The
cyst wall of the resting
cyst of the ciliated protozoan Colpoda cucullus (Nag-1 strain) is composed of several layers of endocyst, a single layer of ectocyst associated with a mucous layer and lepidosomes composed of a fibrous or crystal-like structure.
Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis showed that the ectocyst associated with lepidosomes and mucous materials contained
proteins corresponding to 27, 31, 45 kDa and smear bands ranging from 50 to 60 kDa. Liquid chromatography-tandem mass spectrometry of these
proteins revealed that the 45-kDa
protein (p45) was
elongation factor Tu (
EF-Tu). Immunofluorescence microscopy with an anti-
EF-Tu polyclonal antibody showed that Colpoda
EF-Tu (p45) was localized in the lepidosomes. The lepidosomes were stained vividly with
Congo red, which is bound to the stacked β-sheets of
amyloid protofibrils. In the presence of
puromycin, no
cyst wall components including lepidosomes were formed, indicating that
cyst wall formation requires synthesis of
proteins including
EF-Tu. Electron microscopy of encysting cells implied that vesicles which were presumably budded from endoplasmic reticula possibly fuse with a lepidosome-precursor vacuole containing electron-dense fine particles or fibrous structures, and followed by the subsequent fusion with other electron-lucent granules.