Transport of microinjected alcohol oxidase from Pichia pastoris into vesicles in mammalian cells: involvement of the peroxisomal targeting signal.
This report describes the microinjection of a purified peroxisomal protein, alcohol oxidase, from Pichia pastoris into mammalian tissue culture cells and the subsequent transport of this protein into vesicular structures. Transport was into membrane-enclosed vesicles as judged by digitonin-permeabilization experiments. The transport was time and temperature dependent. Vesicles containing alcohol oxidase could be detected as long as 6 d after injection. Coinjection of synthetic peptides containing a consensus carboxyterminal tripeptide peroxisomal targeting signal resulted in abolition of alcohol oxidase transport into vesicles in all cell lines examined. Double-label experiments indicated that, although some of the alcohol oxidase was transported into vesicles that contained other peroxisomal proteins, the bulk of the alcohol oxidase did not appear to be transported to preexisting peroxisomes. While the inhibition of transport of alcohol oxidase by peptides containing the peroxisomal targeting signal suggests a competition for some limiting component of the machinery involved in the sorting of proteins into peroxisomes, the organelles into which the majority of the protein is targeted appear to be unusual and distinct from endogenous peroxisomes by several criteria. Microinjected alcohol oxidase was transported into vesicles in normal fibroblasts and also in cell lines derived from patients with Zellweger syndrome, which are unable to transport proteins containing the ser-lys-leu-COOH peroxisomal targeting signal into peroxisomes (Walton et al., 1992). The implications of this result for the mechanism of peroxisomal protein transport are discussed.
Walton
P A
PA
UCSD Cancer Center, La Jolla.
Gould
S J
SJ
Rachubinski
R A
RA
Subramani
S
S
Feramisco
J R
JR
D016475:3T3 Cells; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008830:Microbodies / Q000378:metabolism*; D008845:Microinjections; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology*; D013696:Temperature
D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase