| Abstract: | Due in part to the increasing number of links between autophagy malfunction and human diseases, this field has gained tremendous attention over the past decade. Our increased understanding of the molecular machinery involved in macroautophagy (hereafter autophagy) seems to indicate that the most complex step, or at least the stage of the process where the majority of the autophagy-related (Atg) proteins participate, is in the formation of the double-membrane sequestering vesicle. Thus, it is important to establish reliable approaches to monitor this specific process. One of the most commonly used methods is morphological analysis by electron microscopy of the cytosolic vesicles used in the cytoplasm-to-vacuole targeting (Cvt) pathway and autophagy, or the single-membrane intralumenal products, termed Cvt or autophagic bodies, that are formed after the fusion of these vesicles with the yeast vacuole. This method, however, can be costly and time consuming, and reliable analysis requires expert input. Furthermore, it is extremely difficult to detect an incomplete autophagosome by electron microscopy because of the difficulty of obtaining a section that randomly cuts through the open portion of the phagophore. The primary Cvt pathway cargo, precursor amminopeptidase I (prApe1), is enwrapped within either a Cvt vesicle or autophagosome depending on the nutritional conditions. The proteolytic sensitivity of the prApe1 propeptide can therefore serve as a useful tool to determine the completion status of double-membrane Cvt vesicles/autophagosomes in the presence of exogenously added proteinase. Here, we describe an assay that examines the proteinase protection of prApe1 for determining the completion of Cvt vesicles/autophagosomes. |
