Transport of microinjected proteins into peroxisomes of mammalian cells: inability of Zellweger cell lines to import proteins with the SKL tripeptide peroxisomal targeting signal.
Previous work has shown that the firefly (Photinus pyralis) luciferase contains a C-terminal peroxisomal targeting signal consisting of the tripeptide Ser-Lys-Leu. This report describes the microinjection of two proteins, (i) luciferase and (ii) albumin conjugated to a peptide ending in the sequence Ser-Lys-Leu, into mammalian cells grown in tissue culture. Following microinjection, incubation of the cells at 37 degrees C resulted in peroxisomal transport of these exogenous proteins into catalase-containing vesicles. The translocation was both time and temperature dependent. The transport could be inhibited by coinjection of synthetic peptides bearing various peroxisomal targeting signal motifs. These proteins could be transported into peroxisomes in normal human fibroblast cell lines but not in cell lines derived from patients with Zellweger syndrome. These results demonstrate that microinjection of peroxisomal proteins yields an authentic in vivo system with which to study peroxisomal transport. Furthermore, these results reveal that the process of peroxisomal transport does not involve irreversible modification of the protein, that artificial hybrid substrates can be transported and used as tools to study peroxisomal transport, and that the defect in Zellweger syndrome is indeed the inability to transport proteins containing the Ser-Lys-Leu targeting signal into the peroxisomal lumen.
Walton
P A
PA
UCSD Cancer Center, University of California at San Diego, La Jolla 92093-0322.
Gould
S J
SJ
Feramisco
J R
JR
Subramani
S
S
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008830:Microbodies / Q000378:metabolism*; D008845:Microinjections; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013696:Temperature; D015211:Zellweger Syndrome / Q000378:metabolism*
D021382:Protein Sorting Signals; D011506:Proteins; D008156:Luciferases