An oligomeric protein is imported into peroxisomes in vivo

James A. McNew, Joel M. Goodman

Research output: Contribution to journalArticle

261 Citations (Scopus)

Abstract

The mechanism of translocation of peroxisomal proteins from the cytoplasm into the matrix is largely unknown. We have been studying this problem in yeast. We show that the peroxisomal targeting sequences SKL or AKL, with or without a spacer of nine glycines (G9), are sufficient to target chloramphenicol acetyltransferase (CAT) to peroxisomes of Saccharomyces cerevisiae in vivo. The mature form of CAT is a homotrimer, and complete trimerization of CAT was found to occur within a few minutes of synthesis. In contrast, import, measured by immunoelectron microscopy and organellar fractionation, occurred over several hours. To confirm that import of preassembled CAT trimers was occurring, we coexpressed CAT-G9-AKL with CAT lacking a peroxisomal targeting sequence but containing a hemagglutinin- derived epitope tag (HA-CAT). We found that HA-CAT was not imported unless it was co-expressed with CAT-G9-AKL. Both proteins were released from the organelles under mild conditions (pH 8.5) that released other matrix proteins, indicating that import had occurred. These results strongly suggested that HA-CAT was imported as a heterotrimer with CAT-G9-AKL. The process of oligomeric import also occurs in animal cells. When HA-CAT was co- expressed with CAT-G9-AKL in CV-1 cells, HA-CAT co-localized with peroxisomes but was cytoplasmic when expressed alone. It is not clear whether the import of globular proteins into peroxisomes occurs through peroxisomal membrane pores or involves membrane internalization. Both possibilities are discussed.

Original languageEnglish (US)
Pages (from-to)1245-1257
Number of pages13
JournalJournal of Cell Biology
Volume127
Issue number5
DOIs
StatePublished - Dec 1994

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Chloramphenicol O-Acetyltransferase
Peroxisomes
Proteins
Membranes
Immunoelectron Microscopy
Hemagglutinins
Protein Transport
Organelles
Glycine
Saccharomyces cerevisiae
Epitopes

ASJC Scopus subject areas

  • Cell Biology

Cite this

An oligomeric protein is imported into peroxisomes in vivo. / McNew, James A.; Goodman, Joel M.

In: Journal of Cell Biology, Vol. 127, No. 5, 12.1994, p. 1245-1257.

Research output: Contribution to journalArticle

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