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Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon
97291-1000
Alcohol oxidase (AOX), the first enzyme in
the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic 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, we first identified its peroxisomal targeting signal (PTS). We found that sequences essential
for targeting AOX are primarily located within the four
COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine COOH (LARF). To examine whether AOX can oligomerize before import, we
coexpressed AOX without its PTS along with wild-type
AOX and determined whether the mutant AOX could
be coimported into peroxisomes. To identify the mutant
form of AOX, the COOH-terminal LARF sequence of
the protein was replaced with a hemagglutinin epitope
tag (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 octameric active AOX, suggesting that newly synthesized AOX-HA
cannot oligomerize with AOX-WT in the cytoplasm.
Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before
assembly begins.
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