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 filers and Schatz , (Eilers, M., and Schatz, G. (1986) Nature 322, 228-232) to investigate pr otein import into mitochondria. The susceptibility toward proteases of mous e dihydrofolate reductase (DHFR), synthesized in a coupled transcription-tr anslation system with rabbit reticulocytes, decreased in the presence of it s substrate analogue, methotrexate. This analogue complexes with high affin ity with the in vitro synthesized DHFR and locks it into a protease-resista nt folded conformation. DHFR was taken up by freshly isolated rat liver lys osomes and methotrexate reduced this uptake by about 80%. A chimeric DHFR p rotein, which carries the N-terminal presequence of subunit 9 of ATP syntha se 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 me thotrexate 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 t reated with lysosomal inhibitors, suggests that DHFR is degraded by chapero ne-mediated autophagy, Competition with ribonuclease A and stimulation by A TP/Mg2+ and the heat shock cognate protein of 73 kDa show that the lysosoma l uptake of the fusion protein also occurs by this pathway. It is concluded that the lysosomal uptake of cytosolic proteins by chaperone-mediated auto phagy mainly occurs by passage of the unfolded proteins through the lysosom al membrane. Therefore, this mechanism is different from protein transport into peroxisomes, but similar to the import of proteins into the endoplasmi c reticulum and mitochondria.