Because the identification afterward of the specific cytosolic components required for fusion is often difficult, we adopted instead a reverse approach in which individual components were added to the fusion reaction until the minimal requirements to reproduce vesicular fusion were identified. have been explained in mammals, depending on the mechanisms that mediate delivery of cargo to lysosomes: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) (1, 2). Whereas in these two last autophagic pathways, cargo is usually delivered directly from the cytosol to lysosomes, either through invaginations in the lysosomal membrane (in microautophagy) or by directly crossing the membrane through a translocation complex (in CMA), the unique characteristic of macroautophagy is usually that cytosolic components are first sequestered in a double-membrane vesicle [autophagosome (APG)]. This double-membrane vesicle acquires the hydrolases required for cargo degradation upon fusion with lysosomes. Macroautophagic cargo can also reach the lysosomal lumen through fusion of PF 431396 APGs with early or late endosomes (to form amphisomes) (3). Different molecular components of lysosomes and endosomes, such as Rab7 or AAA ATPase SKD, have been reported to be necessary for fusion of APGs with lysosomes or early/late endosomes in mammalian cells (4,5,6). In addition, APG/lysosome fusion can be inhibited by treatment with inhibitors of the lysosomal proton pump (bafilomycin A1) or poor bases (hydroxychloroquine), which neutralize the lysosomal acidic pH. This obtaining suggests that heterotypic fusion depends on luminal pH (7). However, the specific requirements for vesicular fusion in the autophagic pathway are poorly understood, mainly due to the difficulty of separating fusion itself from other cellular events directly related to this process, such Rabbit Polyclonal to FES as vesicular docking into the cytoskeleton network and directional vesicular trafficking. A further level of complexity in the autophagic fusion arises from the fact that what have been classically cataloged as lysosomes include a heterogeneous pool of intracellular vesicles, possibly at different maturation stages and with different functions (8). In fact, we have reported previously the isolation of different lysosomal subgroups with different morphological and biochemical properties, a distinctive proteomic signature and with different functions in autophagy (8). Only one of these lysosomal subgroups is usually competent to perform CMA, as they contain the luminal chaperone required for lysosomal uptake of cytosolic proteins by this pathway (8). However, nothing is known about the participation of the different lysosomal subgroups in macroautophagy. Thus, whether different lysosomal types can undergo fusion with APGs or whether only a particular lysosomal subpopulation is usually dedicated to macroautophagy remains unknown. Understanding the vesicular fusion events that take PF 431396 place between the different subcompartments involved in macroautophagy is important to further elucidate the basis of the crosstalk between autophagic and heterophagic pathways. In addition, alterations in vesicular fusion may be behind the increased quantity of autophagic vacuoles reported in human disorders of very different nature, including, among others, neurodegenerative diseases, myopathies, metabolic disorders, and some infectious diseases (9,10,11). Defects in degradation could arise from both impaired proteolytic activity of the lysosomal hydrolases or from alterations in fusion of APGs with the lytic compartments, either endosomes or lysosomes. Consequently, distinguishing whether improper fusion is a primary defect or an indirect result of alterations in vesicular traffic is essential to the design of future interventions aimed at fixing or improving vesicular fusion. To analyze the characteristics of APGs/lysosome fusion independently of vesicular trafficking, we have developed a fusion assay using isolated APGs and different lysosomal subpopulations from both rodent tissues and cells in culture. By using this assay, we have characterized the basic requirements for heterotypic fusion of APGs with lysosomal compartments. Furthermore, we have identified that defective vesicular fusion is usually responsible, at least in part, for the decrease in autophagic activity that we have explained previously to occur in mouse liver on exposure to a chronic lipid challenge such as a high-fat diet (HFD) (12). Our results support that changes in the intracellular lipid content in conditions such as metabolic PF 431396 disorders may have pronounced effects on.