Dynamin- and clathrin-dependent endocytic pathway in unicellular eukaryote Paramecium.
The first evidence of dynamin presence and its colocalization with clathrin in the compartment involved in Paramecium receptor-mediated endocytosis is presented. We identified dynamin by cloning, Western blotting, and immunodetection in confocal and electron microscopy. The partial genes, which we have designated ParDyn1 and ParDyn2, are 1091 bp long, 90% identical to one another and encode the N-terminal and middle domains of Paramecium dynamin isoform 1 and isoform 2. The deduced amino acid sequences contain all three guanosine 5'-triphosphate (GTP)-binding motifs and show 67% homology to mammalian dynamins. Antibodies generated against the cloned GTPase domain revealed dynamin association with endosomes containing transferrin, the marker of receptor-mediated endocytosis. In Western blotting a strong immunoreactive polypeptide of approximately 116 kDa, which seems to be phosphorylated, was accompanied by a faint one of approximately 90 kDa in cytosolic fraction (S2). Dynamin level was correlated with internalization of transferrin and it was significantly decreased upon inhibition of this process. Immunogold labeling in electron microscopy revealed colocalization of dynamin and clathrin in coated pits and endocytic vesicles. Moreover, the polypeptide cross-reaction with 2 different antibodies against mammalian clathrin was identified by immunoblotting. These results indicate that dynamin- and clathrin-dependent pathway exists in this evolutionary ancient cell.
Wiejak
Jolanta
J
Laboratory of Cell Membrane Physiology, Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Str., 02-093 Warsaw, Poland.
Surmacz
Liliana
L
Wyroba
Elzbieta
E
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism*; D003001:Cloning, Molecular; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D034281:Dynamins / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010247:Paramecium / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002966:Clathrin; D015800:Protozoan Proteins; D011990:Receptors, Transferrin; D034281:Dynamins