Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer.
The active conformation of native peroxisomal 3-ketoacyl-CoA thiolases (EC 2.3.1.16) is homodimeric. We have previously shown that a truncated Saccharomyces cerevisiae thiolase lacking its first 16 N-terminal amino acids fails to be translocated into peroxisomes but assembles into an enzymatically active form in the cytoplasm of a strain with a disrupted nuclear thiolase gene. We now report that when truncated thiolase is cosynthesized with full-length thiolase, approximately 50% of truncated thiolase cofractionates with the full-length thiolase to fractions enriched for peroxisomes and is translocated into peroxisomes as shown by its protection from the action of external proteases. We constructed an immunologically distinct cytosolic variant of thiolase by adding an influenza hemagglutinin epitope tag to the N terminus of the truncated thiolase. In a strain simultaneously expressing the full-length, truncated, and epitope-tagged truncated thiolases, we demonstrated that normally untargeted thiolase subunits are efficiently translocated into peroxisomes by dimerization with full-length thiolase subunits. Even though truncated and epitope-tagged truncated thiolase subunits are translocated into peroxisomes in this strain, only the full-length thiolase subunit can be coimmunoprecipitated with the epitope-tagged truncated thiolase subunit from the peroxisomal matrix. This observation suggests that interactions between thiolase subunits are not disrupted during translocation.
Glover
J R
JR
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Andrews
D W
DW
Rachubinski
R A
RA
D000101:Acetyl-CoA C-Acetyltransferase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D015825:Chromosomes, Fungal; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005838:Genotype; D015151:Immunoblotting; D046911:Macromolecular Substances; D008830:Microbodies / Q000201:enzymology*; D008969:Molecular Sequence Data; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D046911:Macromolecular Substances; D000101:Acetyl-CoA C-Acetyltransferase