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First published online 6 February 2007
doi: 10.1242/jcs.001073

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Altered dynamics of the lysosomal receptor for chaperone-mediated autophagy with age

Roberta Kiffin^*, Susmita Kaushik^*, Mei Zeng, Urmi Bandyopadhyay, Cong Zhang, Ashish C. Massey, Marta Martinez-Vicente and Ana Maria Cuervo

Department of Anatomy and Structural Biology, Department of Developmental and Molecular Biology and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA

View larger version (25K): [in this window] [in a new window]   Fig. 1. Changes in the mRNA levels of the different LAMP-2 splicing variants with age. Semi-quantitative real-time PCR was used to compare mRNA expression levels for LAMP-2A (A), LAMP-2B (B) and LAMP-2C (C) in total mRNA isolated from the livers of normally fed (Fed) or 48-hour starved (Starved) 4-, 12- and 22-month-old rats. mRNA expression levels for the three isoforms in fed rats exposed to mild oxidative stress (treated with two injections of 40 mg/kg body weight of paraquat every 24 hours) are shown as a positive control for LAMP-2A transcriptional upregulation. Values were corrected for actin amplification in each sample and are expressed as fold increase compared with values for the different isoforms in 4-month-old rats (given an arbitrary value of 1). Values are mean + s.e.m. from four different experiments. Significant differences were observed compared with values from 4-month-old rats (*P<0.05, **P<0.01).

View larger version (43K): [in this window] [in a new window]   Fig. 2. Age-related changes in the dynamic distribution of LAMP-2A in lysosomes. (A) Distribution of LAMP-2A between lysosomal membrane and matrix. Membranes (MB) and matrices (MTX) from lysosomes isolated from livers of 4-, 12- and 22-month-old rats were subjected to SDS-PAGE and immunoblot against LAMP-2A. The percentage of total lysosomal LAMP-2A present in each compartment was calculated by densitometry of the immunoblots. Values are mean + s.e.m. of four to five different experiments. (B) Mobilization of LAMP-2A between the lysosomal membrane and matrix. Intact lysosomes from livers of 4-, 12- and 22-month-old rats were incubated in the presence of GAPDH (25 µg) for 15 minutes at 37°C and then the substrate was rapidly removed by centrifugation (arrows) and lysosomes were incubated in a substrate-free isotonic medium. At the indicated times, lysosomal membranes were isolated and subjected to immunoblot against LAMP-2A. Values are expressed as the percentage of LAMP-2A present at the lysosomal membrane at the beginning of the incubation and are the mean + s.e.m. of three different experiments. Right panels show representative immunoblots. (C) Rates of LAMP-2A internalization (left) and retrieval back to the lysosomal membrane after removal of GAPDH (right) were calculated from the data in B. Values are expressed as changes in the percentage of membrane LAMP-2A per minute. (D) Mobilization of recombinant LAMP-2A into lysosomal membranes. Purified recombinant HA-tagged LAMP-2A was placed into resealed lysosomal membranes isolated from 4-, 12- and 22-month-old rats as described under Materials and Methods. Vesicles were incubated at 37°C for 15 minutes and the amount of HA-LAMP-2A associated with the lysosomal membrane was quantified by immunoblot against HA of the membranes isolated by hypotonic shock and centrifugation (protease protection assay was used to determine that all the HA-LAMP-2A associated with the membrane had a cytosolic exposed C-terminus). Values are expressed as percentage of the total LAMP-2A incorporated in each group of resealed membranes and are mean + s.e.m. of three different experiments. Significant differences were observed compared with values from 4-month-old rats (*P<0.05; **P<0.01).

View larger version (59K): [in this window] [in a new window]   Fig. 3. Changes in the lysosomal degradation of LAMP-2A with age. (A) Half-life of LAMP-2A in cultured primary mouse fibroblasts. Early [population doubling level (PDL) 4-6] and late (PDL 12-15) passage primary mouse fibroblasts were labeled with [35S]methionine:cysteine mixture and at the indicated times cells were lysed and subjected to immunoprecipitation with the antibody against the LAMP-2A cytosolic tail. Immunoprecipitates at different times from cells maintained in the presence (serum+) or absence (serum–) of serum were subjected to SDS-PAGE and fluorography, which were quantified by exposure to a PhosphorImager screen. Values are expressed as a percentage of radiolabeled LAMP-2A at time 0 and are mean of three different experiments. The best exponential decay curve was calculated by linear regression analysis and the half-life of LAMP-2A was calculated by the formula t1/2=ln2/degradation rate. (B) Degradation of LAMP-2A in lysosomes. Liver lysosomes from 4- and 22-month-old rats were incubated at 37°C in an isotonic medium. At the indicated times lysosomes were pelleted and membranes and matrices were isolated after hypotonic shock by centrifugation. Samples were divided into two halves and subjected to SDS-PAGE and immunoblot with the antibody that recognizes the cytosolic tail of LAMP-2A or an antibody against the luminal region of LAMP-2, common to all spliced variants (shown here). The graph shows the changes in the amount of cytosolic tail at different times expressed as percentage of the amount present before the incubation. Values are mean ± s.e.m. of three different experiments. Filled arrowhead indicates the truncated form of LAMP-2A generated by cleavage of the protein at the lysosomal membrane, while open arrowhead indicates an unusual lower molecular weight LAMP-2A detected only in the lumen of lysosomes from the oldest animals.

View larger version (59K): [in this window] [in a new window]   Fig. 4. Changes in the LAMP-2A-related protease complex at the lysosomal membrane with age. (A,B) Homogenates (HOM), Lysosomes (LYS), lysosomal membranes (L. MB) and matrices (L. MTX) (100 µg protein) from 4- and 22-month-old rats were subjected to SDS-PAGE and immunoblotted for cathepsin A or cathepsin D (as labeled). White arrowhead indicates the precursor protein while black arrowheads indicate the 32 and 22 kDa catalytic subunits. Only the 32 kDa catalytic subunit is shown in A. (C) Carboxypeptidase activity in lysosomes from 4- and 22-month-old rats was measured. Values are shown as total activity in the lysosomal fraction (right) or specific activity (corrected per protein amount; left) and are means + s.e.m. of three different experiments. (D) Intact lysosomes from 4- and 22-month-old rats were incubated in isotonic buffer supplemented or not with 1 mM CaCl2 for 15 minutes. At the end of the incubation, lysosomal membranes were isolated after hypotonic shock and centrifugation and were subjected to SDS-PAGE and immunoblot for cathepsin A. (E) Lysosomal membranes from 4-, 12- and 22-month-old rats were subjected to zymography (zymog) in a gelatin-embedded gel and the areas of proteolytic cleavage were identified after staining with Amido Black. LMMP, lysosomal membrane metalloprotease. Immunoblot (I.B.) of the same membranes for LAMP-1 is shown in the bottom panel.

View larger version (59K): [in this window] [in a new window]   Fig. 5. Age-related changes in the lipid microdomains at the lysosomal membrane involved in CMA regulation. (A) Lysosomes from livers of fed or 48-hour-starved 4- and 22-month-old rats were extracted with 1% Triton X-114 and then subjected to sucrose gradient centrifugation. Aliquots collected from top to bottom were grouped as the detergent-resistant (DR), intermediate (Int) and detergent-soluble (Sol) fractions, which were then subjected to SDS-PAGE and immunoblotted for LAMP-2A. The graph on the right shows the mean value of the densitometric quantification of the LAMP-2A immunoblots from three experiments. Values are expressed as a percentage of the total LAMP-2A at the lysosomal membrane present in each region. (B) Flotillin-1 (Flot-1) content in lysosomes (Lys) and detergent-resistant regions (DR) isolated and processed as in A from livers of fed 4- and 22-month-old rats. Because Flotillin-1 localizes preferentially in the DR region, levels in the soluble fraction were undetectable. (C) Lysosomal membranes from 4- and 22-month-old rats and their corresponding Triton X-114-resistant (DR) and soluble (Sol) regions were subjected to SDS-PAGE and staining with Sypro Ruby. The densitometric electrophoretic profile of the detergent-resistant regions is shown (bottom). Arrowheads indicate decreased (white) or increased (black) levels compared with 4-month-old rats. (D) Quantification of cholesterol content in homogenates (Top) and lysosomes (bottom) from 4-, 12- and 22-month-old rat livers was carried out by the Amplex Red cholesterol assay. Values are expressed as total units in the fraction and are the mean + s.e.m. of triplicate samples in two different experiments. Significant differences were observed compared with values from 4-month-old rats (*P<0.05, **P<0.01).

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