AN OLIGOMERIC PROTEIN IS IMPORTED INTO PEROXISOMES IN-VIVO

The mechanism of translocation of peroxisomal proteins from the cytopl asm into the matrix is largely unknown. We have been studying this pro blem in yeast. We show that the peroxisomal targeting sequences SKL or AKL, with or without a spacer of nine glycines (G9), are sufficient t o target chloramphenicol acetyltransferase (CAT) to peroxisomes of Sac charomyces cerevisiae in vivo. The mature form of CAT is a homotrimer, and complete trimerization of CAT was found to occur within a few min utes of synthesis. In contrast, import, measured by immunoelectron mic roscopy and organellar fractionation, occurred over several hours. To confirm that import of preassembled CAT trimers was occurring, we coex pressed CAT-G9-AKL with CAT lacking a peroxisomal targeting sequence b ut containing a hemagglutinin-derived epitope tag (HA-CAT). We found t hat HA-CAT was not imported unless it was co-expressed with CAT-G9-AKL . Both proteins were released from the organelles under mild condition s (pH 8.5) that released other matrix proteins, indicating that import had occurred. These results strongly suggested that HA-CAT was import ed as a heterotrimer with CAT-G9-AKL. The process of oligomeric import also occurs in animal cells. When HA-CAT was coexpressed with CAT-G9- AKL in CV-1 cells, HA-CAT co-localized with peroxisomes but was cytopl asmic when expressed alone. It is not clear whether the import of glob ular proteins into peroxisomes occurs through peroxisomal membrane por es or involves membrane internalization. Both possibilities are discus sed.