Fluorescence microscopy-based assays for monitoring yeast Atg protein trafficking.
For several years, the yeast Saccharomyces cerevisiae has been the leading model organism for the study of autophagy. The amenability of this unicellular eukaryote to genetic and biochemical approaches has allowed the isolation and characterization of most of the genes specifically involved in autophagy, which are known as ATG (Reggiori, 2006; Reggiori and Klionsky, 2005). These pioneering studies have been of crucial relevance because most of the yeast ATG genes possess orthologs in all eukaryotic organisms. The experimental advantages, all the available reagents, and the established assays still maintain yeast in a prominent position in the study of autophagy and autophagy-related pathways. In this chapter, we describe fluorescent protein-based methodologies that permit one to readily assay the functionality of the autophagic pathway and to assess the trafficking of one of the key protein of this degradative process, Atg9.
Shintani
Takahiro
T
Laboratory of Bioindustrial Genomics, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
Reggiori
Fulvio
F
D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism; D001343:Autophagy / Q000502:physiology*; D000071183:Autophagy-Related Proteins; D001681:Biological Assay / Q000379:methods*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
C434065:ATG9 protein, S cerevisiae; D000071183:Autophagy-Related Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000626:Aminopeptidases; C019689:APE1 protein, S cerevisiae