An oligomeric protein is imported into peroxisomes in vivo.
The mechanism of translocation of peroxisomal proteins from the cytoplasm into the matrix is largely unknown. We have been studying this problem in yeast. We show that the peroxisomal targeting sequences SKL or AKL, with or without a spacer of nine glycines (G9), are sufficient to target chloramphenicol acetyltransferase (CAT) to peroxisomes of Saccharomyces cerevisiae in vivo. The mature form of CAT is a homotrimer, and complete trimerization of CAT was found to occur within a few minutes of synthesis. In contrast, import, measured by immunoelectron microscopy and organellar fractionation, occurred over several hours. To confirm that import of preassembled CAT trimers was occurring, we co-expressed CAT-G9-AKL with CAT lacking a peroxisomal targeting sequence but containing a hemagglutinin-derived epitope tag (HA-CAT). We found that HA-CAT was not imported unless it was co-expressed with CAT-G9-AKL. Both proteins were released from the organelles under mild conditions (pH 8.5) that released other matrix proteins, indicating that import had occurred. These results strongly suggested that HA-CAT was imported as a heterotrimer with CAT-G9-AKL. The process of oligomeric import also occurs in animal cells. When HA-CAT was co-expressed with CAT-G9-AKL in CV-1 cells, HA-CAT co-localized with peroxisomes but was cytoplasmic when expressed alone. It is not clear whether the import of globular proteins into peroxisomes occurs through peroxisomal membrane pores or involves membrane internalization. Both possibilities are discussed.
McNew
J A
JA
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Goodman
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008830:Microbodies / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D008969:Molecular Sequence Data; D011108:Polymers / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D011108:Polymers; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase