TY - JOUR
T1 - Degradation of overexpressed wild-type and mutant uricase proteins in cultured cells
AU - Yokota, Sadaki
AU - Kamijo, Keiju
AU - Oda, Toshiaki
PY - 1999/9
Y1 - 1999/9
N2 - Wild-type and mutated urate oxidase (UO) proteins were overexpressed in Cos-1 and HEK293 cells and were analyzed by Western blotting and several morphological methods. By immunoelectron microscopy, wild-type UO formed large aggregates in the cytoplasm and nucleoplasm and exhibited a crystalloid structure. Mutated UO (UOdC), from which 28 amino acids, including peroxisomal targeting signal at the C-terminus, were deleted, formed dispersed aggregates in the cytoplasm and nucleus. Chimeric UO (MUOdC), which was made by addition of the mitochondrial targeting signal of serine:pyruvate/glyoxylate aminotransferase to the N-terminus of UOdC, attached to ER to form a complicated MUOdC-ER complex. These three structures were immunostained for ubiquitin- and p32-subunits of proteasomes. Western blotting showed strong signal for UO and UOdC but very weak signal for MUOdC. The results suggest that overexpressed UO and UOdC accumulate in the cells because their synthesis rate is higher than the degradation rate, whereas MUOdC forming a complex with ER is degraded very rapidly. The ubiquitin- proteasome pathway may be involved in the degradation of these proteins.
AB - Wild-type and mutated urate oxidase (UO) proteins were overexpressed in Cos-1 and HEK293 cells and were analyzed by Western blotting and several morphological methods. By immunoelectron microscopy, wild-type UO formed large aggregates in the cytoplasm and nucleoplasm and exhibited a crystalloid structure. Mutated UO (UOdC), from which 28 amino acids, including peroxisomal targeting signal at the C-terminus, were deleted, formed dispersed aggregates in the cytoplasm and nucleus. Chimeric UO (MUOdC), which was made by addition of the mitochondrial targeting signal of serine:pyruvate/glyoxylate aminotransferase to the N-terminus of UOdC, attached to ER to form a complicated MUOdC-ER complex. These three structures were immunostained for ubiquitin- and p32-subunits of proteasomes. Western blotting showed strong signal for UO and UOdC but very weak signal for MUOdC. The results suggest that overexpressed UO and UOdC accumulate in the cells because their synthesis rate is higher than the degradation rate, whereas MUOdC forming a complex with ER is degraded very rapidly. The ubiquitin- proteasome pathway may be involved in the degradation of these proteins.
KW - Degradation
KW - Immunocytochemis try
KW - Mutated uricase
KW - Overexpression
KW - Proteasome
KW - Ubiquitination
UR - http://www.scopus.com/inward/record.url?scp=0032823327&partnerID=8YFLogxK
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U2 - 10.1177/002215549904700905
DO - 10.1177/002215549904700905
M3 - Article
C2 - 10449534
AN - SCOPUS:0032823327
VL - 47
SP - 1133
EP - 1139
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
SN - 0022-1554
IS - 9
ER -