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

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 aSchizosaccharomyces pombe protein and the C-terminal half of the β-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.

Intracellular binding of wheat germ agglutinin by Golgi complexes, phagosomes, and lysosomes of Paramecium multimicronucleatum.

The compartments of the Paramecium digestive system were investigated with wheat germ agglutinin (WGA). By use of cryosectioning or Lowicryl K4M embedding combined with pulse-chase studies and WGA-gold labeling, WGA binding sites were located on membranes of the phagosome-lysosome system, including all four stages of digestive vacuoles, the discoidal vesicles, acidosomes, and lysosomes. In addition, the contents of lysosomes, cisternae at the trans face of Golgi stacks, and coated and uncoated blebs and vesicles at the putative trans Golgi network bind to WGA. Crystal-containing vacuoles characteristic of mid-log to stationary-phase cultures are enclosed by heavily labeled membranes. Alveoli underlying the plasma membrane sometimes contain binding sites, particularly on their outer membranes. Ciliary membranes previously shown to be labeled with WGA-FITC are negative in frozen thin and Lowicryl K4M sections. The presence of WGA binding sites on the trans face of the Golgi stack is the first indication in ciliated protozoa, such as Paramecium, of probable Golgi complex involvement in glycosylation similar to that in higher organisms. WGA-labeled coated vesicles in the endoplasm apparently lose their coats and coalesce to form lysosomes. Our study shows that WGA can be used as a specific intracellular marker of all digestive system membranes and of lysosomal content. These results support and extend our published scheme of membrane flow and recycling in Paramecium by providing another means of demonstrating membrane relationships.

Digestive system membranes: freeze-fracture evidence for differentiation and flow in Paramecium.

Freeze-fractured membranes of digestive vacuoles of randomly feeding Paramecium caudatum exhibit dramatic differences in intramembrane particle (IMP) number and distribution on both E- and P-fracture faces. By pulse-feeding latex spheres to cells we have demonstrated that these differences are related to the age of the digestive vacuoles, and that the membranes of such vacuoles undergo a specific sequence of changes during the digestive cycle. Young digestive vacuoles (DV-I; less than or equal to 6 min), nascent vacuoles still connected to the cytopharynx, and discoidal vesicles, from which vacuole membrane is derived, all have a highly particulate E face and a less particulate P face. As early as 3 min after feeding, a second category of digestive vacuoles (DV-II) can be recognized, which are both considerably smaller in diameter and lack particles on their E face. These findings suggest that the endocytic removal of DV-I membrane material associated with the formation of DV-II vacuoles involves a concomitant and selective removal of E-face particles, as essentially no changes are seen in the density of P-face particles on the two types of vacuoles. Beginning at 10 min the first DV-III vacuoles are encountered. These are both larger than the DV-II vacuoles and possess very prominent E-face particles, which resemble those on the E face of the numerous lysosomes bordering the digestive vacuoles. DV-III vacuoles also exhibit a substantial increase in P-face particles. These membrane changes closely parallel, and are probably correlated with, the physiological events occurring within the vacuole lumen: concentration of food, killing of prey, and digestion. Calculations of the amount of membrane removed from DV-I to form DV-II and of the increase in membrane surface area during the transition from DV-II to DV-III indicate that as much as 90% of the initial phagosome (DV-I) membrane can be removed before digestion begins. The enlargment of DV-II must be caused by fusion with adjacent lysosomes which also contribute the new populations of IMPs to the DV-III membrane. The appearance of numerous endocytic structures on older DV-III vacuoles suggests that membrane is retrieved from DV-III before defecation.

Morphometric analysis of the translocation of lumenal membrane between cytoplasm and cell surface of transitional epithelial cells during the expansion-contraction cycles of mammalian urinary bladder

The flow of membrane between the cytoplasm and the lumenal surface during the expansion-contraction cycle of urinary bladder was estimated by stereological examination of electron micrographs of urothelial cells from guinea pigs, gerbils, hamsters, rabbits, and rats. The quantitative data obtained allowed an approximation of the surface area, volume, and numbers of lumenal membranelike vesicles and infoldings per unit volume of cytoplasm. Depending upon the species, approximately 85 to approximately 94% of the membrane surface area translocated into and out of the cytoplasm was in the form of discoidal vesicles. The remainder was accounted for by infoldings of the lumenal plasma membrane. The density of vesicles involved in transfer of membrane was quite similar in all the species examined, except guinea pigs which yielded lower values. In contrast, the densities of the total cytoplasmic pools of discoidal vesicles potentially available for translocation varied greatly among the different species. In general, species of animals with a highly concentrated urine had a greater density of discoidal vesicles than species with a less concentrated urine. This correlation may indicate an authentic relationship between lumenal membranes and the tonicity of urine, such as increased membrane recycling or turnover with increasingly hypertonic urine; or it may signify the existence of some other, more obscure relationship.