Introducing an H113A mutation into Atg10 Y73Q to reduce autophagic activity and understand the effect of Atg on autophagosome size and number
Open Access Senior Honors Thesis
Department or School
Steven K. Backues, Ph.D.
Hedeel Evans, Ph.D.
Deborah Heyl-Clegg, Ph.D.
Autophagy is an essential recycling process that occurs within eukaryotic cells, however, the individual functions of the current thirty-two known autophagic proteins are not yet entirely understood. At this time, it is known that the autophagic protein Atg7 works upstream of both Atg3 and Atg10. Atg3 is affected by Atg7 and allows for the attachment of Atg8 and the lipid PE which is part of the autophagosome membrane. The Atg10 pathway is also affected by Atg7 but attaches Atg12 to Atg5 which upregulates the function of Atg3 and ultimately Atg8 lipidation. These protein interactions are necessary for forming autophagosomes, large double membrane vesicles that carry cytoplasmic cargo to the lysosome or vacuole by way of autophagy. Previous research has documented that Atg7 affects both the size and the number of autophagosomes formed, while Atg8 affects primarily the size of the autophagosomes. It is our hypothesis that some step in either pathway might be affecting the number of autophagosomes or possibly both size and number like Atg7. Our lab is currently focusing on the Atg10 pathway, and therefore creating Atg10 mutants that show a partial loss of autophagic activity; comparing these to the wild type will allow us to analyze the differences in autophagosome size and number that result from the reduction of the Atg10 function. We created a double mutant (H131A and Y73Q) in Atg10 and used western blots of pre-Ape1 and mature-Ape1, a specific autophagic cargo, along with the Pho8Δ60 assay of bulk autophagy, to determine if it retains ~30-40% of autophagic activity, suitable for further testing and eventually TEM.
Lautenschlager, Konrad L., "Introducing an H113A mutation into Atg10 Y73Q to reduce autophagic activity and understand the effect of Atg on autophagosome size and number" (2022). Senior Honors Theses and Projects. 746.