Different subcellular localizations of discoidin I monomer and tetramer in Dictyostelium discoideum cells: using conformation-specific monoclonal antibodies.
We characterized four monoclonal antibodies that recognize monomeric and/or tetrameric forms of Dictyostelium lectin (discoidin I) to study intracellular localization of this lectin in early development. Three different PAGEs (native-, urea-, and SDS-PAGE) following immunoblot showed that three of the four mAbs exhibit preference for the tetrameric form while mAb DC2 reacts only with unreduced monomer. By immunofluorescence studies of Dictyostelium NC-4, we found that the anti-tetramer antibodies mainly stain multilamellar bodies, which are food vacuoles to be externalized from cells. In contrast, mAb DC2 stains the cytosol weakly but not the multilamellar bodies. The same pattern of distribution was confirmed by immunoelectron microscopy. These results clearly indicate that, upon packaging discoidin I into multilamellar bodies, the tetrameric form is selectively packaged from the cytosolic pool. In addition, to clarify the relation between the multilamellar bodies and the tetramer, we examined the localization of tetramer using both the cells fed by Escherichia coli and the A3 cells axenically grown. When the cells were fed by E. coli, the cells made multilamellar bodies that contained no lectin; instead the tetramer was found in the cytoplasm and the cells still exclude the lectin around the cells. In contrast, A3 cells grown axenically without bacteria do not make multilamellar bodies but the tetramer also was found in the cytoplasm evenly (also, see W.F. Loomis, Dictyostelium Discoideum, pp. 160-161, Academic Press, New York, 1975). The data suggest that, under these conditions, the tetramer can be excluded from cells via some organelle such as normal secretory vacuole but not multilamellar bodies. Moreover this represents the first example of differential localization of tetramer and monomer of discoidin I in the cellular slime mold cells.
Fukuzawa
M
M
Department of Botany, Faculty of Science, Hokkaido University, Sapporo, Japan.
Ochiai
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D002451:Cell Compartmentation; D002460:Cell Line; D003600:Cytosol / Q000737:chemistry; D004023:Dictyostelium / Q000737:chemistry*; D000070821:Discoidins; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005656:Fungal Proteins / Q000032:analysis* / Q000276:immunology; D005856:Germ-Free Life; D015151:Immunoblotting; D037102:Lectins; D016253:Microscopy, Immunoelectron; D015388:Organelles / Q000737:chemistry; D015800:Protozoan Proteins
D000911:Antibodies, Monoclonal; D000070821:Discoidins; D005656:Fungal Proteins; D037102:Lectins; D015800:Protozoan Proteins