Additional file 1 of The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin

Additional file 1: Figure S1. Schematic representation of the thermal proteome profiling temperature range (TPP-TR) workflow. HeLa wt cells were treated with 100 nM prodigiosin or DMSO for 6 h. After the incubation, cells were harvested, washed and aliquots of the cell suspensions were exposed to short (3 min) treatments at different temperatures in the range between 36.5 °C and 67 °C. Cells were lysed and the non-denatured protein fraction was recovered after centrifugation. Quantitative protein analysis was performed by immunoblotting (CETSA) or MS (TPP). For MS, proteins underwent tryptic digest and the resulting peptides were labeled using TMT 10plex. The samples were combined such that prodigiosin treated and corresponding control samples belonging to the same temperature were analyzed within the same TMT set (similarly as described before for RTSA), allowing for studying not only thermal stability but also abundance effects upon prodigiosin treatment

Systematic analysis of ATG13 domain requirements for autophagy induction

<p>Macroautophagy/autophagy is an evolutionarily conserved cellular process whose induction is regulated by the ULK1 protein kinase complex. The subunit ATG13 functions as an adaptor protein by recruiting ULK1, RB1CC1 and ATG101 to a core ULK1 complex. Furthermore, ATG13 directly binds both phospholipids and members of the Atg8 family. The central involvement of ATG13 in complex formation makes it an attractive target for autophagy regulation. Here, we analyzed known interactions of ATG13 with proteins and lipids for their potential modulation of ULK1 complex formation and autophagy induction. Targeting the ATG101-ATG13 interaction showed the strongest autophagy-inhibitory effect, whereas the inhibition of binding to ULK1 or RB1CC1 had only minor effects, emphasizing that mutations interfering with ULK1 complex assembly do not necessarily result in a blockade of autophagy. Furthermore, inhibition of ATG13 binding to phospholipids or Atg8 proteins had only mild effects on autophagy. Generally, the observed phenotypes were more severe when autophagy was induced by MTORC1/2 inhibition compared to amino acid starvation. Collectively, these data establish the interaction between ATG13 and ATG101 as a promising target in disease-settings where the inhibition of autophagy is desired.</p