Protease Trafficking in Two Primitive Eukaryotes Is Mediated by a Prodomain Protein Motif*
- Jorge A. Huete-Pérez‡,
- Juan C. Engel‡,
- Linda S. Brinen§,
- Jeremy C. Mottram¶‖ and
- James H. McKerrow‡**‡
- From the Departments of ‡Pathology,§Biochemistry and Biophysics, and **Pharmaceutical Chemistry, University of California, San Francisco, California 94121 and the ¶Wellcome Unit of Molecular Parasitology, The Anderson College, University of Glasgow, Glasgow, G11 6NU, Scotland, United Kingdom
Abstract
Trypanosome protozoa, an early lineage of eukaryotic cells, have proteases homologous to mammalian lysosomal cathepsins, but the precursor proteins lack mannose 6-phosphate. Utilizing green fluorescent protein as a reporter, we demonstrate that the carbohydrate-free prodomain of a trypanosome cathepsin L is necessary and sufficient for directing green fluorescent protein to the lysosome/endosome compartment. A proper prodomain/catalytic domain processing site sequence is also required to free the mature protease for delivery to the lysosome/endosome compartment. A nine-amino acid prodomain loop motif, implicated in prodomain-receptor interactions in mammalian cells, is conserved in the protozoa. Site-directed mutagenesis now confirms the importance of this loop to protease trafficking and suggests that a protein motif targeting signal for lysosomal proteases arose early in eukaryotic cell evolution.
Footnotes
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↵* This work was supported by TDRU Grant AI35707 from the National Institutes of Health (to J. H. M.) and by Pew Fellowship PO114SC (to J. A. H.-P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ Medical Research Council (UK) Senior Research Fellow.
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↵‡ Recipient of a Burroughs Wellcome Scholar Award in Molecular Parasitology. To whom correspondence should be addressed: Dept. of Pathology, University of California San Francisco, VAMC-113B, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-476-2940; Fax: 415-750-6947; E-mail: jmck{at}cgl.ucsf.edu.
- Abbreviations:
- M6P
-
mannose 6-phosphate
- GFP
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green fluorescent protein
- FBS
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fetal bovine serum
- PCR
-
polymerase chain reaction
- Ctd
-
carboxyl-terminal domain
- L/E
-
lysosome/endosome
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- Received December 16, 1998.
- Revision received January 27, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
