Import of a cytosolic protein into lysosomes by chaperone-mediated autophagy depends on its folding state.
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/Mg(2+) 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.
Salvador
N
N
Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Amadeo de Saboya, 4, Valencia 46010, Spain.
Aguado
C
C
Horst
M
M
Knecht
E
E
D000818:Animals; D001343:Autophagy; D001692:Biological Transport; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D011487:Protein Conformation; D017510:Protein Folding; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate