,594, a value similar to that determined by SDS-PAGE . Sequence comparisons uncovered a low but significant homology with a Schi zosaccharomyces pombe protein and the C-terminal half of the beta-fructofur anosidase protein of Zymomonas mobilis. Lacking an obvious transmembrane do main or a possible signal sequence at the N terminus, C5 was predicted to b e a soluble protein, whereas immunofluorescence data showed that it was pre sent on the membranes of vesicles and digestive vacuoles (DVs). In cells th at were minimally permeabilized but with intact DVs, C3 was found to be loc ated on the cytosolic surface of the DV membranes. Immunoblotting of protei ns from the purified and KCl-washed DVs showed that C5 was tightly bound to the DV membranes. Cryoelectron microscopy also confirmed that C5 was on th e cytosolic surface of the discoidal vesicles, acidosomes, and lysosomes, o rganelles known to fuse with the membranes of the cytopharynx, the DVs of s tages I (DV-I) and II (DV-II), respectively. Although C5 was concentrated m ore on the mature than on the young DV membranes, the striking observation was that the cytopharyngeal membrane that is derived from the discoidal ves icles was almost devoid of C5. Approximately 80% of the C5 was lost from th e discoidal vesicle-derived membrane after this membrane fused with the cyt opharyngeal membrane. Microinjection of the mAb to C5 greatly inhibited the fusion of the discoidal vesicles with the cytopharyngeal membrane and thus the incorporation of the discoidal vesicle membranes into the DV membranes . Taken together, these results suggest that C5 is a membrane protein that is involved in binding and/or fusion of the discoidal vesicles with the cyt opharyngeal membrane that leads to DV formation.