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Supporting_Information - updated – Supporting_Information.pdf – Table S1.xlsx – Table S2.xlsx – Table S3.xlsx, Peer reviewed

(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, Peer reviewed

(A) Orthologues of S. cerevisiae Sec3 in the indicated fungal species were identified by PSI-BLAST searches, and a CLUSTAL O multiple sequence alignment was performed. Conserved NDR/LATS phosphorylation consensus is marked with a red square following consensus in S. cerevisiae [47] and in S. pombe [54]. Putative Cbk1 phosphorylation sites were identified in Sec3 by scanning the sequence (S18, S32, S43, and S66). (B) To confirm that Sec3 was essential in the budding yeast strain used in this work, we deleted SEC3 in diploid cells (YMF4159). Spores that lack Sec3 were able to grow extremely deficiently, although they formed tiny colonies (i). To conditionally inactivate Sec3, we planned to follow the same strategy as we did for the study of Cbk1 mutants that is explained in detail in S2E Fig. We added the “auxin inducible degron” (“aid”) cassette [38] to the C-terminal end of SEC3. Then, control cells (ADH-TIR1; YJW15) and sec3-aid ADH-TIR1 cells (YMF4612) were grown at 24 °C on YPD medium before serial dilutions of 50,000, 5,000, 500, and 50 cells were plated on the indicated media and incubated for 3 days (ii). sec3-aid ADH-TIR1 cells were able to grow under restrictive conditions at 24 °C (ii), which showed that Sec3 depletion was not completely effective. This prevented us from the use of sec3-aid mutant, in combination with cdc15-2 to inactivate Cdc15 function at 37 °C, and test whether phosphomimetic version of Sec3 (Sec3-4E, S18E, S32E, S43E, and S66E) was able to rescue cell separation defects associated to cdc15-2 cells. (C) To be able to follow version of t-SNARE Sso1 that lacks N-terminal autoinhibition domain, we deleted DNA sequence that corresponds to amino acids 1–146. At the same time, we fused the tag 5xFLAG at the N-terminal of the truncated Sso1 to be able to follow the protein dynamics. As control, we tagged the N-terminal end of wt Sso1 with 5xFLAG. To determine whether 5xFLAG-Sso1 and 5xFLAG-Sso1Δ1–146 were functional, we combined 5xFLAG-SSO1 (i) or 5xFLAG-sso1Δ1–146 (ii) with sso2Δ. In both cases, harbouring 5xFLAG version of Sso1 and sso2Δ was lethal, which indicates that addition of 5xFLAG disturbed Sso1 function. Individual deletions of SSO1 and SSO2 are alive, but inactivation of both t-SNARE Sso proteins is lethal., pbio.3002263.s009.pdf, Peer reviewed

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