Tripeptidyl peptidase I, the late infantile neuronal ceroid lipofuscinosis gene product, initiates the lysosomal degradation of subunit c of ATP synthase.
The specific accumulation of a hydrophobic protein, subunit c of ATP synthase, 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 peptidase 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 directly to the lysosomal accumulation of subunit c. The inclusion of a specific 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 proteolysis of subunit c, but not the b-subunit of ATP synthase. The use of two antibodies that recognize the aminoterminal and the middle portion of subunit c revealed that the subunit underwent aminoterminal proteolysis, when TPP-I, purified from rat spleen, was added to the mitochondrial fractions. The addition of both purified TPP-I and the soluble lysosomal fractions, which contain various proteinases, to the mitochondrial fractions resulted in rapid degradation of the entire molecule of subunit c, whereas the degradation 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 mitochondrial-lysosomal fractions from cells of a patient with LINCL was degraded on incubation with purified TPP-I. The presence of TPP-I led to the sequential cleavage of tripeptides from the N-terminus of the peptide corresponding to the amino terminal sequence of subunit c.
Ezaki
J
J
Department of Biochemistry, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. kominami@med.juntendo.ac.jp
Takeda-Ezaki
M
M
Kominami
E
E
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000626:Aminopeptidases; D000818:Animals; D003574:Cytochrome c Group / Q000378:metabolism*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D003576:Electron Transport Complex IV / Q000302:isolation & purification / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D007223:Infant; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology; D009472:Neuronal Ceroid-Lipofuscinoses / Q000201:enzymology* / Q000235:genetics; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D057057:Serine Proteases; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013154:Spleen / Q000201:enzymology; D000091346:Tripeptidyl-Peptidase 1
D000590:Amino Acid Chloromethyl Ketones; D003574:Cytochrome c Group; D010455:Peptides; D015842:Serine Proteinase Inhibitors; C000717269:Tpp1 protein, rat; D000091346:Tripeptidyl-Peptidase 1; C055604:alanyl-alanyl-phenylalanine chloromethyl ketone; D003576:Electron Transport Complex IV; D010450:Endopeptidases; D010447:Peptide Hydrolases; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C000717267:TPP1 protein, human; D006180:Proton-Translocating ATPases