TY - JOUR
T1 - Aggregate formation and degradation of overexpressed wild-type and mutant urate oxidase proteins. Quality control of organelle-destined proteins by the endoplasmic reticulum
AU - Yokota, Sadaki
AU - Kamijo, Keiju
AU - Oda, Toshiaki
N1 - Funding Information:
Acknowledgements We are grateful to Dr. Jun-ichi Miyazaki (Osaka University Medical School) for generously donating pCAGGS vector, to Drs. Masataka Mori (Kumamoto University School of Medicine) and Arata Ichiyama (Hamamatsu University School of Medicine) for useful advice and help during this study. We also greatly thank Dr. Shinji Asano (Molecular Genetics Research Center, Toyama Medical and Pharmaceutical University) for donating HEK293 cells. This work was supported in part by Grants-in-aid for Scientific Research (number 03833013) to S.Y. and (number 10480165) to T.O. from the Ministry of Education, Science, Sport and Culture of Japan, and by a grant to T.O. from the Naito Foundation, Japan.
PY - 2000
Y1 - 2000
N2 - To analyze the cellular response caused by the overexpression of proteins in subcellular compartments, we constructed four expression clones encoding wild-type peroxisomal urate oxidase (UO), truncated UO lacking the peroxisomal targeting signal (UOdC), and chimeric UOs with a mitochondrial targeting signal (MTS) at the N-terminus of UOdC (MUOdC) or UO (MUO). After transfection, we examined COS-1 and HEK293 cells by immunofluorescence and immunoelectron microscopy, transmission electron microscopy, and pulse-chase experiments. The overexpressed UO and UOdC formed large electron-dense aggregates with no limiting membrane in both the cytoplasm and the nucleus. The UO aggregates exhibited the crystalloid structure quite similar to that of rat liver peroxisomal cores, whereas the UOdC aggregates formed a loose mass consisting of small dense substructures. The overexpressed MUOdC and MUO, on the other hand, formed other types of aggregates which were distributed in the cytoplasm. They consisted of tubular and circular membrane structures, which were morphologically confirmed to be derived from the endoplasmic reticulum (ER). No immunolabeling signals for MUOdC and MUO were present free in the cytoplasm and most of them were associated with membrane structures, suggesting that overexpressed UO containing the MTS attached to the ER membranes soon after synthesis and segregated from the cytosolic compartment. All the UO aggregates were stained for ubiquitin antigen. Pulse-chase experiments in combination with proteasome inhibitors suggested that proteasomes did not contribute to the degradation of these products.
AB - To analyze the cellular response caused by the overexpression of proteins in subcellular compartments, we constructed four expression clones encoding wild-type peroxisomal urate oxidase (UO), truncated UO lacking the peroxisomal targeting signal (UOdC), and chimeric UOs with a mitochondrial targeting signal (MTS) at the N-terminus of UOdC (MUOdC) or UO (MUO). After transfection, we examined COS-1 and HEK293 cells by immunofluorescence and immunoelectron microscopy, transmission electron microscopy, and pulse-chase experiments. The overexpressed UO and UOdC formed large electron-dense aggregates with no limiting membrane in both the cytoplasm and the nucleus. The UO aggregates exhibited the crystalloid structure quite similar to that of rat liver peroxisomal cores, whereas the UOdC aggregates formed a loose mass consisting of small dense substructures. The overexpressed MUOdC and MUO, on the other hand, formed other types of aggregates which were distributed in the cytoplasm. They consisted of tubular and circular membrane structures, which were morphologically confirmed to be derived from the endoplasmic reticulum (ER). No immunolabeling signals for MUOdC and MUO were present free in the cytoplasm and most of them were associated with membrane structures, suggesting that overexpressed UO containing the MTS attached to the ER membranes soon after synthesis and segregated from the cytosolic compartment. All the UO aggregates were stained for ubiquitin antigen. Pulse-chase experiments in combination with proteasome inhibitors suggested that proteasomes did not contribute to the degradation of these products.
KW - Aggregate formation
KW - ER
KW - Protein quality control
KW - Ubiquitination
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U2 - 10.1007/s004180000208
DO - 10.1007/s004180000208
M3 - Article
C2 - 11201604
AN - SCOPUS:0034526106
VL - 114
SP - 433
EP - 446
JO - Zeitschrift für Zellforschung und Mikroskopische Anatomie. Abteilung Histochemie
JF - Zeitschrift für Zellforschung und Mikroskopische Anatomie. Abteilung Histochemie
SN - 0948-6143
IS - 6
ER -