SPECIFIC DELAY IN THE DEGRADATION OF MITOCHONDRIAL ATP SYNTHASE SUBUNIT C IN LATE INFANTILE NEURONAL CEROID-LIPOFUSCINOSIS IS DERIVED FROM CELLULAR PROTEOLYTIC DYSFUNCTION RATHER THAN STRUCTURAL ALTERATION OF SUBUNIT-C

Previously we indicated that a specific delay in subunit c degradation causes the accumulation of mitochondrial ATP synthase subunit c in ly sosomes from the cells of patients with the late infantile form of neu ronal ceroid lipofuscinosis (NCL), To explore the mechanism of lysosom al storage of subunit c in patient cells, we investigated the mechanis m of the lysosomal accumulation of subunit c both in cultured normal f ibroblasts and in in vitro cell-free incubation experiments. Addition of pepstatin to normal fibroblasts causes the marked lysosomal accumul ation of subunit c and less accumulation of Mn2+-superoxide dismutase (SOD), In contrast, E-64-d stimulates greater lysosomal storage of Mn2 +-SOD than of subunit c. Incubation of mitochondrial-lysosomal fractio ns from control and diseased cells at acidic pH leads to a much more r apid degradation of subunit c in control cells than in diseased cells, whereas other mitochondrial proteins, including Mn2+-SOD, beta subuni t of ATP synthase, and subunit IV of cytochrome oxidase, are degraded at similar rates in both control and patient cells. The proteolysis of subunit c in normal cell extracts is inhibited markedly by pepstatin and weakly by E-64-c, as in the cultured cell experiments. However, th ere are no differences in the lysosomal protease levels, including the levels of the pepstatin-sensitive aspartic protease cathepsin D betwe en control and patient cells, The stable subunit c in mitochondrial-ly sosomal fractions from patient cells is degraded on incubation with mi tochondrial-lysosomal fractions from control cells. Exchange experimen ts using radiolabeled substrates and nonlabeled proteolytic sources fr om control and patient cells showed that proteolytic dysfunction, rath er than structural alterations such as the posttranslational modificat ion of subunit c, is responsible for the specific delay in the degrada tion of subunit c in the late infantile form of NCL.