Fig. 1. PPCA associates with lamp2a in rat liver lysosomes. (A) Solubilized lysosomes cross-linked with DSP were subjected to affinity chromato graphy with the antibody against the cytosolic tail of lamp2a (lanes 1 and 2). Eluted proteins were subjected to SDS–PAGE and silver stained (lane 1), or immunoblotted for lamp2a (lane 2). The sequence of the N-terminus of the 32 kDa band is shown. Part of the solubilized lysosomes were directly subjected to non-reducing SDS–PAGE (5%) and immunoblotted for lamp2a (lane 3) or PPCA (lane 4). Arrowheads indicate lamp2a multimeric complexes. The 600 kDa (*) and 200 kDa (**) molecular weight complexes of lamp2a were excised and subjected to reducing SDS–PAGE (12%) and silver staining (lanes 5 and 6). Molecular weight (kDa) is indicated on the left. (B) Immunoblot analysis of PPCA in rat liver lysosomes, their membranes (MB) and matrices (MTX) (100 µg of protein) incubated alone (None) or with 5 mM MgCl_2. Arrowheads indicate the precursor (54 kDa) and heavy chain (32 kDa) of the mature PPCA. (C) Immunoblot analysis of hsc73 (upper) and PPCA (lower) in membranes from rat liver lysosomes incubated alone (None) or with 5 mM MgCl₂, CaCl₂, MnCl₂ or 2 mM EDTA. (D) Immunoblot analysis of lamp2a co-immunoprecipitated with PPCA in the absence of chemical cross-linkers from membranes of liver lysosomes incubated as in (B). Light and heavy chains of IgG are shown. Lane 1 contains 1/10 of the total amount of lysosomal membranes used for immunoprecipitation. (E) Membranes from lysosomes treated as in (D) were subjected to immunoprecipitation with an antibody specific for lamp2a or with a pre-immune serum. Carboxypeptidase activity in total lysosomes and in the immunoprecipitates was measured as described under Materials and methods. Values are expressed as percentage of total carboxypeptidase activity in lysosomes present in the immunoprecipitates and are the mean ± SE of three different experiments.