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

Fernando A. Agarraberes* and J. Fred Dice{ddagger}

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



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  		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.

 


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  		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).

 


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  		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.

 


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  		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.

 


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  		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.

 


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  		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.

 

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