Different accumulations of 3-ketoacyl-CoA thiolase precursor in peroxisomes of Chinese hamster ovary cells harboring a dysfunction in the PEX2 protein.
The peroxisomal localization of 3-ketoacyl-CoA thiolase (hereafter referred to as thiolase) was characterized in five Chinese hamster ovary (CHO) mutant cell lines each harboring a dysfunction in the PEX2 protein. PT54 (Pex2pN100) cells carry a nonsense mutation that results in the PEX2 protein truncated at amino acid position 100. SK24 (Pex2pC258Y) cells carry a missense mutation resulting in the amino acid substitution of a cysteine residue by a tyrosine residue at amino acid position 258 of the PEX2 protein. The WSK24 (Pex2pC258Y/+wild) cell line is a stable transformant of SK24 (Pex2pC258Y) cells transfected with wild-type rat PEX2 cDNA. The SPT54 (Pex2pN100/+Pex2pC258Y) and WPT54 (Pex2pN100/+wild) cell lines are stable transformants of PT54 (Pex2pN100) cells transfected with the mutant PEX2 cDNA from SK24 (Pex2pC258Y) cells and wild-type rat PEX2 cDNA, respectively. In these cell lines, except PT54 (Pex2pN100), thiolase appeared to be localized in peroxisomes, as it is in the wild-type cells. When the molecular size of the enzyme was examined on SDS-polyacrylamide gel electrophoresis, the peroxisome-localized enzyme exhibited a larger precursor form in these mutant cells. The characterizations with salt wash, sodium carbonate extraction and proteinase K digestion indicated that the precursor forms of the enzyme were accumulated at different states in peroxisomes of these mutant cells. The dispositions on the peroxisomal membrane were further sustained by differential permeabilization using digitonin, followed by immunocytochemical fluorescence. These results suggest that PEX2 protein functions differently on two processes of the maturation and the disposition in the import pathway of thiolase.
Huang
Yuan
Y
Department of Biology, Saga Medical School, Nabeshima, Japan.
Ito
Ritsu
R
Imanaka
Tsuneo
T
Usuda
Nobuteru
N
Ito
Masaki
M
D000102:Acetyl-CoA C-Acyltransferase / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002458:Cell Fractionation; D006224:Cricetinae; D004072:Digitonin; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000074428:Peroxisomal Biogenesis Factor 2; D020675:Peroxisomes / Q000378:metabolism*; D011498:Protein Precursors / Q000032:analysis / Q000378:metabolism*; D014170:Transformation, Genetic
D008565:Membrane Proteins; C000614232:Pex2 protein, rat; D011498:Protein Precursors; D000074428:Peroxisomal Biogenesis Factor 2; D000102:Acetyl-CoA C-Acyltransferase; D004072:Digitonin