Sea urchin prosome: characterization and changes during development.

Abstract

A cytoplasmic particle displaying properties in common with a structure present in duck erythroblasts, termed the prosome, has been isolated from eggs and embryos of two species of sea urchin. This particle was partially purified by sedimentation in sucrose gradients containing 0.5 M KCI, and some of its physical properties and its behavior during early development were determined. The prosome sediments between 16 and 19 S and has a buoyant density of 1.30 g/cm3 in Cs2SO4 gradients. Biochemically, the particle is characterized as 20-25 polypeptides of molecular size 24-35 kDa with about 10 small RNAs. A monoclonal antibody directed against the 27-kDa protein of duck erythroblast prosome recognizes a 27-kDa protein of the sea urchin prosome. We have used this protein, as representative of the prosome, to immunologically determine the level and the subcellular localization of the particle during development. Immunoblotting and cellular fractionation studies show that the 27-kDa prosome polypeptide is present almost entirely in the postribosomal supernatant of unfertilized egg lysates. After fertilization and during early development, the total amount of 27-kDa protein per embryo remains constant, but the amount in the postribosomal supernatant decreases; there is a concomitant increase in the level of the 27-kDa protein in a rapidly sedimenting, particulate fraction containing nuclei. Immunofluorescence studies further show that the 27-kDa protein is located mainly in the cytoplasm of eggs and two-cell embryos. The subcellular location of the prosome, therefore, appears to change during development. In vivo labeling experiments have failed to detect the synthesis of either the prosome proteins or RNAs in eggs and embryos up to 48 hr of development, suggesting that this cytoplasmic particle is not synthesized de novo in early embryogenesis and thus is metabolically stable. The prosome is thus a normal cellular constituent of the sea urchin and is most likely synthesized during oogenesis and stored in the unfertilized egg.

Cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mRNAs.

“…4, panel 1). These are a class of highly conserved RNP particles originally isolated from vertebrate cells (55) but later on also isolated from D. melanogaster (4,56), sea urchins (1), and plants (31). Their extraordinary stability in the presence of Sarkosyl (55) can be used for a direct purification from the postpolysomal RNPs by two cycles of centrifugation in sucrose gradients containing 1% Sarkosyl (data not shown).…”

Abnormally high expression of proteasomes in human leukemic cells.

“…The c-myc and c-myb oncogene products have been shown to be degraded very rapidly through an energy-dependent pathway (32). The nuclear localization of proteasomal 19-20S particles has been observed in transcriptionally active hepatocytes (17), actively dividing mammalian cells (8,18), and avian erythroblastosis virus-transformed erythroleukemic cells (23) and in cells of the sea urchin (19,20), newt (24), axolotl (25), and Drosophila (14) in certain stages of oogenesis, embryogenesis, and development, but the relation of these particles to proliferation or transformation has not been determined. Another possible explanation for enhanced expression of proteasomes in cancer cells is that these complexes may be responsible for functions related to RNA metabolism, as has been proposed for the 20S ribonucleoprotein particles mentioned in the Introduction.…”

“…Moreover, proteasomes or the related 20S particles have been found in both the cytoplasm and the nuclei of a variety of mammalian cells (8,17,18) and also various lower eukaryotes (14,(19)(20)(21)(22)(23)(24)(25), suggesting that their diverse roles depend on their differential localizations in the cells. However, the exact function of proteasomes is still unknown.…”

Direct evidence for nuclear and cytoplasmic colocalization of proteasomes (Multiprotease complexes) in liver

“…Interestingly, the size, shape, and subunit multiplicity of these proteasomes are remarkably similar to those of eukaryotic ring-shaped 16-22 S-particles containing specific RNA species (Schmid et al, 1984;Schuldt and Kloetzel, 1985;Arrigo et al, 1985Arrigo et al, , 1987Martins de Sa et al, 1986;Kloetzel et al, 1987;Akhayat et al, 1987), certain heat-shock proteins (Schmid et al, 1984;Schuldt and Kloetzel, 19851, aminoacyl transferase activity (Shelton et al, 1970), or tRNA processing activity (Castano et al, 1986). The identity of some, but not all, subcellular 19-20 S particles, with mammalian multicatalytic proteinases (proteasomes) has been reported by the present authors (Arrigo et al, 1988) and by others (Falkenburg et al, 1988).…”