Isolation and characterization of Chinese hamster ovary cell mutants defective in assembly of peroxisomes.
We made use of autoradiographic screening to isolate two Chinese hamster ovary (CHO) cell mutants deficient in peroxisomal dihydroxyacetonephosphate acyltransferase, a key enzyme for the biosynthesis of ether glycerolipids such as plasmalogens. Morphological analysis revealed no evidence of peroxisome in these mutants. Catalase was as active as in the normal cells but was not sedimentable. Pulse-chase radiolabeling experiments and cell-free translation of RNA demonstrated that acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation system, was synthesized as the 75-kD form but was not converted to 53- and 22-kD mature components that were present in the wild-type CHO cells; rather, degradation was apparent. Peroxisomal thiolase was synthesized as in normal cells but remained as a larger, 44-kD precursor, whereas maturation to the 41-kD enzyme was detected in the wild-type cells. The peroxisomal 70-kD integral membrane protein was also equally synthesized, as in the wild-type cells, and was not degraded. These results suggest that assembly of the peroxisomes is defective in the mutants, whereas the synthesis of peroxisomal proteins appears to be normal. Cell-fusion studies revealed that the two mutants are recessive to the wild-type CHO cells and belong to different complementation groups. Thus, these mutants presumably contain different lesions in gene(s) encoding factor(s) required for peroxisome assembly.
Tsukamoto
T
T
Laboratory of Molecular Cell Biology, Meiji Institute of Health Science, Odawara, Japan.
Yokota
S
S
Fujiki
Y
Y
D000101:Acetyl-CoA C-Acetyltransferase / Q000096:biosynthesis; D000217:Acyltransferases / Q000096:biosynthesis; D000818:Animals; D002459:Cell Fusion; D002460:Cell Line; D002469:Cell Separation; D006224:Cricetinae; D003412:Cricetulus; D044943:Fatty Acid Desaturases / Q000096:biosynthesis; D005260:Female; D006822:Hybrid Cells / Q000166:cytology / Q000187:drug effects; D008830:Microbodies / Q000648:ultrastructure*; D008854:Microscopy, Electron; D009154:Mutation; D010042:Ouabain / Q000494:pharmacology; D010053:Ovary; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D013866:Thioguanine / Q000494:pharmacology
D010042:Ouabain; D012313:RNA; D044943:Fatty Acid Desaturases; D000217:Acyltransferases; C020051:glycerone-phosphate O-acyltransferase; D000101:Acetyl-CoA C-Acetyltransferase; D013866:Thioguanine