LAMP-2A exits the membrane microdomains when CMA is activated

The lower levels of LAMP-2A in the DRM of the lysosomal membrane during starvation did not correlate with changes in the total content of cholesterol in lysosomes (Figure 3, Supplementary data), but they could be owing to changes in the lysosomal system independent of changes in CMA. To analyze the consequences of CMA activation in the distribution of LAMP-2A in the lysosomal membrane, we incubated lysosomes from fed rats with different components known to activate CMA in isolated lysosomes (Figure 4, Supplementary data) (Cuervo et al, 1997, 1995). Incubation of lysosomes with two well-characterized CMA substrates, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Figure 6A) or ribonuclease A (data not shown), resulted in partial exclusion of LAMP-2A from the DRM toward the intermediate and soluble fractions (Figure 6A). This effect was considerably more pronounced when lysosomes were incubated with cytosol from starved animals, which contains potential CMA substrates and also, the cytosolic chaperone/cochaperone complexes, ATP and possibly other still unidentified components required for CMA activation (Figure 6A). In fact, as shown in Figure 6A, incubation with purified hsc70 or ATP also decreased the amount of LAMP-2A in DRM, although to a lesser extent, and combination of CMA substrates with these factors showed the closest effect to the one observed with the cytosol, supporting the additive effect of all of these components when added as part of the cytosolic fraction. The contribution of cytosolic ATP to CMA activation and/or LAMP-2A mobilization was confirmed when apyrase was added to the cytosol to deplete it of ATP. After this treatment, the effect observed with untreated cytosol was lost (Figure 6A, bottom right panel; note that apyrase alone does not change the distribution of LAMP-2A in the DRM). None of these treatments considerably changed the amount of flotillin-1 in the DRM (Figure 6A, left), suggesting that they did not result in disruption of these domains but rather, in the selective mobilization of LAMP-2A out of these discrete membrane regions.

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Figure 6

The distribution of LAMP-2A in discrete lysosomal membrane regions changes with changes in CMA. (A) Fed rat liver lysosomes were incubated in an isotonic buffer without additions, or in the presence of rat liver cytosol (25 μg), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (25 μg), GST-hsc70 (10 μg/ml), ATP (5 mM) or the indicated combinations for 15 min at 37°C. Lysosomes incubated directly with apyrase or with cytosol treated with apyrase are shown in the right bottom panel. At the end of the incubation, lysosomes were subjected to Triton X-114 extraction, sucrose density gradient centrifugation and immunoblot as in Figure 2C. Graph shows changes in LAMP-2A distribution expressed as times the value in untreated lysosomes and are the mean of the densitometric quantification of 3–6 experiments as the one shown here. Because all the incubations were performed in the presence of protease inhibitors, total levels of LAMP-2A in lysosomes remained constant throughout the incubation. (B) Lysosomes from livers of fed rats, untreated or treated with paraquat (+PQ), were processed as in (A). Graph shows changes in LAMP-2A distribution induced by the PQ treatment, expressed as times the value in untreated animals and are the mean of three experiments. (C) Lysosomes with high (CMA+) and low (CMA−) ability for CMA isolated from livers of fed or 48 h starved rats were processed as in (A). Top left: Levels of two CMA components (LAMP-2A and hsc70) and of an unrelated lysosomal membrane protein (LAMP-1) in each of the four groups of lysosomes. Bottom left: Percentage of total lysosomal LAMP-2A present in each fraction, determined by densitometric quantification of the immunoblots.

Mild-oxidative stress also activates CMA even under normal nutritional conditions (Kiffin et al, 2004). Consistent with the mobilization of LAMP-2A out of the DRM when CMA is activated in vitro, a larger percentage of LAMP-2A localized outside the DRM in lysosomes isolated from fed rats exposed to sublethal doses of paraquat, a pro-oxidant shown to induce activation of CMA (Kiffin et al, 2004) (Figure 6B). Likewise, when we subdivided the pool of CMA-related lysosomes used throughout this study into two subgroups, based on their CMA activity (Cuervo et al, 1997), we found that LAMP-2A was practically undetectable in DRM of the lysosomes with higher CMA activity (those enriched in the luminal hsc70 and LAMP-2A (as shown in Figure 6C, left)), whereas larger amounts of LAMP-2A could be detected in the DRM of lysosomes latent for CMA (Figure 6C, right). When maximal activation of CMA is needed, such as during prolonged starvation, this second group of lysosomes acquires the lumenal chaperone and become active (Figure 6C, left; Cuervo et al, 1997). Consistently, the amount of LAMP-2A in DRM decreased considerably in this reservoir pool of lysosomes after prolonged starvation (Figure 6C, right). In summary, activation of CMA both in vitro and in vivo associates with reduced levels of LAMP-2A in the DRM of the lysosomal membrane.

Changes in the lipid composition of the lysosomal membrane microdomains alter CMA

To determine how changes in the distribution of LAMP-2A between DR and soluble regions of the lysosomal membrane affect CMA activity, we disrupted these discrete cholesterol-enriched regions in cultured mouse fibroblasts. CMA is activated in these cells in response to removal of serum, increasing the degradation of long-lived cytosolic proteins, typically substrates for this pathway (Cuervo and Dice, 2000a; Cuervo et al, 2003; Massey et al, 2006b). Treatment with the cholesterol-depleting agent, MBCD, increased the proteolysis of long-lived proteins (Figure 7A). This increased degradation was mainly lysosomal, because it was significantly reduced in the presence of ammonium chloride and leupeptin, inhibitors of the lysosomal-dependent proteolysis (Figure 7A). Treatment with 3-methyladenine (3MA), which inhibits macroautophagy but not CMA (Massey et al, 2006b), partially reduced the observed MBCD-mediated increase in protein degradation (Figure 7B), suggesting that part of the increase in protein degradation (38%) was owing to activation of macroautophagy, whereas the other part (62%) was taking place via CMA. Thus, disruption of lipid microdomains resulted in activation of both CMA and macroautophagy. The effect on macroautophagy was, however, independent of changes in LAMP-2A redistribution, as MBCD treatment also increased protein degradation rates in cells stably RNA interfered for LAMP-2A, where the vast majority of the MBCD-induced increase took place via macroautophagy (89% sensitive to 3MA) (Figure 7B) (Massey et al, 2006b).

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Figure 7

Consequences of changes in the lysosomal cholesterol content on CMA activity. (A) Degradation of long-lived proteins in mouse fibroblasts (NIH3T3 cells) treated or not with 25 mM MBCD. Where indicated, 15 mM NH₄Cl/100 μM leupeptin (NH₄Cl+Leup) were added into the medium. Values are the mean+s.e. of three experiments with triplicate samples. (B) Effect of MBCD on total rates of protein degradation, measured as in (A), in wild-type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP-2A (LAMP-2A(−)). The ratio of degradation in MBCD-treated and untreated cells is shown for cells maintained with or without 10 mM 3-methyladenine (3MA). (C) Proteolysis of [¹⁴C]GAPDH or a pool of [³H]labeled cytosolic proteins (pool proteins) by isolated rat liver lysosomes, treated or not with MBCD. Broken: lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins. Values are expressed as percentage of the degradation by untreated lysosomes and are the mean+s.e. of three experiments with triplicate samples. (D) Lysosomal–mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol (50 μM). Top: Distribution of LAMP-2A after Triton X-114 extraction and sucrose density gradient centrifugation. Bottom: Ability of intact or disrupted lysosomal–mitochondrial fractions to proteolize [¹⁴C]GAPDH. Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean±s.e. of triplicate samples.

We further confirmed the effect of modifying the membrane distribution of LAMP-2A on CMA activity using isolated lysosomes. Lysosomes previously treated with MBCD, and consequently with higher percent of LAMP-2A in the soluble fraction of the membrane, showed higher rates of degradation of GAPDH and of a pool of radiolabeled cytosolic proteins than untreated lysosomes (Figure 7C). The increased degradation was not owing to leakage of lysosomal enzymes (we did not find changes in β-hexosaminidase latency (data not shown)) or to an increase in the activity of the lysosomal proteases (the MBCD-mediated increase in proteolysis was no longer evident when the lysosomal membrane was disrupted) (broken lysosomes; Figure 7C), suggesting that the enhanced degradation was, indeed, the result of higher rates of substrate binding and uptake inside lysosomes.

To determine whether sequestration of LAMP-2A in the DRM decreased CMA rates, we loaded cells with cholesterol by administering it directly to the culture medium. This treatment increases the concentration of cholesterol in lysosomes (Lusa et al, 2001). Because changes in the cholesterol content in lysosomes could modify their flotation in the discontinuous density gradients used for the isolation of the CMA-related lysosomes, we utilized instead differential centrifugation, which renders a more heterogeneous fraction of lysosomes with some light mitochondria. As in the fraction enriched in CMA-related lysosomes, we verified that (1) a percentage of LAMP-2A was located in the DRM in these lysosomes, (2) the amount of LAMP-2A associated to DRM was lower when CMA was activated by removal of serum and (3) treatment with cholesterol increased (4.5-folds) the amount of LAMP-2A in DRM (Figure 7D, top). CMA activity in this fraction is lower than in the highly purified lysosomes used in the rest of this study, but it can still be measured. As shown in Figure 7D (bottom), the ability to degrade GAPDH was significantly reduced (almost 70% less than control) in the lysosomes from cholesterol-loaded cells. This treatment did not affect the activity of the lysosomal proteases, as rates of GAPDH proteolysis were similar in lysosomes from treated and untreated cells, once the lysosomal membrane was disrupted (Figure 7D, bottom).

These results support that changes in the lipid composition of the lysosomal membrane alter LAMP-2A distribution in DRM and have an effect on CMA activity.

Exclusion of LAMP-2A from the membrane microdomains enhances lysosomal CMA activity

To directly analyze the effect on CMA of changes in the association of LAMP-2A with DRM, we carried out directed mutagenesis in LAMP-2A, in search of a mutation in the cytosolic or transmembrane region of LAMP-2A that altered its ability to associate to the DRM. We first verified that human LAMP-2A transfected into mouse fibroblasts behaved similarly to the endogenous protein: (1) 30% of the protein associated to lysosome membrane DRM and (2) serum removal reduced (0.6 times) the amount of LAMP-2A in DRMs (Figure 8A). As depicted in Figure 8B, we replaced with alanines (1) a proline residue (proline 378) close to the transmembrane/lumenal boundary (P/A), aiming to induce a pronounced conformational change; (2) two glycine residues in the transmembrane region (GG/AA), which resemble a previously described dimerization motif (GXXXG); (3) a tyrosine residue near the transmembrane/cytosolic tail boundary (Y/A), for possible phosphorylation events and (4) four positively charged residues in the cytosolic tail (4A), required for substrate binding to LAMP-2A (Cuervo and Dice, 2000b). The former three mutations did not alter the ability of LAMP-2A to bind CMA substrates (Figure 5, Supplementary data), or the targeting of LAMP-2A to lysosomes. Replacement of proline 378 by alanine markedly decreased association of LAMP-2A with DRM (Figure 8C and D). Furthermore, removal of serum from the culture medium to activate CMA, which reduced DRM levels of endogenous LAMP-2A and overexpressed wild-type, GG/AA and Y/A hLAMP-2A mutants, did not affect the membrane distribution of P/A hLAMP-2A (Figure 8E). Consistently with the lower sequestration of P/A hLAMP-2A into DRM, lysosomes from cells overexpressing this mutant showed higher CMA than lysosomes from cells overexpressing equivalent amounts of wild-type hLAMP-2A (Figure 8F shows higher degradation rates of GAPDH by intact but not by broken lysosomes), and lower increase in CMA activity after treatment of lysosomes with MBCD (Figure 8G) (the observed stimulatory effect is, for the most part, a consequence of the mobilization of the endogenous mLAMP-2A outside of the DRM). Although the 4A hLAMP-2A mutant unable to bind CMA substrates was detected in lysosomal DRM, levels of this protein in these regions remained unchanged upon serum-induced activation of CMA (Figure 8C–E), suggesting that binding of substrate proteins to the cytosolic tail of LAMP-2A could contribute to its mobilization from the DRM. As previously reported, the high levels of endogenous mLAMP-2A in lysosomes prevented any significant dominant-negative effect on CMA activity for this mutant.

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Figure 8

Lysosomes containing a mutant LAMP-2A unable to move into discrete lysosomal membrane regions show enhanced CMA activity. (A) Lysosomes from mouse fibroblasts transfected with wild-type hLAMP-2A and maintained in the presence (Serum+) or absence (Serum−) of serum were collected by centrifugation and subjected to Triton X-114 extraction, sucrose density gradient centrifugation and immunobloting for human (hL2A) or mouse (mL2A) LAMP-2A. Lane 1: homogenate. (B) Amino-acid sequence of the transmembrane and cytosolic region of hLAMP-2A and the mutations performed. Replaced amino acids are shadowed. (C) Lysosomes from mouse fibroblasts transfected with the indicated forms of hLAMP-2A and maintained in the presence of serum were processed as in (A). (D) The percentage of total lysosomal hLAMP-2A present in each fraction was determined by densitometric quantification of immunoblots as the ones shown here. Values, corrected for the amount of mLAMP-2A in each fraction, are mean+s.e. of 3–4 experiments. (E) Changes in hLAMP-2A distribution induced by serum removal. Values are expressed as times the values in cells maintained in the presence of serum and are the mean of two experiments. Changes in the three nonDR regions are shown together (no DR). (F) Proteolysis of a pool of ³[H]labeled cytosolic proteins by intact (left) or disrupted (right) lysosomes isolated from untransfected mouse fibroblasts (none) or fibroblasts transfected with wild-type (WT) or the indicated mutant forms of hLAMP-2A. Values are the mean+s.e. of two experiments with triplicate samples. (G) Part of the lysosomes from (F) was treated with MBCD before the incubation with the radiolabeled proteins. Values are expressed as percentage of degradation by untreated lysosomes and are the mean+s.e. of two experiments with triplicate samples.

Chemicals

Sources of chemicals were as described previously (Cuervo and Dice, 2000a; Cuervo et al, 2003; Kiffin et al, 2004). The antibodies against the cytosolic tail of rat and mouse LAMP-2A and -2B were prepared in our laboratory (Cuervo and Dice, 1996; Massey et al, 2006b). The antibodies against human LAMP-2 and mouse LAMP-1 were from the Developmental Studies Hybridoma Bank (Iowa University), against cathepsin D was from Santa Cruz Biotechnology, against hsc70 and hsp90 from Stressgen and against flotillin-1 from BD Transduction Laboratories. The polyclonal antibody against cathepsin A was a gift from Dr A D'Azzo (SJCRH, Memphis). MBCD, filipin, cholesterol and apyrase were from Sigma. Texas-Red-conjugated cholera toxin, Amplex Red Cholesterol Assay kit and SyproRuby were from Molecular Probes (Invitrogen).

Isolation of subcellular fractions

Rat liver lysosomes were isolated from a light mitochondrial–lysosomal fraction in a discontinuous metrizamide density gradient, and lysosomal fractions with different activities for CMA were further separated by differential centrifugation as described (Cuervo et al, 1997). Lysosomes from cultured cells were isolated as described (Storrie and Madden, 1990). A crude fraction containing lysosomes and mitochondria was prepared after cell lysis by differential centrifugation (2500 g 15 min; 17 000 g 10 min). Lysosomal matrices and membranes were isolated after hypotonic shock (Cuervo et al, 1995).

Isolation of detergent resistant lysosomal membrane microdomains

Lysosomal membranes (150 μg protein) from rat liver or mouse cultured fibroblasts were incubated with 1% of nonionic detergents in 150 mM NaCl, 50 mM Tris–HCl and 5 mM EDTA pH 7.4 (incubation buffer) on ice for 30 min and then subjected to centrifugation in a step-wise discontinuous sucrose gradient (5–35%). Gradient fractions, collected from top to bottom, were concentrated by precipitation in acid or by high-speed centrifugation and then subjected to SDS–PAGE and immunoblot (for details see Supplementary material and methods). The LAMP-2A complexes in the DR and soluble fractions were resolved by centrifugation on a continuous density (10–80%) sucrose gradient as described before (Cuervo and Dice, 2000b).

We are grateful to Frank Macaluso and Leslie Gunther (Analytical Imaging Facility) for their valuable assistance with cryoelectron microscopy and immunogold procedures, to Ms Urmi Bandyopadhyay for her generous help isolating lysosomes, and to Dr Nancy Carrasco for her assistance in the interpretation of the mutation-induced conformational changes in LAMP-2A. This work was supported by grants NIH/NIA AG021904 and AG25355 and EMF AG-NS-0163.