SUMMARY Macroautophagy is a lysosomal degradative pathway essential for neuron survival. Here we show that macroautophagy requires the Alzheimer's disease (AD) - related protein, presenilin-1 (PS1). In PS1-null blastocysts, neurons from mice hypomorphic for PS1 or conditionally depleted of PS1, substrate proteolysis and autophagosome clearance during macroautophagy are prevented due to a selective impairment of autolysosome acidification and cathepsin activation. These deficits are caused by failed PS1-dependent targeting of the v-ATPase subunit to lysosomes. N-glycosylation of the V0a1 subunit, essential for its efficient ER-to-lysosome delivery, requires the selective binding of PS1 holoprotein to the unglycosylated subunit and the Sec61alpha/oligosaccharyltransferase complex. PS1 mutations causing early-onset AD produce a similar lysosomal/autophagy phenotype in fibroblasts from AD patients. PS1 is therefore essential for v-ATPase targeting to lysosomes, lysosome acidification, and proteolysis during autophagy. Defective lysosomal proteolysis represents a basis for pathogenic protein accumulations and neuronal cell death in AD and suggests novel therapeutic targets.

Chaperone-mediated autophagy (CMA) is a selective pathway for the degradation of cytosolic proteins in lysosomes. CMA declines with age because of a decrease in the levels of lysosome-associated membrane protein (LAMP) type 2A, a lysosomal receptor for this pathway. We have selectively blocked the expression of LAMP-2A in mouse fibroblasts in culture and analyzed the cellular consequences of reduced CMA activity. CMA-defective cells maintain normal rates of long-lived protein degradation by up-regulating macroautophagy, the major form of autophagy. Constitutive upregulation of macroautophagy is unable, however, to compensate for all CMA functions. Thus, CMA-defective cells are more sensitive to stressors, suggesting that, although protein turnover is maintained, the selectivity of CMA is necessary as part of the cellular response to stress. Our results also denote the existence of crosstalk among different forms of autophagy.lysosome membrane proteins ͉ lysosomes ͉ proteases ͉ protein degradation ͉ macroautophagy I n mammalian cells, three different mechanisms contribute to the degradation of intracellular components inside lysosomes (autophagy) (1, 2). Two of these mechanisms, macroautophagy and microautophagy, are high-capacity processes that allow the simultaneous sequestration of multiple cytosolic constituents (soluble proteins and organelles) and their degradation, all at once, in the lysosomal lumen (1-3). In contrast, chaperone-mediated autophagy (CMA) allows the lysosomal degradation of specific cytosolic proteins on a molecule-by-molecule basis (4, 5). The selectivity of this pathway is conferred by means of the recognition of a pentapeptide amino acid motif in the CMA substrates by a cytosolic chaperone [heat shock cognate (hsc) protein of 70 kDa] (6). The substrate-chaperone complex is targeted to the lysosomal surface, where it interacts with the lysosome-associated membrane protein (LAMP) type 2A, a lysosomal membrane receptor for this pathway (7). After unfolding (8), the substrate translocates into the lysosomal lumen, assisted by a luminal chaperone (lys-hsc70), where it is rapidly degraded (4, 5). Binding of substrate proteins to LAMP-2A is a limiting step for CMA. Levels of LAMP-2A at the lysosomal membrane are tightly controlled and constitute a regulatory mechanism for CMA (4, 5). Up-regulation of CMA occurs during prolonged nutritional stress (starvation), exposure to toxic compounds, and mild oxidative stress (9) (reviewed in refs. 4 and 5), suggesting a role for this pathway in the selective removal of abnormal or damaged proteins under these conditions. In addition, a role for CMA in antigen presentation has recently been described (10). CMA activity decreases during aging (11), and a blockage of this pathway by mutant forms of synuclein also occurs in familial forms of Parkinson's disease (PD) (12). However, because of the complexity of the phenotypes associated with both aging and PD, the direct consequences of blockage of CMA in these systems are difficult to infer.The LAMP-2 gene und...

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Molecular chaperones are pivotal in folding and degradation of the cellular proteome but their impact on the conformational dynamics of near-native membrane proteins with disease relevance remains unknown. Here we report the effect of chaperone activity on the functional conformation of the temperature-sensitive mutant cystic fibrosis channel (∆F508-CFTR) at the plasma membrane and after reconstitution into phospholipid bilayer. Thermally induced unfolding at 37 °C and concomitant functional inactivation of ∆F508-CFTR are partially suppressed by constitutive activity of Hsc70 and Hsp90 chaperone/co-chaperone at the plasma membrane and post-endoplasmic reticulum compartments in vivo, and at single-molecule level in vitro, indicated by kinetic and thermodynamic remodeling of the mutant gating energetics toward its wild-type counterpart. Thus, molecular chaperones can contribute to functional maintenance of ∆F508-CFTR by reshaping the conformational energetics of its final fold, a mechanism with implication in the regulation of metastable ABC transporters and other plasma membrane proteins activity in health and diseases.