(A) CBK1-GFP cdc15-2 (YMF3566) and CBK1-GFP TOR1-1 cdc15-2 (YMF3565) cells were grown in YPD and arrested in late anaphase by shifting the temperature to 37 °C before the addition of rapamycin for 20 min to both strains. In addition, DMSO was added to one half of CBK1-GFP cdc15-2. Untagged cdc15-2 (YMF3580) and TOR1-1 cdc15-2 cells (YMF3578) were used as controls. Cell extracts were prepared before the immunoprecipitation of Cbk1-GFP on ChromoTek GFP-Trap Magnetic beads. Isolated material was subjected to analysis by mass spectrometry. Numbers represent spectral average of key Cbk1 functional-related interactors in 3 independent replicates of the experiment. The average is calculated as the sum of all spectral counts for the 3 replicates divided by 3. We found Cbk1 interactions previously described: Mob2 [48] or components of the RAM pathway such as Tao3 [97], Sog2 [98], and Kic1 [99]. Besides, Cbk1 negative regulators Lre1 and Fir1 were also identified [5,43]. Besides, MS found Myo1, a component of the actomyosin ring [43]. Our results showed that Cbk1 interacts with the transcription factor Ace2 [47,48]. Moreover, Cbk1 was bound to Ssd1, an RNA-binding protein that represses the translation of cell wall remodelling proteins until Ssd1 is phosphorylated by Cbk1 [48,100]. Interestingly, TORC1 and Ssd1 have been described to collaborate to maintain cellular integrity [73]. We found interactions with paralogs serine/threonine kinases Kin1 and Kin2 [74,75]. Finally, we detected protein Yol036w of unknown function interacting with Cbk1 [40,74]. Details of other known Cbk1 interactors are shown in S2 Table. (B) CBK1-5FLAG ACE2-HA cdc15-2 (YMF3585) and control cells (YMF3587) (i), CBK1-5FLAG MOB2-9MYC cdc15-2 (YMF3838) and control cells (YMF3835) (ii), or CBK1-TAP SOG2-6HA cdc15-2 (YMF4235) and control cells (YMF4236) cells (iii) were grown in YPD and arrested in late anaphase by shifting to 37 °C before the addition of rapamycin for 20 min when indicated. Subsequently, protein extracts were prepared and immunoprecipitations of Cbk1 on FLAG-beads or IgG beads (as denoted) were performed before the detection of the indicated proteins by immunoblotting. Raw data for blots can be found in Supporting information (S7 Raw Images). (C) Components of TORC1 were found in the same mass spectrometry analysis as in A. Numbers represent spectral average of 3 independent replicates of the experiment.

pbio.3002263.s008.pdf

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