Tripeptidyl peptidase I, the late infantile neuronal ceroid lipofuscinosisgene product, initiates the lysosomal degradation of subunit c of ATP synthase

The specific accumulation of a hydrophobic protein, subunit c of ATP syntha se, in lysosomes from the cells of patients with the late infantile form of NCL (LINCL) is caused by a defect in the CLN2 gene product, tripeptidyl pe ptidase I (TPP-I), The data here show that TPP-I is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads d irectly to the lysosomal accumulation. of subunit c, The inclusion of a spe cific inhibitor of TPP-I, Ala-Ala-Phe-chloromethylketone (AAF-CMK), in the culture medium of normal fibroblasts induced the lysosomal accumulation of subunit c, In an in vitro incubation experiment the addition, of AAF-CMK to mitochondrial-lysosomal fractions from normal cells inhibited the proteoly sis of subunit c, but not the beta-subunit of ATP synthase, The use of two antibodies that recognize the aminoterminal and the middle portion of subun it c revealed that the subunit underwent aminoterminal proteolysis, when TP P-I, purified from rat spleen, was added to the mitochondrial fractions. Th e addition of both purified TPP-I and the soluble lysosomal fractions, whic h contain various proteinases, to the mitochondrial fractions resulted in r apid degradation of the entire molecule of subunit c, whereas the degradati on of subunit c was markedly delayed through the specific inhibition of TPP -I in lysosomal extracts by AAF-CMK, The stable subunit c in the mitochondr ial-lysosomal fractions from cells of a patient with LINCL was degraded on incubation with purified TPP-I, The presence of TPP-I led td the sequential cleavage of tripeptides from the N-terminus of the peptide corresponding t o the amino terminal sequence of subunit c.