title: GFP-Atg8 protease protection as a tool to monitor autophagosome biogenesis.
abstract: Perhaps the most complex step of macroautophagy is the formation of the double-membrane autophagosome. The majority of the autophagy-related (Atg) proteins are thought to participate in nucleation and expansion of the phagophore, and/or the completion of this compartment. Monitoring this part of the process is difficult, and typically involves electron microscopy analysis; however, unless three-dimensional tomography is performed, even this method cannot be used to easily determine if the phagophore is completely enclosed. Accordingly, a complementary approach is to examine the accessibility of sequestered cargo to exogenously added protease. This type of protease protection analysis has been used to monitor the formation of cytoplasm-to-vacuole targeting (Cvt) vesicles and autophagosomes by examining the protease sensitivity of precursor aminopeptidase I (prApe1). For determining the status of autophagosomes formed during nonselective autophagy, however, prApe1 is not the best marker protein. Here, we describe an alternative method for examining autophagosome completion using GFP-Atg8 as a marker for protease protection.
authors: Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.
authors: Usha
authors: U
authors: Nair
authors: Michael
authors: M
authors: Thumm
authors: Daniel J
authors: DJ
authors: Klionsky
authors: Roswitha
authors: R
authors: Krick
mesh_terms: D000626:Aminopeptidases / Q000378:metabolism*; D001343:Autophagy; D000071190:Autophagy-Related Protein 8 Family; D001681:Biological Assay / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D010588:Phagosomes / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: C113757:ATG8 protein, S cerevisiae; D000071190:Autophagy-Related Protein 8 Family; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D000626:Aminopeptidases