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Originally published In Press as doi:10.1074/jbc.M001394200 on June 20, 2000

J. Biol. Chem., Vol. 275, Issue 35, 27447-27456, September 1, 2000
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Import of a Cytosolic Protein into Lysosomes by Chaperone-mediated Autophagy Depends on Its Folding State*

Natalia SalvadorDagger §, Carmen AguadoDagger §||, Martin Horst**, and Erwin KnechtDagger Dagger Dagger

From the Dagger  Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Amadeo de Saboya, 4, Valencia 46010, Spain and the ** Faculty Phil. II, University of Basel, Missionstr. 64, CH-4055 Basel, Switzerland

We have analyzed the folding state of cytosolic proteins imported in vitro into lysosomes, using an approach originally developed by Eilers and Schatz, (Eilers, M., and Schatz, G. (1986) Nature 322, 228-232) to investigate protein import into mitochondria. The susceptibility toward proteases of mouse dihydrofolate reductase (DHFR), synthesized in a coupled transcription-translation system with rabbit reticulocytes, decreased in the presence of its substrate analogue, methotrexate. This analogue complexes with high affinity with the in vitro synthesized DHFR and locks it into a protease-resistant folded conformation. DHFR was taken up by freshly isolated rat liver lysosomes and methotrexate reduced this uptake by about 80%. A chimeric DHFR protein, which carries the N-terminal presequence of subunit 9 of ATP synthase preprotein from Neurospora crassa fused to its N terminus, was taken up by lysosomes more efficiently. Again, methotrexate abolished the lysosomal uptake of the fusion protein, which was partially restored by washing of methotrexate from DHFR or by adding together methotrexate and dihydrofolate, the natural substrate of DHFR. Immunoblot analysis with anti-DHFR of liver lysosomes and of other fractions, isolated from rats starved for 88 h and treated with lysosomal inhibitors, suggests that DHFR is degraded by chaperone-mediated autophagy. Competition with ribonuclease A and stimulation by ATP/Mg2+ and the heat shock cognate protein of 73 kDa show that the lysosomal uptake of the fusion protein also occurs by this pathway. It is concluded that the lysosomal uptake of cytosolic proteins by chaperone-mediated autophagy mainly occurs by passage of the unfolded proteins through the lysosomal membrane. Therefore, this mechanism is different from protein transport into peroxisomes, but similar to the import of proteins into the endoplasmic reticulum and mitochondria.

* This work was supported by Ministerio de Educación y Ciencia Grants PB97-1445 and PM98-0041 and Fundació La Caixa Grant 97/131-00.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.

§ Contributed equally to the results of this work.

Predoctoral fellow of the Consellería de Cultura Educación y Ciencia de la Generalitat Valenciana.

|| Postdoctoral fellow of the Fundación Bancaixa.

Dagger Dagger To whom correspondence should be addressed: Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Amadeo de Saboya, 4, 46010 Valencia, Spain. Tel.: 346-3391250; Fax: 346-3601453; E-mail: knecht@ochoa.fib.es.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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