PEROXISOMAL TARGETING, IMPORT, AND ASSEMBLY OF ALCOHOL OXIDASE IN PICHIA-PASTORIS

Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilizat ion pathway is a homooctameric peroxisomal matrix protein, In peroxiso me biogenesis-defective (per) mutants of the yeast Pichia pastoris, AO X fails to assemble into active octamers and instead forms inactive cy toplasmic aggregates, The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import, To further investigate the import of AOX, w e first identified its peroxisomal targeting signal (PTS), We found th at sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-argi nine-phenylalamine(COOH) (LARF). To examine whether AOX can oligomeriz e before import, we coexpressed AOX without its PTS along with wild-ty pe AOX and determined whether the mutant AOX could be coimported into peroxisomes. To identify the mutant form of AOX, the COOH-terminal LAR F sequence of the protein was replaced with a hemagglutinin epitope ta g (AOX-HA), Coexpression of AOX-HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX-HA present in octameri c active AOX, suggesting that newly synthesized AOX-HA cannot oligomer ize with AOX-WT in the cytoplasm. Thus, AOX cannot initiate oligomeriz ation in the cytoplasm, but must first be targeted to the organelle be fore assembly begins.