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First published online 14 November 2006
doi: 10.1242/jcs.03283

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The 46-kDa mannose 6-phosphate receptor does not depend on endosomal acidification for delivery of hydrolases to lysosomes

¹ Institut für Angewandte Genetik und Zellbiologie, Universität für Bodenkultur Wien, Muthgasse 18, 1190 Vienna, Austria
² Department of Biochemistry, University of Western Australia, Nedlands, WA 6907, Australia
³ Institut für Physiologische Chemie und Pathobiochemie, Universität Münster, Waldeyerstr. 15, 48149 Münster, Germany

View larger version (49K): [in this window] [in a new window]   Fig. 1. Effects of tunicamycin and monensin on cathepsin B biosynthesis in SCC-VII cells. Confluent monolayers of SCC-VII cells were metabolically labeled for 1 hour with 100 µCi/ml [35S]methionine and subsequently chased for 4 hours in the absence (-) or continuous presence (+) of 10 µg/ml tunicamycin (A) or 1 µM monensin (B). Cathepsin B was then immunoprecipitated from equivalent amounts of cell and medium extracts and analyzed by SDS-PAGE and fluorography. proCB, procathepsin B; proCB(3), fully glycosylated procathepsin B carrying 3 N-linked oligosaccharide side chains; proCB(0), unglycosylated procathepsin B; scCB, mature cathepsin B (single-chain form). Experiments using cathepsin-B-specific murine embryonic fibroblasts (MEFs) revealed that the band labeled with an asterisk represents a polypeptide unrelated to cathepsin B, which is sometimes nonspecifically co-precipitated from cell extracts by the antiserum used in these studies. Note that this polypeptide is not present in cathepsin B immunoprecipitates from culture media. The migration positions of 14C-labeled molecular mass standards are indicated.

View larger version (58K): [in this window] [in a new window]   Fig. 2. Effects of NH4Cl and monensin on acidic organelles in SCCVII cells and L-M(TK-) fibroblasts. SCC-VII cells and M6P/IGF2R-positive L-M(TK-) fibroblasts were incubated in complete culture medium in the absence (control) or presence of either 10 mM NH4Cl or 1 µM monensin for 2 hours at 37°C. The cells were then labeled with LysoSensor Yellow/Blue (which accumulates in acidic compartments; pseudocolored in green) and DAPI (which stains nuclei; pseudocolored in red) before analysis by confocal laser-scanning microscopy. Bars, 10 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 3. Effect of NH4Cl on the biosynthesis of cathepsin B, D and L in NIH/3T3 cells and M6P/IGF2R-negative mouse embryonic fibroblasts. (A) Igf2r-/- MEFs and receptor-positive NIH/3T3 cells were metabolically labeled with [35S]methionine and chased for 5 hours in the absence (-) or continuous presence (+) of 10 mM NH4Cl as described in Fig. 1. Cathepsin B was then immunoprecipitated from equivalent amounts of cell and medium extracts and analyzed by SDS-PAGE followed by fluorography. Note that the nonspecific band described in Fig. 1 was not observed in this experiment. proCB, procathepsin B; scCB, mature cathepsin B (single-chain form). (B) Igf2r-/- MEFs were metabolically labeled with [35S]methionine and chased for 5 hours in the absence (-) or continuous presence (+) of 10 mM NH4Cl as described in Fig. 1. Cathepsin D and L were then sequentially immunoprecipitated from equivalent amounts of cell and medium extracts and analyzed by SDS-PAGE followed by fluorography. proCL, procathepsin L; scCL, mature cathepsin L (single-chain form); tcCL, heavy-chain of mature cathepsin L (two-chain form); proCD, procathepsin D; scCD, mature cathepsin D (single-chain form).

View larger version (43K): [in this window] [in a new window]   Fig. 4. Biosynthesis of cathepsin B, D and L in MPR-negative mouse embryonic fibroblasts. (A) MPR-negative MEFs lacking both M6/IGF2R and MPR46 were metabolically labeled with [35S]methionine for 1 hour and then chased for up to 5 hours as described in Fig. 1. Procathepsin B (40-42 kDa), procathepsin D (46 kDa) and procathepsin L (37 kDa) were then sequentially immunoprecipitated from equivalent amounts of cell and medium extracts and analyzed by SDS-PAGE and fluorography. The mature forms of the proteinases were not detected in this experiment. The band labeled with an asterisk is nonspecific as described in Fig. 1. proCB, procathepsin B; proCD, procathepsin D; proCL, procathepsin L. (B) Microsomal extracts (15 µg total protein) of L-M(TK-) cells (lane 1), Igf2r-/- MEFs (lane 2) and MPR-negative MEFs (lane 3) were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to cathepsin B, cathepsin D and cathepsin L. Band intensities determined by densitometry were used to compare the antigen contents of the samples. proCB, procathepsin B (42 kDa); scCB, mature cathepsin B (single-chain form; 30-32 kDa); scCD, mature cathepsin D (single-chain form; 43 kDa); tcCD, heavy-chain of mature cathepsin D (two-chain form; 29 kDa); proCL, procathepsin L (34-36 kDa); scCL, mature cathepsin L (single-chain form; 29 kDa); tcCL, heavy-chain of mature cathepsin L (two-chain form; 21 kDa).

View larger version (39K): [in this window] [in a new window]   Fig. 5. Effects of NH4Cl and monensin on the endosomal/lysosomal distribution of cathepsin D in SCC-VII cells. SCC-VII cells were incubated in complete culture medium in the absence (control) or presence of either 10 mM NH4Cl or 1 µM monensin for 10 hours at 37°C. Post-nuclear supernatants were obtained and fractionated by density-gradient centrifugation. Pooled heavy (lane 1), intermediate (lane 2) and light (lane 3) gradient fractions were then tested for ß-N-acetylhexosaminidase (Hex) activity and subjected to immunoblotting with antibodies to cathepsin D (CD). Band intensities were determined by densitometric analysis of the respective films. Data are presented as the mean values of two to four experiments. One representative blot is shown in each case. The band labeled with an asterisk represents a polypeptide unrelated to cathepsin D that was nonspecifically detected in these experiments. Markers for the Golgi apparatus and the endoplasmic reticulum were largely confined to the light gradient fractions. scCD, mature cathepsin D (single-chain form; 43 kDa).

View larger version (46K): [in this window] [in a new window]   Fig. 6. Effects of NH4Cl and monensin on the subcellular localization of cathepsin D and LAMP-1 in SCC-VII cells. SCC-VII cells were incubated in complete culture medium in the absence (control) or presence of either 1 µM monensin or 10 mM NH4Cl for 10 hours at 37°C. The cells were then fixed, permeabilized and incubated with antibodies to cathepsin D and LAMP-1. Bound primary antibodies were then detected with FITC- and Cy3-labeled secondary antibodies before analysis of the immunostained cells by confocal laser-scanning microscopy. Bars, 10 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 7. Effects of NH4Cl and monensin on the subcellular localization of MPR46 and its ligands in SCC-VII cells. SCC-VII cells were incubated in complete culture medium in the absence (control) or presence of either 1 µM monensin or 10 mM NH4Cl for 10 hours at 37°C. The cells were then fixed, permeabilized and incubated with antibodies to cathepsin D and TGN38 (panel A), or antibodies to MPR46 and the lysosomal marker LAMP-1 (panel B). Bound primary antibodies were then detected with FITC- and Cy3-labeled secondary antibodies before analysis of the immunostained cells by confocal laser-scanning microscopy. Bars, 10 µm.