Chemical modulators of autophagy provide useful pharmacological tools for examination of autophagic processes, and also may lead to new therapeutic agents for diseases in which control of cellular sequestration and degradation capacity are beneficial. We have identified that timosaponin A-III (TAIII), a medicinal saponin reported to exhibit anticancer properties and improve brain function, is a pronounced activator of autophagy. In this work, the salient features and functional role of TAIIIinduced autophagy were investigated. In TAIII-treated cells, autophagic flux with increased formation of autophagosomes and conversion into autolysosomes is induced in association with inhibition of mammalian target of rapamycin activity and elevation of cytosolic free calcium. The TAIII-induced autophagy is distinct from conventional induction by rapamycin, exhibiting large autophagic vacuoles that appear to contain significant contents of endosomal membranes and multivesicular bodies. Furthermore, TAIII stimulates biosynthesis of cholesterol, which is incorporated to the autophagic vacuole membranes. The TAIII-induced autophagic vacuoles capture ubiquitinated proteins, and in proteasome-inhibited cells TAIII promotes autophagy of aggregation-prone ubiquitinated proteins. Our studies demonstrate that TAIII induced a distinct form of autophagy, and one of its pharmacological actions is likely to enhance the cellular quality control capacity via autophagic clearance of otherwise accumulated ubiquitinated protein aggregates.Autophagy, or precisely referred to macroautophagy, is a distinct lysosomal degradation pathway for intracellular materials (1, 2). Although the ubiquitin proteasome system (UPS) 2 usually degrades short-lived soluble proteins, autophagy complements UPS to degrade long-lived proteins, protein aggregates, or organelles (3-5). Autophagy is essentially a regulated intracellular vacuolization process consisting of two key steps: formation of autophagosomes that capture the cytosolic substrates, and fusion of autophagosomes and lysosomes to form autolysosomes in which the autophagic content is degraded. The membrane trafficking processes in autophagy are specifically controlled by proteins encoded by highly conserved autophagy related genes, ATGs, as well as the signaling lipid phosphatidylinositol 3-phosphate that recruits effector proteins to the membranes of autophagic vacuoles (6 -8).A major function of autophagy is to recycle free amino acids, lipids, and carbohydrates for resynthesis of biomolecules or energy production, particularly under starvation conditions. In this regard, autophagy was thought to be a nonspecific process involving random sequestration and degradation of cytoplasmic components, but it is now evident that cargo can also be selectively incorporated into autophagosomes (9). Emerging evidence has shown that autophagy may play a role in selective sequestration and degradation of aggregated protein complexes and/or damaged organelles, thereby serving as a cellular quality control system for ...