Aggregate formation and degradation of overexpressed wild-type and mutant urate oxidase proteins. Quality control of organelle-destined proteins by the endoplasmic reticulum

Sadaki Yokota, Keiju Kamijo, Toshiaki Oda

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)433-446
Number of pages14
JournalHistochemistry and Cell Biology
Volume114
Issue number6
DOIs
Publication statusPublished - 2000

Keywords

  • Aggregate formation
  • ER
  • Protein quality control
  • Ubiquitination

ASJC Scopus subject areas

  • Histology
  • Molecular Biology
  • Medical Laboratory Technology
  • Cell Biology

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