Cloning and Sequencing of a Protein Involved in Phagosomal Membrane Fusion inParamecium

Abstract

An mAb was raised to the C5 phagosomal antigen in Paramecium multimicronucleatum. 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 Schizosaccharomyces pombe protein and the C-terminal half of the beta-fructofuranosidase protein of Zymomonas mobilis. Lacking an obvious transmembrane domain or a possible signal sequence at the N terminus, C5 was predicted to be a soluble protein, whereas immunofluorescence data showed that it was present on the membranes of vesicles and digestive vacuoles (DVs). In cells that were minimally permeabilized but with intact DVs, C5 was found to be located on the cytosolic surface of the DV membranes. Immunoblotting of proteins 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 the cytosolic surface of the discoidal vesicles, acidosomes, and lysosomes, organelles known to fuse with the membranes of the cytopharynx, the DVs of stages I (DV-I) and II (DV-II), respectively. Although C5 was concentrated more on the mature than on the young DV membranes, the striking observation was that the cytopharyngeal membrane that is derived from the discoidal vesicles was almost devoid of C5. Approximately 80% of the C5 was lost from the discoidal vesicle-derived membrane after this membrane fused with the cytopharyngeal 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 cytopharyngeal membrane that leads to DV formation.

The Vacuolar‐ATPase of Paramecium multimicronucleatum: Gene Structure of the B Subunit and the Dynamics of the V‐ATPase‐rich Osmoregulatory Membranes

“…The lysosomal and microsomal pellets obtained by differential centrifugation were solubilized using 1.0% NP‐40 and incubated with the mAb‐solid matrix. After incubation and extensive washing, the bound antigen was eluted from the solid matrix and subjected to SDS‐PAGE under a non‐reducing condition (Yamauchi et al 1999). Protein bands were fixed, stained with Coomassie brilliant blue, excised and each digested with trypsin.…”

“…Amino acid sequences were determined directly by protein sequencing. Codon selections were based on the highest frequency of codon usage for paramecia (Yamauchi et al 1999). Two amino acid residues from the tryptic fragments that did not match those of the cloned sequence are enclosed in parentheses.…”

DNA Fluorescencein situHybridization in Paramecium: Telomere Localization in Macronucleus

“…C5 antigen is a 33 kDa phagosomal membrane protein, involved in binding and/or fusion of the donor vesicles with the cytopharyngeal membrane that leads to digestive vacuole formation (Yamauchi et al, 1999). Telomeres, the specialized structures found at the end of chromosomes, consist of simple, tandem DNA repeats and the proteins which bind them (reviewed in Zakian, 1989;Biessmann and Mason, 1992).…”

Calmodulin Localization and its Effects on Endocytic and Phagocytic Membrane Trafficking in Paramecium multimicronucleatum

“…The processes of membrane transport and recycling in the digestive cycle have been studied extensively in Paramecium multimicronucleatum (Allen and Fok 2000). A digestive cycle is initiated by the movement of the discoidal vesicles to the cytopharyngeal membrane (Allen 1974) where they fuse to form the nascent vacuole (Allen 1974; Yamauchi et al 1999). As a nascent vacuole grows in size, larger vesicles, the acidosomes, move to and dock at its cytosolic surface (Allen and Fok 1983a).…”