Cell Culture and Isolation of Lysosomes—Human embryonic fibroblasts (IMR-90) were obtained from Corriel Cell Repositories (Camden, NJ). Cells were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% newborn calf serum and 1% antibiotic (penicillin/streptomycin with fungicide; Invitrogen). IMR-90 cells were used for experiments at 20–40 population doubling levels (PDLs). To deprive cells of serum, cultures were washed several times with Hanks' balanced salts' solution (Invitrogen) and then placed in media without serum. Lysosomes from IMR-90 cells were isolated as described previously (21). Lysosomal matrix preparations were prepared by subjecting the lysosomes to hypotonic shock, as reported previously (22).
Lysosomal Latency Assay—To assess the integrity of the lysosomal membrane, we performed a -hexosaminidase latency study as described previously (13). Lysosomal preparations that contained >10% broken lysosomes, as determined by -hexosaminidase latency, were not used.
Chemicals and Antibodies—All chemicals were obtained from Sigma unless otherwise indicated. GAPDH was obtained from Roche Applied Science, and RNase A was obtained from Worthington Biochemical Company (Lakewood, NJ). Antibodies against lamp2a were obtained from Zymed Laboratories Inc.. Antibodies against GAPDH were obtained from Biodesign (Saco, ME), and antibodies against hsc70 were obtained from Maine Biotechnology Services (Portland, ME). Detection of the formation of carbonyl groups in proteins was performed by following the manufacturer's instructions using the OxyBlotTM oxidized protein detection kit supplied by Chemicon International (Temecula, CA). [14C]GAPDH and [14C]RNase A were radiolabeled by using [14C]formaldehyde reductive methylation as described previously (23).
Cellular Protein Degradation—Confluent cells were labeled with [3H]leucine (2 µCi/ml) for 48 h in media containing 10% newborn calf serum. Cells were washed twice with Hanks' balanced salt solution, and the media were replaced with either complete media or media without serum both containing excess (2.8 mM) unlabeled leucine (24). Aliquots of media (200 µl) were taken at the indicated time intervals and precipitated in 20% trichloroacetic acid. Proteolysis was determined by measuring the percent of acid soluble radioactivity compared with the total radioactivity of cell lysates prepared by the addition of 0.1 N NaOH and 0.1% sodium deoxycholate (24).
In Vitro Lysosomal Import and Protease Protection Assays— [14C]GAPDH and [14C]RNase A were incubated in the presence of isolated lysosomes for 1.5 h as described previously (13). Degradation was calculated as the percent of trichloroacetic acid-soluble radioactivity converted from the trichloroacetic acid-precipitable radioactivity (13). The protease protection assay was performed as described previously (7). Briefly, GAPDH was incubated with purified lysosomes treated with 100 µM chymostatin A. The GAPDH was incubated with the lysosomes for 30 min and then washed and treated with 10 µg of proteinase K and 1 µM CaCl2 for 15 min. The reaction was stopped by incubation with 4-(2-aminoethyl)-benzenesulfonyl fluoride. Lysosomes were solubilized and processed for SDS-PAGE.
Immunoprecipitation of GAPDH—Cells were placed in conditions of serum deprivation for 24 h. The cells were washed twice in ice-cold phosphate-buffered saline and incubated in Nonidet P-40 lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5 µg/ml phenylmethylsulfonyl fluoride, and 0.01 µg/ml each aprotinin, leupeptin, and pepstatin for 15 min The lysate was collected by centrifuging the samples at 14,000 x g for 15 min. Aliquots were then taken and precleared with protein A-Sepharose beads (Amersham Biosciences) for 1 h. Antibody (8 µg) was added to the precleared lysate and incubated overnight at 4 °C. The antibody-antigen complex was removed from the lysate by centrifuging the samples at 10,000 x g for 30 s to pellet the protein A-Sepharose beads. The supernatant was discarded, and beads washed an additional three times. The beads were resuspended in Laemmli sample buffer and subjected to SDS-PAGE followed by immunoblot analysis.
General Methods—Protein determination was performed using the Lowry method with bovine serum albumin as a standard (25). Protein detection methods such as SDS-PAGE (26) and immunoblotting (27) were visualized using chemiluminescence detection methods (Western Lightning; PerkinElmer Life Sciences). Radioactivity was determined by counting samples in a Packard liquid scintillation analyzer with quenching detected by an automatic external standard (Packard Tri-Carb 2100 TR). Densitometric analysis was performed by using Adobe Photoshop 7.0. Statistical analyses were preformed using the two-tailed Student's t test.