A lysosomal proteinase, the late infantile neuronal ceroid lipofuscinosis gene (CLN2) product, is essential for degradation of a hydrophobic protein,the subunit c of ATP synthase

The specific accumulation of the hydrophobic protein, subunit c of ATP synt hase, in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by lysosomal proteolyti c dysfunction. The defective gene in LINCL (CLN2 gene) has been identified recently. To elucidate the mechanism of lysosomal storage of subunit c, ant ibodies against the human CLN2 gene product (Cln2p) were prepared. Immunobl ot analysis indicated that Cln2p is a 46-kDa protein in normal control skin fibroblasts and carrier heterozygote cells, whereas it was absent in cells from four patients with LINCL. RT-PCR analysis indicated the presence of m RNA for CLN2 in cells from the four different patients tested, suggesting a low efficiency of translation of mRNA or the production of the unstable tr anslation products in these patient cells. Pulse-chase analysis showed that Cln2p was synthesized as a 67-kDa precursor and processed to a 46-kDa matu re protein (t(1/2) = 1 h). Subcellular fractionation analysis indicated tha t Cln2p is localized with cathepsin B in the high-density lysosomal fractio ns. Confocal immunomicroscopic analysis also revealed that Cln2p is colocal ized with a lysosomal soluble marker, cathepsin D. The immunodepletion of C ln2p from normal fibroblast extracts caused a loss in the degradative capac ity of subunit c, but not the beta subunit of ATP synthase, suggesting that the absence of Cln2p provokes the lysosomal accumulation of subunit c.