Fig 2: Atg1/Ulk1-mediated phosphorylation of Hsp90 is essential for autophagy induction(A) Yeast expressing yHsp90-His6-WT, S25A, or S25E was transformed with GFP-Atg8. Cells were grown to mid-log phase and treated with rapamycin (200 ng/mL) for the indicated times. Induction of autophagy was evaluated by immunoblotting for GFP to detect cleavage of Atg8. a-Tubulin was used as loading control.(B) Yeast expressing yHsp90-His6-WT, S25A, or S25E and GFP-Atg8 was grown to mid-log phase and treated with rapamycin (200 ng/mL) for 2 h. Cells were stained with 8 mM vacuole marker FM4–64 (red) for 30 min prior to imaging. Induction of autophagy was evaluated by visualizing localization of GFP-Atg8 to the vacuole. Scale bar is 5 µm.(C) ATG1 was knocked out of yeast expressing yHsp90-His6-WT, S25A, or S25E and GFP-Atg8. Cells were grown to mid-log phase and treated with rapamycin (200 ng/mL) for the indicated times. Induction of autophagy was evaluated by immunoblotting for GFP to detect cleavage of Atg8. a-Tubulin was used as loading control.(D) Yeast with ATG1 KO expressing yHsp90-His6-WT, S25A, or S25E and GFP-Atg8 was treated with rapamycin (200 ng/mL) for 2 h. Cells were stained with 8 mM vacuole marker FM4–64 (red) for 30 min prior to imaging. Induction of autophagy was evaluated by visualizing localization of GFP-Atg8 to the vacuole. Scale bar is 5 µm.(E) Yeast expressing yHsp90-His6-WT, S25A, or S25E and GFP-Atg17 was grown to mid-log phase and treated with rapamycin (200 ng/mL) for 2 h. Yeast with ATG1 KO expressing yHsp90-His6-WT was used as a control. Cells were stained with 8 mM vacuole marker FM4–64 (red) for 30 min prior to imaging. Pre-autophagosomal structure formation was evaluated by visualizing localization of GFP-Atg17 to the vacuole. Scale bar is 5 µm.(F) Pho8?60 gene was transformed into yeast expressing yHsp90-His6-WT or the phosphomutant S25A or S25E. Cells were grown to mid-log phase and left untreated (-) or treated with 200 ng/mL rapamycin (rapa) for 4 h. ALP activity is presented as emission per the amount of protein in the reaction (mg) and the reaction time (mins). Data are presented as mean ± standard deviation derived from three independent replicates. Tukey’s multiple comparisons test was used to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.(G) HEK293 cells were transfected with hHsp90a-FLAG-WT, S39A, S39E, or EV as a control. Induction of autophagy was determined by immunoblotting for LC3B. GAPDH was used as a loading control.(H) HEK293 cells transiently expressing hHsp90a-FLAG-WT, S39A, S39E, or EV was treated with Bafilomycin A (100 nM) for 16 h. Induction of autophagy was determined by immunoblotting for LC3B. GAPDH was used as a loading control.

Fig 3: Ulk1-mediated phosphorylation of a conserved serine in amino domain of Hsp90(A) Schematic representation of yHsp90 domain architecture. A PreScission Protease cleavage motif was inserted between the N-terminal domain and the charged linker. Amino acid residues within the yHsp90 N-terminal domain fitting the consensus phosphorylated (phos)-(Ser/Thr) Phe motif are colored red, with the surrounding motif highlighted yellow.(B) WT-yHsp90-His6 or indicated yHsp90 mutants were expressed as the sole copy of Hsp90 in yeast. yHsp90-His6 was isolated by Ni-NTA pulldown. The N-domain was further isolated by cleavage with PreScission Protease, and phosphorylation of N-yHsp90-His6 was determined by immunoblotting with an anti-Phos-(Ser/Thr) Phe antibody.(C) yHsp90-His6-WT or S25A was expressed in yeast with ATG1 expressed (WT) or ATG1 knocked out (?atg1). yHsp90-His6 was isolated by Ni-NTA pull-down, and phosphorylation was evaluated by immunoblotting with antibodies against phos-Thr, phos-Ser, or phos-(Ser/Thr) Phe.(D) DLD-1 cells stably expressing WT ULK1 or kinase-dead ULK1 (K46I) were transiently transfected with hHsp90a-FLAG-WT or S39A. hHsp90-FLAG was isolated by immunoprecipitation (IP), and levels of phos-(Ser/Thr) Phe and phos-Ser were determined by western blot.(E) Recombinant hHsp90alpha-WT or S39A was incubated with Ulk1 and with or without ATP. hHsp90alpha was isolated by Ni-NTS pulldown, and levels of phos-(Ser/Thr) Phe were determined by immunoblot.

Fig 4: Phosphorylation of S25-Hsp90 is essential for Atg1/Ulk1 activity(A) HEK293 cells were transfected with hHsp90a-FLAG-WT, S39A, S39E, or EV as a control. Immunofluorescence staining was used to detect total hHsp90a-FLAG protein (green). Duolink proximity ligation assay was used to detect hHsp90a-FLAG:Ulk1 complexes (red). DAPI was used for nuclear staining. Scale bar is 10 µm.(B) hHsp90a-FLAG-WT, S39A, or S39E was transiently expressed and isolated from HEK293 cells. coIP of endogenous Ulk1 was evaluated by immunoblot. EV was used as a control. GAPDH was used as a loading control.(C) Yeast expressing yHsp90-His6-WT, S25A, or S25E in the ?atg1 strain was transformed with Atg1-FLAG. Cells were grown to mid-log phase and left untreated (-) or treated with 200 ng/mL rapamycin (+) for 20 min. Atg1-FLAG was isolated by IP, and Atg1 phosphorylation was evaluated by immunoblot for phos-Ser and phos-Thr. coIP of yHsp90-His was determined by immunoblot. a-Tubulin was used as a loading control.(D) LiP-MS was performed on Ulk1:hHsp90a and Ulk1:hHsp90a+ATP to identify regions of altered protease sensitivity. Ribbon structure of the Ulk1 kinase domain (AlphaFold-O75385) with the conformotypic peptide (F118-L129) is highlighted magenta (left). The proximity of the conformotypic peptide to functionally important regions of Ulk1 was visualized using Chimera Chimera v.1.14 (UCSF). Conformotypic peptide: magenta; activation loop: yellow; catalytic loop: red; PTM (phos-T180, acK162): green.(E) Yeast expressing yHsp90-His6-WT in the ?atg1 strain was transformed with Atg1-FLAG-WT or conformotypic peptide mutants. Cells were grown to mid-log phase and left untreated (-) or treated with 200 ng/mL rapamycin (+) for 20 min. Atg1-FLAG was isolated by IP, and Atg1 phosphorylation was evaluated by immunoblot for phos-Ser and phos-Thr. coIP of yHsp90-His was determined by immunoblot. a-Tubulin was used as a loading control.