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A molecular chaperone complex at the lysosomal membrane is required for protein translocation

Fernando A. Agarraberes^* and J. Fred Dice

The Sackler School of Graduate Biomedical Sciences, Department of Cellular and Molecular Physiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA
^* Present address: Howard Hughes Medical Institute, Department of Molecular Genetics, Yale University School of Medicine, New Haven, CT 06510, USA

View larger version (16K): [in a new window]   Fig. 1. Characterization of lymHsc70. (a) Two-dimensional gel electrophoresis of lysosomal Hsc70. The numbers at the top refer to isoelectric points. Top, untreated IMR-90 human fibroblast lysosomes (100 µg protein); middle, lysosomes treated with trypsin (100 µg); bottom, untreated purified lysosomal membranes (100 µg). Blots were probed with mAb 13D3 (anti-Hsc70) and developed with chemiluminescense (see Materials and Methods). (b) Lysosomal membranes (30 ug) were treated with 0.5 M NaCl (lane 1), 0.1 M Na2CO3, pH 11.0 (lane 2), or 5.0 M urea (lane 3). Samples were analyzed by SDS-PAGE and transferred to Immobilon-P membranes. Blots were probed with anti-Hsc70 and anti-Lamp2a antibodies and developed with chemiluminescense (see Materials and Methods). (c) Purified lysosomes (100 µg) were incubated as indicated without additions, with ADP/Mg2+, the protein substrate, RNase A, or RNase A plus ADP/Mg2+. Samples were incubated or not for 20 minutes with proteinase K (10 µg/ml). Samples were separated by SDS-PAGE, transferred to Immobilon-P membranes, probed with mAb 13D3, and developed with chemiluminescense (see Materials and Methods). Blots were quantitated by densitometry.

View larger version (42K): [in a new window]   Fig. 2. Immunoprecipitation analysis of solubilized lysosomal membranes. Cytosolic proteins (15 µg) from fibroblasts deprived of serum for 18 hours were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Solubilized lysosomal membrane proteins (100 µg) from fibroblasts deprived of serum for 18 hours were immunoprecipitated with anti-Hsc70, anti-Hip and anti-p19 as a negative control. Samples were treated as described in Materials and Methods. Blots were probed with specific antibodies against the indicated proteins and developed with chemiluminescense (see Materials and Methods).

View larger version (78K): [in a new window]   Fig. 3. Cellular localization of Hsc70 and other molecular chaperones and cochaperones. Serum-starved IMR-90 fibroblasts were fixed in methanol at -20°C. and then incubated with mAb 13D3 followed by FITC-conjugated secondary antibodies, anti-DNA antibodies followed by Cy5-conjugated secondary antibodies, and antibodies against either Hip, Hop, BAG-1 or Hsp40 followed by Texas Red-conjugated secondary antibodies. Incubation with anti-Hsp90 antibodies was followed by Rhodamine-conjugated secondary antibodies. Bar, 10 µm.

View larger version (20K): [in a new window]   Fig. 4. Levels of chaperones and cochaperones on the lysosomal membranes and in the cytosol. (a) Lysosomes were from cells maintained under serum-supplemented (+S) or serum-starved (-S) conditions for 18 hours. Lysosomal membrane proteins (30 µg) were separated by SDS-PAGE, transferred to Immobilon-P membranes, probed with corresponding antibodies, developed with chemiluminescense (see Materials and Methods), and quantified by densitometry. Values shown (±s.d.) are averages of three separate experiments (top). Cytosolic proteins (15 µg) were analyzed similarly (bottom). (b) Purified lysosomes (50 µg) were incubated at different times with an ATP-regenerating system without an added protein substrate. After incubation for increasing times, lysosomal membranes were purified and analyzed by SDS-PAGE and western blot. Levels of the indicated proteins were determined by densitometry and expressed as percentage of the value at the beginning of the experiment.

View larger version (27K): [in a new window]   Fig. 5. Inhibition of protein transport into lysosomes with specific antibodies. Lysosomes (100 µg protein) in a total volume of 0.1 ml were incubated with increasing amounts of mouse serum containing anti-Hip, nonimmune mouse serum, rabbit serum containing anti-Hop or anti-Hsp40, or nonimmune rabbit serum as indicated. Samples were treated as described in Materials and Methods, and membranes were probed with either rabbit anti-RNase A or mouse anti-GAPDH, and developed with chemiluminescense (see Materials and Methods). The lower panels are the densitometric quantitation of the results.

View larger version (31K): [in a new window]   Fig. 6. A working model for the steps in the chaperone-mediated autophagy. Although Hsp90 is present in substoichiometric amounts, we speculate that it may be required for activity of the complex. Step 1: the protein substrate is recognized in the cytosol by the Hsc70 chaperone system. It binds substrates at a KFERQ motif. Step 2: either the substrate binds to the lymHsc70 chaperone system or the cytosolic Hsc70 chaperone system-substrate complex docks at the lysosomal membrane lamp2a. This interaction between Hsc70, substrate, and lamp2a has been previously documented (Cuervo and Dice, 1996). Step 3: the substrate interacts with Lamp2a and is inserted into the translocation complex. Binding of the substrate protein to intralysosomal Hsc70 is required for the substrate protein’s translocation. Step 4: the substrate protein is degraded in the lumen of the lysosome.