A phospho-regulated ensemble

acetyla- the molecular mechanisms that regulate multi-tasking for dynamic acetylation obscure. Here we identified a signal motif in the intrinsically disordered C-terminus of α -TAT1, which comprises three functional elements - nuclear export, nuclear import and cytosolic retention. Their balance is tuned via phosphorylation by serine- kinases to determine subcellular localization of α -TAT1. the phosphorylated form binds to 14-3-3 adapters and cytosol maximal substrate form is sequestered inside the nucleus, thus microtubule acetylation minimal. mutations have reported to this motif, the unique ensem- ble regulation of α -TAT1 localization may hint at a role of microtubule acetylation aberrant Kinase A (PKA) and Casein kinase 2 (CK2) and identify 14-3-3 proteins as binding partners of 60 α -TAT1. Our findings establish a novel role of the intrinsically disordered C-terminus in 61


Discussion: 218 219
One of the bottlenecks in elucidating the role of microtubule acetylation in biological 220 phenomena is the knowledge gap of how upstream molecular signaling pathways control α-221 TAT1 function to modulate microtubule acetylation. Our study demonstrates that intracellular 222 α-TAT1 localization is a dynamically regulated process, orchestrated by a balance of nuclear 223 export and import, which modulates microtubule acetylation levels (Fig. 5e). To our 224 knowledge, this is the first study to identify the molecular mechanisms that spatially regulate 225 α-TAT1. We have demonstrated a hitherto unknown role of the inherently disordered α-TAT1 226 C-terminus and identified novel interactions with 14-3-3 proteins and several kinases. TAK1 227 dependent phosphorylation of α-TAT1 Serine-237 has been reported to stimulate its catalytic 228 property 62 . In neurons, p27 kip1 directly binds to α-TAT1 and stabilizes it against proteasomal 229 degradation 24 , thus enhancing α-tubulin acetylation. Our observation that spatial 230 sequestration of α-TAT1 from microtubules modulates acetylation dynamics suggests a role of 231 the nucleus as a reservoir or sequestration chamber to control protein access of substrates. 232 Regulated spatial sequestration of biomolecules can control their action [63][64][65][66][67] and aberrant 233 localization of proteins have been reported in many diseases 68-71 . Our study further highlights 234 the role of spatial signaling processes in controlling protein function. 235 We have demonstrated active nuclear export of α-TAT1 by Exp1 through an NES rich 236 in hydrophobic residues, which was critical for efficient microtubule acetylation. In addition, we 237 have identified an NLS consistent with non-canonical class IV NLS 44 . Interestingly, position 7 238 of this NLS, which should not be an acidic residue, is occupied by Threonine-322. Since 239 phosphorylation of threonine can significantly increase its net negative charges, it is ideally 240 situated to act as an ON/OFF switch for the NLS. Although we have identified Threonine-322 241 to be the critical phospho-residue that inhibits nuclear import, Serine-315 appears to provide 242 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.23.310235 doi: bioRxiv preprint additional inhibition. The increased nuclear localization of ST/A mutant over T322A mutant 243 raises the possibility that S315 and T322 may aggregate signals from different signaling 244 pathways to fine-tune α-TAT1 localization. 245 Our data demonstrate that nuclear localization of α-TAT1 is inhibited by kinase action, 246 possibly on Threonine-322 and Serine-315. Specifically, our study shows a role of CDKs, PKA 247 and CK2 in coordinating spatial distribution of α-TAT1. Such phospho-regulation of α-TAT1 248 provides a possible mechanism for the changes in α-TAT1 localization and microtubule 249 acetylation observed at different stages of the cell cycle 72 . We identified Threonine-322 to be 250 a putative binding site for 14-3-3 proteins and demonstrated that α-TAT1 binds to 14-3-3-β 251 and 14-3-3-ζ proteins. 14-3-3s typically interact with phospho-serines or phospho-threonines 252 in intrinsically disordered regions and may mediate nuclear transport of proteins by masking 253 NES or NLS 73 . Furthermore, 14-3-3 proteins may significantly alter the structure of their 254 binding partners to align along their rigid α-helical backbone, to expose or hide critical binding 255 sites 74 . Comparable kinase-mediated regulation of nuclear export and nuclear import has 256 previously been reported in a few transcription regulators 75-78 . In particular, regulation of 257 Cdc25 localization by Checkpoint kinase1 (Chk1) mediated phosphorylation of and 258 subsequent recruitment of 14-3-3-β to an NLS-proximal phosphosite is virtually the same as 259 our proposed model (Fig. 5e) of α-TAT1 localization 78,79 , suggesting that such kinase-260 mediated balancing of nuclear export and import is a general strategy for protein localization. 261 α-TAT1 is unique in this aspect in that unlike the other proteins that are spatially regulated in 262 this manner, α-TAT1 has no known substrates in the nucleus and that its nuclear localization 263 appears to be primarily to sequester it from microtubules. Of course, it is possible that nuclear 264 import of α-TAT1 facilitates interactions with presently unidentified substrates located in the 265 nucleus. In a similar vein, 14-3-3 proteins and Exp1 are also acetylated 80,81 , and it is intriguing 266 to speculate that these might be substrates of α-TAT1. 267 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.23.310235 doi: bioRxiv preprint It is worthwhile to consider that a significant number of post-translational modifications 268 of α-TAT1 appear on its intrinsically disordered C-terminus ( Supplementary Fig. 7b). In conclusion, we propose a new model for regulation of microtubule acetylation 284 through spatial sequestration of α-TAT1 (Fig. 5e), which include three key aspects: presence 285 of an NES that facilitates Exp1 mediated nuclear export, presence of an NLS to mediate 286 nuclear import and finally, modulation of this nuclear import by kinases. Further investigation 287 into the role of specific kinases on α-TAT1 localization may yield a better understanding of its 288 function in cellular processes and pathologies and help identify new therapeutic targets. 289 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. subcloned into the pTriEx-4 vector (Novagen) using PCR and restriction digestion with 302 mVenus at the N terminus and α-TAT1 at the C terminus. H2B-mCherry and CFP-PKI 303 constructs were respectively subcloned into mCherry-C1 and mCer3-C1 vectors (Clontech). 304 GFP-αTAT1 construct was a gift from from Dr. Philippe Chavrier and Dr. Guillaume 305 Montagnac. HA-14-3-3 plasmids were a generous gift from Dr. Michael Yaffe. As indicated in 306 the results and figure legends, tags of compatible fluorescent proteins including Cerulean, 307 mVenus and mCherry were appended to facilitate detection. Unless specified otherwise, the 308 termini of tagging were positioned as in the orders they were written. Truncations of α-TAT1 309 were generated by PCR. Point mutations of α-TAT1 were generated using overlapping PCR. 310 The open reading frames of all DNA plasmids were verified by Sanger sequencing. 311 Sequence alignment: Protein sequence alignment was performed using Clustal-W 84 312 (https://www.ebi.ac.uk/Tools/msa/clustalo/). 313 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020.  Tubulin (Sigma Aldrich, catalog # T7451). Next day, the samples were washed thrice with cold 334 PBS and incubated with secondary antibodies (Invitrogen) for one hour at room temperature, 335 after which they were washed thrice with PBS and images were captured by microscopy. For 336 LMB treatment, HeLa cells were dosed with 100 nM LMB or equal volume of vehicle (EtOH), 337 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.23.310235 doi: bioRxiv preprint incubated for 4 hours, after which methanol fixation and immunostaining was performed as 338 described above. 339 Immunoprecipitation assays: HEK293T cells were transiently transfected with pEGFP-c1 340 (GFP-Ctl) or GFP-αTAT1 with HA-14-3-3β or HA-14-3-3ζ using the calcium phosphate 341 method. Cell lysates were prepared by scraping cells using 1X lysis buffer (10X recipe-50 342 mM Tris pH 7.5, triton 20%, NP40 10%, 2 M NaCl, mixed with cOmplete protease inhibitor (Andor), driven by NIS Elements software (Nikon). Time lapse imaging was performed at 15 361 min intervals for 10-15 hours. All live cell imaging was conducted at 37°C, 5% CO2 and 90% 362 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.23.310235 doi: bioRxiv preprint humidity with a stage top incubation system (Tokai Hit). Vitamin and phenol red-free media 363 (US Biological) supplemented with 2% fetal bovine serum were used in imaging to reduce 364 background and photobleaching. Inhibitors and vehicles were present in the imaging media 365 during imaging. All image processing and analyses were performed using Metamorph 366 (Molecular Devices, Sunnyvale, CA, USA) and FIJI software (NIH, Bethesda, MD, USA). 367 For categorical analysis of mVenus-α-TAT1 localization, images were visually 368 inspected and classified as displaying either cytosolic, diffused, or nuclear localization of 369 mVenus fluorescence signal. For ratiometric analysis, the ratio of the fluorescence intensity 370 from region of interest (≈ 10 µm diameter) in the nucleus to that in a perinuclear area was 371 used to minimize any volumetric artifacts (Fig. 1c). To determine the baseline Nuc/cyto ratio 372 for cytosolic (<0.8) and nuclear (>1.2), we visually identified WT mVenus-α-TAT1 cells, with or 373 without LMB treatment, that showed distinctly cytosolic or nuclear localization and used the 374  Supplementary Table T1. 399 Data availability: All relevant data and source codes are included. Plasmid constructs will be 400 available through Addgene. 401 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.23.310235 doi: bioRxiv preprint

Acknowledgements 402
We thank Allen Kim for discussions that led to initiation of this project and Amy F. Peterson for 403 help with the kinase inhibitor assays. We thank Yuta Nihongaki and Helen D. Wu for 404 constructive discussions, as well as Robert DeRose for proofreading. This

Competing interests 418
The authors declare no competing interests. 419 420 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.23.310235 doi: bioRxiv preprint      (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.23.310235 doi: bioRxiv preprint 52. Madeira, F. et al. 14-3-3-Pred: improved methods to predict 14-3-3-binding 594 phosphopeptides. Bioinforma. Oxf. Engl. 31, 2276-2283 (2015). (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.23.310235 doi: bioRxiv preprint Supplementary Figure S1. αTAT1 C-terminus is disordered. a) amino acid sequence of αTAT1 isoform 7 used as the query, the putative NES and NLS are in bold, b) IUPred2 (and ANCHOR2) and c) PrDOS results suggesting that αTAT1 C-terminus is disordered. The threshold to be considered disordered is 0.5 for both.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Figure S2. NetNES prediction for NES in αTAT1 identifying the putative NES, the predicted NES from NetNES optimized algorithm (pink) and that from Hidden Markov Model (blue) are shown with the hydrophobic residues indicated in bold.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020.  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. Diffused Nuclear . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.23.310235 doi: bioRxiv preprint Supplementary Figure S10. Normal probability plot of nuclear/cytosolic ratio of mVenus-α-TAT1 expressed in HeLa cells.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted September 23, 2020. ; https://doi.org/10.1101/2020.09.23.310235 doi: bioRxiv preprint