bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 available under aCC-BY-NC-ND 4.0 International license.

1 Time-resolved proteomic profiling of the ciliary Hedgehog

2 response reveals that GPR161 and PKA undergo regulated co-

3 exit from cilia 4 5 Elena A. May1,#, Marian Kalocsay2,3,#, Inès Galtier D’Auriac4, Steven P. Gygi3, Maxence V. 6 Nachury4,* and David U. Mick1,5,* 7 8 1- Center of Human and Molecular Biology (ZHMB), Saarland University School of Medicine, 9 Homburg, Germany 10 2- Department of Systems Biology, Laboratory of Systems Pharmacology, Harvard Medical 11 School, Boston, Massachusetts, USA 12 3- Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA

13 4- Department of Ophthalmology, University of California San Francisco, CA 94143, USA 14 5- Center for Molecular Signaling (PZMS), Department of Medical Biochemistry and Molecular 15 Biology, Saarland University School of Medicine, Homburg, Germany 16 17 # authors contributed equally 18 19 * Correspondence: [email protected], [email protected] 20 21 Key words: 22 Primary Cilium, protein trafficking, proximity labeling, proteomics, hedgehog signaling 23

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24 ABSTRACT 25 The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of 26 its protein, lipid and second messenger compositions. Here, we combine proximity labeling of cilia 27 with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary 28 proteome in response to Hedgehog. This approach correctly identifies the three factors known to 29 undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we 30 find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together 31 with the G protein-coupled receptor GPR161 in response to Hedgehog; and we propose that the 32 GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, 33 we identify the putative phosphatase Paladin as a cell type-specific regulator of Hedgehog signaling 34 that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary 35 proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling 36 malfunctions.

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37 INTRODUCTION 38 The primary cilium is a solitary, microtubule-based protrusion of the cell that organizes 39 developmental, sensory and homeostatic signaling pathways inside a functionally distinct 40 compartment. Cilia defects cause multi-system pathologies named ciliopathies with symptoms 41 including kidney cysts, retinal degeneration, obesity, brain malformations and skeletal anomalies 42 (Reiter and Leroux, 2017; Hildebrandt et al., 2011). The vast range of symptoms underscores the 43 broad physiological importance of cilium-based signaling. Our understanding of how cilia transduce 44 signals is based in large part on studies of the developmental morphogen Hedgehog (Hh) (Gigante and 45 Caspary, 2020; Anvarian et al., 2019; Kong et al., 2019). In vertebrates, Hh signaling is strictly 46 dependent on an intact primary cilium. The core Hh machinery comprises the Hh receptor Patched 47 (PTCH1), the G-protein coupled receptors (GPCR) GPR161 and Smoothened (SMO), and the GLI 48 transcription factors, all of which dynamically localize to primary cilia in response to Hh (Fig. 1A). 49 PTCH1 and GPR161, two molecules that restrain Hh pathway activation inside cilia in unstimulated 50 cells, undergo ciliary exit upon pathway stimulation while the central pathway activator SMO becomes 51 enriched inside cilia when activated. It has been proposed that PTCH1 pumps a lipidic activator of 52 SMO out of the ciliary membrane and that the PTCH1 lipid extruder activity is directly suppressed 53 upon liganding Hh. Ciliary exit of PTCH1 further reduces the inhibitory effect exerted by PTCH1 on 54 SMO. Downstream of SMO and GPR161 lies the cAMP-dependent protein kinase (PKA) which 55 phosphorylates GLI2 and GLI3 and commits them to processing into transcriptional repressors. It is 56 now believed that it is the PKA activity within cilia that is important for transduction of Hh signals 57 (Kong et al., 2019; Gigante and Caspary, 2020). SMO blocks PKA activity, either directly (Arveseth

58 et al., 2020) or via its Gαi-mediated inhibition of adenylyl cyclases (Riobo, 2014) and GPR161

59 increases PKA activity within cilia via its tonic activation of adenylyl cyclases though Gαs. By 60 triggering the ciliary exit of GPR161, the accumulation of activated SMO in cilia thus results in a 61 drastic decrease in ciliary PKA activity (Mukhopadhyay et al., 2013; Pusapati et al., 2018a), and a 62 switch in the processing of the transcription factors GLI2/3 from repressor to activator forms. The 63 dynamic redistribution of signaling molecules thus plays an integral part in the transduction of Hh 64 signals inside the cilium. Despite the recognized importance of dynamic ciliary localization in the Hh 65 response, the extent of ciliary proteome remodeling during Hh signaling remains unknown and several 66 of the key steps remain incompletely characterized. For example, the exact mechanism of how ciliary 67 SMO triggers the exit of GPR161 from cilia remains to be determined. 68 An unbiased, systematic description of the mammalian primary cilia proteome has proven 69 challenging because the isolation of mammalian cilia remains fraught with severe limitations (Ishikawa

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70 et al., 2012). To overcome this technical barrier, we previously established a proximity labeling-based 71 method using cilia-localized ascorbate peroxidase (cilia-APEX) (Mick et al., 2015). Cilia-targeted 72 APEX fusion proteins allow labeling of ciliary components with biotin-derivatives that are readily 73 enriched by streptavidin chromatography (Mick et al., 2015; Kohli et al., 2017). Proteomics of cilia 74 using cilia-APEX has contributed to our molecular understanding of Hh signaling (Mick et al., 2015), 75 of extracellular vesicles shedding from cilia (Nager et al., 2017) and of regulated ciliary GPCR 76 trafficking (Shinde et al., 2020). While APEX approaches in cilia have identified several ciliary 77 signaling proteins (Mick et al., 2015; Kohli et al., 2017), they failed to robustly detect signaling proteins 78 of high importance and low abundance, such as PTCH1, GPR161 and SMO. The scarcity of these 79 factors in cilia combined with the high hydrophobicity of these multi-pass membrane proteins makes 80 mass spectrometric analysis a considerable challenge. The limited sensitivity of cilia-APEX thus 81 hampers a systematic and quantitative assessment of the proteomic changes in cilia in response to 82 signals and other perturbations. 83 Here, we profile the changes of the ciliary proteome during Hh signaling in a systematic and 84 time-resolved manner by combining improvements in the cilia-APEX2 proximity labeling scheme with 85 state-of-the-art quantitative mass spectrometry using tandem-mass-tags (TMTs) (Paek et al., 2017). 86 These technical advances enabled us to uncover the extent of the ciliary proteome remodeling in 87 response to Hh ligand and gain novel mechanistic insights into Hh signaling. 88 89 RESULTS AND DISCUSSION 90 91 The cilia-APEX2 expression system limits perturbations of ciliary dynamics 92 To investigate why cilia-APEX had failed to identify ciliary membrane proteins of the Hh pathway, 93 we surveyed the ciliary accumulation of these Hh signaling molecules by immunofluorescence 94 microscopy in the inner medullary collecting duct (IMCD3) cell line stably expressing the cilia-APEX 95 transgene (Fig. 1B). Surprisingly, while endogenous GPR161 was readily detected in cilia of parental 96 IMCD3 cells, ciliary GPR161 was nearly undetectable in IMCD3-[cilia-APEX] cells (Figs. 1C and 97 1D). The relatively high level of cilia-APEX expression required to support efficient biotinylation may 98 have altered ciliary composition. Even though a weak promoter (pEF1α) was used to stably express 99 cilia-APEX and expression was limited by integration at a defined genomic locus, we previously found 100 that pEF1α-driven expression of a ciliary GPCR from the same locus produced nearly 50,000 101 molecules per cilium and led to a drastic lengthening of cilia (Ye et al., 2018). 102 We thus reduced expression levels of the transgene by switching to the truncated CMV(∆6) 103 promoter (Morita et al., 2012), a considerably weaker promoter than pEF1α (Ye et al., 2018). To

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104 maintain robust biotinylation levels, we leveraged APEX2, an APEX variant with increased turnover 105 rates (Lam et al., 2015). As for the original cilia-APEX, ciliary targeting was carried out by the N- 106 terminus of Nphp3 (Wright et al., 2011; Nakata et al., 2012), and the presence of a GFP moiety in both 107 cilia-APEX and cilia-APEX2 allowed us to compare their relative abundance (Fig. 1B). The total levels 108 of cilia-APEX2 were reduced nearly 5-fold compared to cilia-APEX in the respective cell lines (Figs. 109 1E and 1F) while the abundance of the ciliary protein IFT88 was not affected (Figs. 1E and S1A). 110 Congruently, the ciliary intensity of cilia-APEX2 was reduced about 5-fold compared to cilia-APEX 111 (Figs. 1G and 1H). Despite the drastic reduction in ciliary abundance of cilia-APEX2 compared to 112 cilia-APEX, ciliary biotinylation efficiency in the presence of the APEX substrates biotin tyramide and

113 H2O2 nearly doubled in cilia-APEX2 compared to cilia-APEX (Figs. 1G and 1I). Importantly, the 114 ciliary abundance of GPR161 was nearly indistinguishable between parental and cilia-APEX2 cells 115 (Figs. 1C and 1D). Furthermore, GPR161 was efficiently removed from cilia in response to Hh 116 pathway activation by Sonic Hedgehog (Shh) in the cilia-APEX2 cell line, confirming that the IMCD3- 117 [cilia-APEX2] cell line recapitulates the physiological ciliary dynamics of the Hh signaling molecules 118 in response to Hh pathway activation. Together, these results predict that cilia-APEX2 improves 119 sensitivity of ciliary proteomics while minimizing perturbations of ciliary protein dynamics. 120 121 Tandem mass tag analyses of cilia-APEX2 samples extend coverage of the ciliary 122 proteome 123 We next sought to compare the performance of cilia-APEX2 to that of cilia-APEX in quantitative 124 mass spectrometric analysis. After labeling, biotinylated proteins were isolated via streptavidin 125 chromatography and quantified by tandem mass tag labeling and mass-spectrometry. In addition to 126 proteins biotinylated by the APEX enzyme, we expected streptavidin enrichment of several 127 endogenously biotinylated proteins as well as proteins biotinylated by endogenous peroxidases during 128 the labeling reaction. To discriminate proteins directly labeled by cilia-APEX2 from other biotinylated 129 proteins, we leveraged several controls. First, we introduced a single amino acid substitution in the 130 myristoylation site of the Nphp3-based ciliary targeting signal to abolish cilia targeting (Wright et al., 131 2011; Nakata et al., 2012), thus establishing control-APEX2. Expressed from the same locus and 132 promoter as cilia-APEX2, control-APEX2 identifies the proteins labeled by the cilia-APEX2 molecules 133 that did not reach the cilium (e.g. biogenesis intermediates). Secondly, to further control for non-ciliary 134 proteins, in particular membrane proteins that may not be labeled by control-APEX2 (a soluble 135 protein), we genetically ablated cilia in the cilia-APEX2 cell line by disrupting the centriole distal 136 appendage protein CEP164 using CRISPR/Cas9-mediated genome editing (Fig. S1B). Basal bodies 137 cannot dock to the plasma membrane in the absence of CEP164, thus precluding cilium assembly

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138 without effecting other cellular processes (Daly et al., 2016; Tanos et al., 2013). Cep164-/- IMCD3- 139 [cilia-APEX2] cells lack cilia (Fig. S1C-D) while retaining expression of the identical fusion protein 140 that is used to label ciliary proteins in wild-type cells. 141 We conducted synchronous precursor selection MS/MS/MS (MS3) analyses of APEX-labeled 142 samples from WT cilia-APEX2, Cep164-/- cilia-APEX2, and control-APEX2 cell lines in triplicate after 143 streptavidin capture and tandem mass tag (TMT) labeling of tryptic peptides (Fig. 2A). The TMT 144 isobaric tagging approach enables precise and reproducible quantification of the relative protein 145 abundances in different samples (Liu et al., 2020; Paek et al., 2017; Li et al., 2020). Hierarchical 146 clustering of each protein’s relative abundance in the ten samples analyzed within one multiplex 147 experiment demonstrates the high reproducibility of the triplicate experiment (Fig. S1E). Proteins that 148 are highly enriched in the cilia-APEX2 data set compared to the controls form two clusters of candidate 149 ciliary proteins (Fig. S1E and Fig. 2B) while non-ciliary proteins fall into separate clusters (Fig. S1F). 150 We defined the cilia-APEX2 proteome via statistical analyses of the relative enrichment between the 151 cilia-APEX2 samples and the controls (Fig. 2C and 2D). To be included in the cilia-APEX2 proteome, 152 proteins had to fulfill four criteria (Figs. 2C-D, blue dots): greater than 2-fold enrichments in the cilia- 153 APEX2 samples over control-APEX2 samples and over the Cep164-/- cilia-APEX2 samples (TMT ratio 154 > 2.0), and statistically significant enrichments in the cilia-APEX2 samples vs. the control-APEX2 155 samples and the Cep164-/- cilia-APEX2 samples (p-value < 0.05). In addition, proteins that fulfilled 156 only 3 out of 4 criteria (Figs. 2C-D, black dots) were included if they were close to matching the fourth 157 criterion (TMT ratio > 1.5, or p-value < 0.1). This set of criteria resulted in the inclusion of 203 158 proteins (Table S1) and represents a compromise between inclusion of false positives and exclusion of 159 false negatives (Fig. 2C-D). It should be noted that these criteria select for proteins significantly 160 enriched in cilia and ciliary proteins that are found at similar levels inside and outside of cilia will be 161 excluded from the cilia-APEX2 proteome. 162 To extend the coverage of the cilia-APEX2 method, we leveraged two additional independent 163 experiments where the cilia-APEX2 Cep164-/- control was replaced by a no labeling control. We 164 grouped the resulting candidate cilia proteins into two tiers, depending upon whether they were 165 identified in all three datasets (Tier 1) or missing from one of the three datasets (Tier 2; Table S2). 166 Application of these criteria resulted in the inclusion of 179 proteins in tier 1 and 91 proteins in Tier 167 2. While cilia-APEX had identified 75% of the IFT motor subunits, 60% of the IFT subunits and none 168 of the BBSome subunits (Mick et al., 2015), the cilia-APEX2 proteome comprises nearly all subunits 169 of the IFT motors kinesin-2 and dynein 2, of the IFT complexes and of the BBSome. (Fig. 2E). Most 170 of the subunits that were not identified –e.g. BBS18 and LC8 – were less than 10 kDa and likely to be 171 missed by MS because of the small number of tryptic peptides produced (Fig. 2E). Unexpectedly, half

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172 of the currently known components of the transition zone (TZ) were identified as hits in in the cilia- 173 APEX2 proteome. The TZ acts as a diffusion barrier that functionally separates the cilium from the 174 rest of the cell and was initially considered to be a static structure of the ciliary base (Garcia-Gonzalo 175 and Reiter, 2017; Gonçalves and Pelletier, 2017). The notion of a static TZ was further reinforced by 176 the near absence of TZ components from prior ciliary APEX studies (Mick et al., 2015; Kohli et al., 177 2017). However, recent studies have revealed that, while some TZ components are static, other TZ 178 components dynamically cycle between the TZ and the cytoplasm or the ciliary shaft (Nachury and 179 Mick, 2019) It is particularly striking to note that TZ components with previously documented 180 dynamicity (e.g. Nphp4 and Cep290) were identified by cilia-APEX2 while TZ components that 181 remain statically associated with the TZ over the timescale of tens of minutes (e.g. MKS2 and 182 TMEM107) were missing from the cilia-APEX2 proteome. It is also conceivable that some TZ 183 components identified by cilia-APEX2 are positioned at the distal side of the TZ, and hence in close 184 proximity to the cilia-APEX2 enzyme to be labeled by biotinyl radicals. This latter hypothesis is 185 consistent with an extension of the TZ proteins NPHP3, INVS, NEK8 into the inversin compartment 186 that spreads from the TZ to the first few µm of the ciliary shaft. 187 Most importantly, cilia-APEX2 combined with TMT labeling enabled previously unachievable 188 identification of central cilia-enriched signaling components, including most Hh signaling components 189 known to localize to cilia (PTCH1, SMO, GPR161, KIFf7, SUFU and GLI3). The Hh transcription 190 factor GLI1 was not identified by cilia-APEX2, consistent with undetectable GLI1 expression in the 191 absence of Hh pathway stimulation and GLI2 could only be quantified in one (out of three) 192 experiments, possibly because of its low abundance in IMCD3 cells. 193 194 Time-resolved cilia-APEX2 proteomics reveals global alterations of the cilia proteome 195 in response to Hh stimulation 196 Encouraged by the detection of the core Hh signaling machinery by cilia-APEX2, we sought 197 to determine the global changes of the cilia proteome in response to Hh by subjecting cells exposed to 198 Sonic Hedgehog (Shh) for 0, 1, 4 or 24 h to the cilia-APEX2/TMT workflow (Fig. 3A). The duplicate 199 experimental repeats showed high reproducibility as judged by hierarchical cluster analysis of relative 200 protein abundances (Fig. S2A). Strikingly, comparison of the cilia-APEX2 proteomes at 0 and 24 h 201 post-Shh treatment revealed only a handful proteins with significantly changed abundance (TMT ratio 202 > 2-fold) (Fig. 3B). Out of the 272 quantified cilia proteins (Table S2), 267 did not experience significant 203 changes in abundance (TMT ratio < 2.0) after 24 h of Shh ligand addition. For example, the 204 normalized protein abundance of the cilia trafficking components IFT88 or BBS1 (components of the 205 IFT-B and BBSome protein complexes, respectively), or of other known cilia proteins, such as the

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206 inositol polyphosphate 5-phosphatase (INPP5E) or Polycystin-2 (PKD2) displayed no significant 207 change over the course of a 24 h treatment with Shh (Fig. 4A). The low variability in relative 208 abundances of >95% of the ciliary proteome between different time points highlights the robust and 209 reproducible quantitation enabled by cilia-APEX2/TMT. 210 Importantly, cilia-APEX2/TMT profiling detected changes in ciliary abundance of the three 211 Hh signaling components known to undergo signal-dependent redistribution in or out of cilia (Fig. 4B). 212 Levels of the Hh receptor PTCH1 and the GPCR GPR161 decreased while SMO increased within 4 213 h after pathway activation. The kinetics revealed by cilia-APEX2 closely matched the kinetics 214 previously defined by immunofluorescence microscopy (Rohatgi et al., 2007; Mukhopadhyay et al., 215 2013). While the transcription factors GLI2 and GLI3, the GLI chaperone SUFU and the GLI 216 regulator KIF7 become enriched at the tip of cilia in response to Hh (Wen et al., 2010; Tukachinsky 217 et al., 2010; Endoh-Yamagami et al., 2009; Liem et al., 2009), the cilia-APEX2 signals for GLI2, GLI3 218 and KIF7 increased by less than 20% and the cilia-APEX2 signal for SUFU did not change during the 219 time course of Shh treatment. We note that while GLI2 failed the stringent inclusion criteria for the 220 cilia-APEX2 proteome, cilia-APEX2/TMT successfully quantified peptides in the 24 h time course 221 experiment. These findings suggest that the total ciliary amounts of GLI2, GLI3, KIF7 and SUFU 222 may not change significantly in response to Hh signal and that these factors may undergo subciliary 223 re-distribution, i.e. mobilization of a broadly distributed ciliary pool to the tip. Similarly, while the 224 BBSome becomes enriched at the tip of cilia upon Hh pathway activation (Ye et al., 2018), cilia- 225 APEX2 signals of BBSome subunits do not change appreciably during the 24 h time course. 226 Since the ciliary changes of PTCH1, SMO and GPR161 were nearly complete after only 1h 227 of pathway activation, we sought to resolve the changes in proteome remodeling during the first 60 228 min after Shh addition (see Fig. S3). While housekeeping ciliary proteins remained largely unchanged, 229 the high temporal resolution and precise TMT-based quantitation of cilia-APEX2 profiling enabled a 230 refined characterization of the redistribution of Hh signaling components (Fig. 4C). The levels of the 231 Hh receptor PTCH1 in cilia started dropping 5 min after Shh addition and reached a minimum after 232 30 min. Meanwhile, ciliary levels of SMO steadily increased during the 60 min time course and until 233 the 4 h time point (Fig. 4B). The removal of GPR161 was preceded by an increase in ciliary β- 234 arrestin2 levels (Fig. 4C), consistent with the proposed role of β-arrestin2 in triggering signal 235 dependent exit of GPR161 from cilia (Pal et al., 2016). 236 To determine if any other proteins besides SMO, PTCH1 and GPR161 undergo changes in 237 ciliary abundance in response to Shh, we searched for proteins that co-clustered with SMO, PTCH1 238 or GPR161 during the 24h time course in a hierarchical two-way cluster analysis. Only one protein

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239 clustered closely with SMO (Fig. 5A), the putative phosphatase Paladin which displayed kinetics of 240 ciliary accumulation closely mirroring those of SMO (Fig. 4B). It should be noted that despite tight 241 clustering of PKD1 and GLI2, the PKD1/GLI2 cluster sits at a substantial distance from the 242 SMO/PALD1 mini-cluster (Fig. 5A). Similarly, hierarchical clustering revealed only one tight cluster 243 of proteins disappearing from cilia in response to Shh (Fig. 5B). This cluster contained GPR161, 244 PTCH1 and the PKA regulatory subunit Iα (PKA-RIα), all of which nearly reached their minimum 245 within the first hour of pathway induction (Fig. 4B). As observed in the 24 h time course, the ciliary 246 exit kinetics of GPR161 and PKA-RIα were nearly identical in the 60 min time course (Fig. 4C). 247 Thus, cilia-APEX2/TMT not only detects known changes in remodeling of the ciliary 248 proteome in response to Hh, the systematic nature of the TMT platform enables discovery of novel 249 dynamic factors, co-enriched and co-depleted with known components, via kinetic profiling. 250 251 Time-resolved cilia-APEX2/TMT illuminates the mechanisms of regulated GPR161 252 removal from cilia 253 Consistent with the current models of Hh signal transduction that predict a drop in PKA- 254 mediated phosphorylation of GLI3 inside cilia upon Hh pathway activation, biosensor-based 255 measurements showed that ciliary PKA activity decreases in response to Hh (Moore et al., 2016). Yet, 256 our understanding of how ciliary PKA activity becomes depressed upon Hh pathway activation

257 remains incomplete. On one hand, GPR161, a tonically active Gαs-coupled GPCR (GsPCR), is 258 thought to maintain high cAMP levels in cilia of unstimulated cells. Ciliary exit of GPR161 will thus

259 lead to a decrease of active Gαs in cilia and a corresponding decrease of ciliary cAMP levels. On the 260 other hand, the regulatory PKA subunit PKA-RIα, a stoichiometric inhibitor of the kinase-bearing 261 catalytic PKA subunit PKA-C (Taylor et al., 2012, 201), is found inside cilia (Mick et al., 2015; 262 Bachmann et al., 2016). PKA-RIα is thus thought to constitutively repress PKA activity inside cilia. 263 To validate the unexpected finding that PKA-RIα may exit cilia upon Hh pathway activation, we 264 established a stable IMCD3 cell line that expresses PKA-RIα fused to the fluorescent protein 265 mNeonGreen (NG; (Shaner et al., 2013)) and imaged NGPKA-RIα by fluorescence microscopy. While 266 unstimulated cells exhibited robust ciliary signals of NGPKA-RIα, addition of Shh triggered a decrease 267 of NGPKA-RIα ciliary fluorescence with kinetics that mirrored those measured by cilia-APEX2/TMT 268 profiling (Figs. 5C and 5D). To pinpoint the step in the pathway that triggers the removal of PKA-RIα 269 from cilia, we directly activated SMO via the SMO agonist SAG (Figs. S2B and S2C). The kinetics of

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270 NGPKA-RIα exit from cilia were nearly identical in cells treated with Shh or SAG and we conclude 271 that PKA-RIα exit from cilia lies downstream of SMO activation. 272 In agreement with our cluster analysis (Fig. 5B), the kinetics of NGPKA-RIα removal from cilia 273 upon Hh pathway activation were closely reminiscent of the exit kinetics of GPR161 (Mukhopadhyay 274 et al., 2013), which we confirmed by imaging of GPR161NG (Nager et al., 2017) (Figs. 5E, 5F, S2D). 275 The concomitant exit of GPR161 and PKA-RIα raises the possibility that PKA-RIα piggybacks onto 276 GPR161 to exit cilia. In support of this hypothesis, the cytoplasmic tail of GPR161 harbors an atypical 277 A kinase anchoring protein (AKAP) motif with exquisite and unprecedented specificity for PKA-RIα 278 (Bachmann et al., 2016). A major function of the 60 different AKAP is to recruit that the PKA 279 regulatory subunits at discrete cellular locations to direct the catalytic subunits to their substrates 280 (Torres-Quesada et al., 2017). We conclude that GPR161 and PKA-RIα form a stable complex that 281 represents a functional unit, most likely together with PKA-C. Meanwhile, the abundances of other 282 AKAPs quantified in the cilia-APEX2 dataset, e.g. AKAP11 and AKAP9, did not change appreciably 283 in response to Hh (Fig. S2E). 284 A remarkable feature of the C-terminal tail of GPR161 is that it encodes both an AKAP motif 285 for PKA-RIα and a PKA phosphorylation consensus site (Bachmann et al., 2016). In most studied 286 instances, AKAPs interact directly with the PKA substrates (Musheshe et al., 2018) and GPR161 may 287 further reduce the complexity of PKA recruitment to its substrate by encoding an AKAP motif within 288 its sequence rather than interact with a separate AKAP. Because phosphomimetic mutations of the 289 PKA site in the C-tail of GPR161 drastically reduce ciliary levels of GPR161 (Bachmann et al., 2016), 290 we hypothesized that PKA-RIα promotes phosphorylation of GPR161 by PKA in response to Hh 291 pathway activation and thus triggers ciliary exit of GPR161. To test this hypothesis, we assessed the 292 Hh-induced removal of GPR161 from cilia after siRNA-mediated depletion of PKA-RIα. While 293 control siRNA did not interfere with GPR161NG exit, GPR161NG failed to exit cilia in response to Hh 294 signal in PKA-RIα-depleted cells (Fig. 5G). It is thus conceivable that the retention of GPR161 in cilia 295 contributes to the previously reported defects in Hh pathway activation in cells depleted of PKA-RIα 296 (Evangelista et al., 2008). A major conundrum then lies in how Hh pathway activation may control the 297 PKA-RIα-dependent phosphorylation of the GPR161 C-tail. In the test tube, PKA regulatory subunits 298 inhibit the activity of the catalytic subunits until the regulatory subunits bind cAMP and release free 299 and active PKA-C. Here, recent findings that intermediate concentrations of cAMP promote PKA 300 activation without dissociation of catalytic from regulatory subunits (Smith et al., 2017) may shed light

301 on the regulation of GPR161 C-tail phosphorylation. While few measurements of [cAMP]cilia have

302 been published, one study found that [cAMP]cilia is about 4 µM in unstimulated cells (Moore et al.,

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303 2016), a concentration sufficient to trigger nearly complete PKA-C/PKA-R dissociation within cilia. 304 Under these circumstances, PKA-C will freely diffuse in the cilium and phosphorylate GLI2/3 and 305 other substrates (Fig. 5H, left). Because SMO entry into cilia is already detectable at the onset of 306 GPR161 exit (Fig. 4C), we consider the ciliary state where GPR161 and activated SMO co-exist inside

307 cilia. We propose that the activation of Gαi by SMO inside cilia will reduce [cAMP]cilia to a level where 308 an active PKA holoenzyme assembles on the C-tail of GPR161 and phosphorylates GPR161 (Fig. 5H,

309 center). This hypothesis of ciliary Gαi activation promoting phosphorylation of the C-tail of GPR161 310 and subsequent exit of GPR161 is supported by our previously published and yet to be explained

311 finding that activation of the ciliary GiPCR SSTR3 leads the exit of GPR161 from cilia (Ye et al., 312 2018). The ultimate exit of the GPR161/PKA-RIα/PKA-C complex (Fig. 5H, right) further amplifies

313 the effect of ciliary Gαi activation via SMO to fully depress [cAMP]cilia to a level where GLI2/3 no 314 longer become phosphorylated by PKA. Similar to Hh ligands dually inhibiting ciliary PTCH1 by 315 blocking its transporter activity and promoting its exit from cilia, our model proposes that PKA activity 316 in cilia is reduced via a two-pronged mechanism that lowers cAMP levels and removes the PKA 317 holoenzyme from cilia. Consistent with the observation that PKA-RIα is required for pathway 318 activation in response to Smoothened agonist (Evangelista et al., 2008), our model predicts that PKA- 319 C will remain in cilia of Hh-stimulated cells in the absence of PKA-RIα, leading to the continued 320 production of Gli3R. 321 This model of regulated GPR161 exit resolves another conundrum raised by [Pal 2016]. 322 Multiple groups have found that β-arrestin2 is required for exit of GPR161 from cilia subsequent to 323 entry of activated Smoothened into cilia. Consistent with the cilia-APEX2/TMT profile of β-arrestin2 324 in response to Shh (Fig. 4C), recent imaging studies found that the ciliary levels of β-arrestin2 rapidly 325 increase upon Hh pathway activation and reach a plateau at 20 min (Shinde et al., 2020). Interestingly, 326 β-arrestin2 is also recruited to cilia upon activation of the ciliary GPCR SSTR3 and β-arrestin2 is 327 required for removal of activated GPCRs from cilia (Green et al., 2015; Pal et al., 2016; Ye et al., 328 2018). Because β-arrestins are rapidly and stably recruited to activated GPCRs after they become 329 phosphorylated, these results suggest that β-arrestin2 recognizes phosphorylated GPR161 inside cilia 330 and instructs the ciliary export machinery to remove GPR161 from cilia. However, GPR161 was 331 shown to be tonically active in the absence of Hh pathway stimulation (Mukhopadhyay et al., 2013). 332 The model proposed in Fig. 5H solves this conundrum as it parsimoniously accounts for increased 333 recruitment of β-arrestin2 to GPR161 when the entry of SMO into cilia promotes PKA-mediated 334 phosphorylation of GPR161 C-tail. Our model also suggests that GPR161 may represent the first 335 instance of a GPCR that is not controlled by extracellular ligands but instead monitors intracellular 336 changes in second messenger concentrations. The GPR161/PKA-RIα module may thus act as a

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337 simple amplifier of the state of ciliary SMO activity. Considering the very recent finding that SMO 338 can directly regulate PKA-C (Arveseth et al., 2020), our model of regulated GPR161 exit may become 339 further refined as new details of cAMP regulation by SMO and GPR161 emerge. 340 While our model accounts for Hh pathway regulation by ciliary PKA-RIα, it is important to 341 consider the extra-ciliary functions of PKA-RIα when interpreting the drastic overproduction of GLI3R 342 in unstimulated Prkar1a-/- mouse embryonic fibroblasts (Jacob et al., 2011). PKA-RIα is the only one 343 of four PKA regulatory subunit essential for embryonic development and partial loss of PKA-RIα leads 344 to a wide range of disease states including cystic kidneys (Veugelers et al., 2004; Amieux et al., 2002; 345 Ye et al., 2017). Accordingly, Taylor and colleagues describe PKA-RIα as the ‘master regulator of 346 PKA signaling’ and conclude that PKA-RIα contributes the bulk of PKA activity restriction in cells 347 (Lu et al., 2019). Cytoplasmic PKA-C activity therefore becomes greatly elevated in the absence of 348 PKA-RIα, and GLI2/3 phosphorylation by PKA can likely take place outside of cilia when PKA-RIα 349 is missing. Congruent with our interpretation, overexpression of a PKA-C variant incapable of binding 350 to the regulatory subunits potently inhibits Hh signaling, presumably by phosphorylating GLI2/3 in 351 the cytoplasm (Iglesias-Bartolome et al., 2015). 352 353 PALD1 accumulates within cilia of selected cell types upon Hh stimulation 354 Cilia-APEX2/TMT profiling detected a steady increase in SMO and PALD1 ciliary signals 355 over the course of 60 min, and the 24 h time course indicates that SMO and PALD1 keep co- 356 accumulating in cilia until 4 h (Figs. 4B, 4C and 5A). To validate this result, we assessed the ciliary 357 accumulation of endogenous PALD1 by immunofluorescence microscopy (Fig. 6A). In agreement with 358 the hierarchical clustering analysis (Fig. 5A), the increase in ciliary PALD1 signals upon Shh 359 stimulation followed very similar kinetics to those of SMO (Fig. 6B). Given the striking Hh signal- 360 dependent co-localization SMO and PALD1 in cilia, we sought to determine the functional interaction 361 of PALD1 with the Hh pathway. Experimentally, activation of SMO by Smoothened agonist (SAG) 362 fully engages the Hh pathway and bypasses PTCH1 (see Fig. 1A). Given that endogenous PALD1 and 363 SMO accumulated efficiently in primary cilia in response to SAG (Figs. 6C-D), we conclude that ciliary 364 accumulation of PALD1 lies downstream of SMO. Considering the nearly identical kinetics of PALD1 365 and SMO ciliary accumulation, we hypothesized that PALD1 may piggyback on SMO entering cilia 366 via a physical interaction. To test this hypothesis, we leveraged cyclopamine, a SMO antagonist that 367 promotes ciliary accumulation of SMO while blocking Hh pathway accumulation (Wilson et al., 2009; 368 Wang et al., 2009; Rohatgi et al., 2009). While cyclopamine promoted ciliary accumulation of SMO 369 in IMCD3 cells, it did not increase the ciliary levels of PALD1 (Figs. 6E and 6F), indicating that ciliary 370 accumulation of SMO is not sufficient to drive PALD1 into cilia and that SMO must be activated for

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371 PALD1 to accumulate in cilia. To test whether PALD1 entry into cilia is sensitive to the reduction in

NG 372 [cAMP]cilia downstream of SMO, we resorted to the ciliary GiPCR somatostatin receptor 3 (SSTR3 ) 373 (Nager et al., 2017). Addition of somatostatin to IMCD3 cells stably expressing mNeonGreen-tagged

NG 374 SSTR3 (SSTR3 ) triggers activation of the GiPCR inside cilia and hallmarks of ciliary Gαi 375 activation (Ye et al., 2018). Strikingly, SSTR3 activation is sufficient to recruit PALD1 to primary cilia 376 (Figs. 6G-H), suggesting that, similarly to GPR161 exit from cilia, ciliary PALD1 accumulation 377 responds to drops in ciliary cAMP levels. 378 We next sought to determine whether PALD1 is generally integrated with Hh signaling, or 379 whether PALD1 performs a more specialized function related to Hh signaling. In addition to IMCD3, 380 PALD1 protein expression was detectable in NIH-3T3 cells, C2C12 myoblasts, MIN6 pancreatic ß 381 cells and human embryonic kidney (HEK) cells (Fig. 7A). Unlike some Hh pathway components such 382 as PTCH1 or GLI1 that are translational targets of the Hh pathway, PALD1 expression did not 383 appreciably change in response to Hh pathway stimulation. Unexpectedly, PALD1 protein expression 384 was not detectable in telomerase immortalized human retinal pigment epithelial (RPE1-hTERT) cells. 385 Since PALD1 was detected in HEK cells, the antibody does recognize the human protein. Because 386 RPE1-hTERT cells may not be capable of mounting a robust Hh response, we turned our attention 387 to cell types where the Hh response has been extensively validated. NIH-3T3 cells constitute a well- 388 accepted cell-based system for Hh signaling (Taipale et al., 2000). Surprisingly, activation of the Hh 389 pathway in 3T3 cells led to the accumulation of SMO in cilia but failed to promote ciliary entry of 390 PALD1 (Fig. 7B). Hedgehog signaling controls muscle differentiation (Hu et al., 2012) and the 391 requirement for primary cilia in myoblast proliferation can be recapitulated in cultured C2C12 cells 392 (Fu et al., 2014). While PALD1 was absent from unstimulated C2C12 cilia, PALD1 became enriched 393 in primary cilia in response to Hh signal (Figs. 7C and 7D). These results indicate that, while not 394 broadly generalizable, the association of PALD1 with Hedgehog signaling is not limited to IMCD3 395 cells. 396 Sequence analysis revealed several key features of PALD1 (Fig. 7E). PALD1 contains a glycine 397 residue at amino acid position 2 that is almost certainly myristoylated as PALD1[Gly2] scores highly 398 in all predictors of N-myristoylation (Bologna et al., 2004; Maurer-Stroh et al., 2002; Xie et al., 2016) 399 and expression of the N-terminus of PALD1 in a cell-free system produced a protein myristoylated at 400 Gly2 (Suzuki et al., 2010). Importantly, PALD1 was recovered in affinity purification of the myristoyl 401 chaperone Unc119 (Wright et al., 2011). Myristoylation is a major determinant in the ciliary targeting 402 of a variety of proteins (e.g. NPHP3, Cystin) and UNC119 mediates the entry of these myristoylated 403 proteins into cilia (Stephen and Ismail, 2016). It is thus conceivable that PALD1’s regulated targeting 404 to cilia involves the regulated unmasking of its attached myristate. PALD1 belongs to the large protein

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405 tyrosine phosphatase (PTP) superfamily (Chen et al., 2017). While PALD1 contains two PTP active 406 site motifs (CXXXXXR), PALD1 is missing the 280 amino acid extended catalytic domain and 407 represents a divergent PTP family member. In agreement with these observations, in vitro assays have 408 thus far failed to detect protein phosphatase activity for PALD1 (Huang et al., 2009). In the absence of 409 demonstrated phosphatase activity or identified substrate, PALD1 has been proposed to be an anti- 410 phosphatase or a pseudophosphatase (Roffers-Agarwal et al., 2012). 411 PALD1 is conserved among all clades of eukaryotic life, from protists to mammals (Fig. 7F). 412 Interestingly, a clustering analysis of pathways based on shared inferred ancestry (Li et al., 2014) 413 grouped PALD1 with the cilia-associated proteins CFAP54 (cilia- and flagella-associated protein 54 414 (McKenzie et al., 2015, 54)), the tubulin detyrosinases vasohibin-1 and -2 (Nieuwenhuis et al., 2017; 415 Aillaud et al., 2017), and the IFT-B subunit IFT25. Although PALD1 is not restricted to ciliated 416 organisms, it displays some co-conservation with cilia (Fig. 7F) and the overlap in phylogenetic co- 417 conservation is most striking with IFT25. Together with the IFT27 protein, IFT25 forms a stable 418 subcomplex of IFT-B that functions as a regulator of BBSome function and thus participates in the 419 regulated removal of membrane proteins from cilia (Bhogaraju et al., 2011; Liew et al., 2014; Eguether 420 et al., 2014). Metazoa that have lost IFT25/27 either lack a Hh response (C. elegans) or transduce 421 Hedgehog signals independently of cilia (Drosophila) (Fig. 7F). Meanwhile, organisms such as 422 Chlamydomonas that have retained IFT25/27 but that do not transduce Hh signals require this IFT-B 423 subcomplex for BBSome export from cilia (Dong et al., 2017). The shared phylogenetic pattern of 424 association of IFT25/27 and PALD1 with Hh signaling and cilia suggests that PALD1 performs a 425 function in cilia that supports efficient Hh signaling while not being absolutely required for either cilia 426 assembly or Hh signaling. In agreement with the tissue-specific expression of PALD1 (Huang et al., 427 2009) and the integration of PALD1 with Hh signaling in a subset of cell lines, we propose that PALD1 428 fulfills a cell-type specific function in multicellular organisms. 429 Besides the aforementioned cilia-related proteins, the CLIME analysis revealed two 430 phosphatases co-conserved with PALD1: Phospho1, an ancient phosphoethanolamine and 431 phosphocholine phosphatase with roles in bone mineralization in vertebrates and Phospho2, a 432 pyridoxal-5-phosphate phosphatase related to Phospho1. Phospho1/2 belong to the haloacid 433 dehalogenase phosphatase superfamily and bear no resemblance to the PTP family or PALD1. The 434 co-retention of Phospho1, Phospho2 and PALD1 in a variety of organism is remarkable as the only 435 common thread between these proteins is the release of inorganic phosphate. In this context, probing 436 the genetic interactions between PALD1, Phospho1 and Phospho2 may reveal an unexpected 437 functional relationship between these proteins.

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438 PALD1-deficient myoblasts exhibit increased levels of insulin receptor –a protein previously 439 detected in primary cilia (Gerdes et al., 2014)– and PALD1 overexpression reduces insulin receptor 440 levels as well as its signal-dependent phosphorylation (Huang et al., 2009). As we observe PALD1 441 accumulation in myoblast primary cilia in response to Shh but also after stimulation of the ciliary 442 GPCR SSTR3, it is tempting to speculate that PALD1 may assist IFT25/27 in the ciliary exit of ciliary 443 receptors and the desensitization of associated signaling pathways. 444 445 PALD1 is an attenuator of Hh signaling in IMCD3 cells 446 To assess a potential role for PALD1 in Hh signaling, we generated PALD1-deficient IMCD3 447 cells bearing biallelic frameshift mutations by CRISPR/Cas9-mediated genome editing (Fig. S4). The 448 morphology of cilia, as assessed by acetylated tubulin or IFT88 staining, was indistinguishable between 449 WT and Pald1-/- cells (Fig. 8A). Immunoblotting confirmed the absence of PALD1 from Pald1-/- cells, 450 while the levels of ciliary proteins PTCH1 and IFT88 were unaffected (Fig. 8B). Consistent with a 451 functional relationship between PALD1 and the Hh pathway, we detected a significant reduction in 452 the levels of GLI3 repressor (GLI3R) as well as an increase in full-length GLI3 (GLI3FL) in unstimulated 453 Pald1-/- cells compared to WT cells (Fig. 8B). Further underscoring the similarities in phylogenetic 454 distribution between PALD1 and IFT25/27, the effect of PALD1 deletion on GLI3 processing is 455 reminiscent of the Hh defect described in IFT27-deficient mouse skin (Yang et al., 2015). The ratio 456 between the full-length and repressor forms of GLI3 (GLI3FL/GLI3R) has been used as a proxy for Hh 457 pathway activation, as this ratio increases severalfold upon Hh pathway activation (Wang et al., 2000; 458 Wen et al., 2010). In wild-type IMCD3 cells, the GLI3FL/GLI3R ratio increased 2-fold after Hh 459 addition (Fig. 8C). Meanwhile, unstimulated Pald1-/- IMCD3 cells exhibit a similar GLI3FL/GLI3R 460 ratio as Shh-treated wild-type cells, suggesting that PALD1 restricts Hh pathway activation in the 461 absence of Hh ligand. The GLI3FL/GLI3R ratio further increased after Hh stimulation of Pald1-/- cells, 462 indicating that PALD1-deficient cells still remained Hedgehog-responsive (Fig. 8C). 463 To investigate the source of spontaneous pathway activation in PALD1-deficient IMCD3 cells, 464 we assessed the cilia localization of SMO and GPR161 in response to Hh pathway stimulation. As in 465 WT cells, SMO was not detected in resting Pald1-/- cilia and accumulated normally in response to 466 pathway activation (Figs. 8D and 8E). Meanwhile, GPR161 levels were reduced in cilia of unstimulated 467 PALD1-deficient IMCD3 cells compared to unstimulated WT cells (Figs. 8D and 8F), consistent with 468 a mild spontaneous activation of the Hh pathway. In agreement with the observed increase in the 469 GLI3FL/GLI3R ratios, GPR161 levels in PALD1-deficient cilia decreased further after Hh pathway 470 activation. In conclusion, PALD1-deficient IMCD3 cells do respond to Hh stimulation but 471 spontaneously activate the pathway in IMCD3 cells. Hence, unlike for negative regulators, the Hh

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472 pathway is not strictly dependent on PALD1 function, which appears to attenuate Hh signals in certain 473 cell types to finetune cellular responses during tissue patterning, as proposed for GPR161 (Pusapati et 474 al., 2018a). 475 Numerous genetic screens identified a large number of components required for Hh signaling, 476 including proteins required for protein trafficking and maintenance of primary cilia (Jacob et al., 2011; 477 Breslow et al., 2018; Pusapati et al., 2018b; Kim et al., 2010; Roosing et al., 2015; Wheway et al., 478 2015). Here, we have identified PALD1 in our proteomic screen as a protein that accumulates in 479 primary cilia in response to Hh signal, similar to SMO. Two aspects might explain why PALD1 had 480 not been previously associated with Hh signaling: i) PALD1 is dispensable for Hh signal transmission 481 in several tissues, as PALD1-deficient IMCD3 cells mount a partial response to Hh (Fig. 8) and Pald1- 482 /- mice only show a mild phenotype (German Mouse Clinic Consortium et al., 2017). ii) PALD1 is a 483 cell-type specific factor that accumulates in primary cilia of select cell types in response to Hh signal. 484 The absence of signal-dependent accumulation of PALD1 in NIH-3T3 cells suggests that PALD1 may 485 not participate in the Hh response in these cells, thus providing a possible explanation for why the 486 functional genomics screen for Hh signaling conducted in 3T3 cells did not identify PALD1 as a 487 regulator of Hh signaling (Breslow et al., 2018; Pusapati et al., 2018b). 488 Starting with the discovery that Hedgehog signaling require cilia for signal transduction in 489 vertebrates (Huangfu et al., 2003), the past 15 years revealed an elaborate choreography of signaling 490 factors entering and exiting cilia: one protein (SMO) that accumulates in cilia upon Hh pathway 491 activation, two proteins (PTCH1 and GPR161) that undergo Hh-dependent exit from cilia and four 492 proteins (GLI2, GLI3, SUFU and KIF7) that accumulate at the tip of cilia in Hh-stimulated cells. 493 Because these findings relied on imaging the core Hh signaling components defined by Drosophila 494 genetics, and since Drosophila transduce Hedgehog signals independently of cilia, one was left to wonder 495 how many proteins redistributing in response to Hh were left to discover. The cilia-APEX2/TMT 496 proteomics workflow addresses this question by enabling a systematic quantitation of individual 497 signaling molecules in cilia in a time-resolved manner. While the discovery of novel ‘tipping’ proteins 498 awaits the development of CiliaTip-APEX2, our analysis of the cilia proteome remodeling during Hh 499 signaling correctly identified the Hh-dependent redistribution of SMO, PTCH1 and GPR161 and 500 revealed two additional factors that undergo Hh-induced ciliary redistribution. PKA-RIα is likely to 501 be part of a universal ciliary cAMP gain modulator together with GPR161 that operates in all cells, 502 with roles possibly extending beyond Hh signaling. Interestingly, PALD1 is only connected to Hh 503 signaling in a subset of cells. This observation highlights a critical limitation of primary cilia proteomics 504 in mammalian cells: all available data sets are either for mouse kidney IMCD3 cells or highly 505 specialized sensory cell types (Ishikawa et al., 2012; Mick et al., 2015; Kohli et al., 2017; Mayer et al.,

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506 2009; Liu et al., 2007; Kuhlmann et al., 2014). Given that cilia-APEX2 only relies on targeting an 507 enzyme to cilia, it should be applicable to specific tissues and cell types, ideally from a live organism or 508 organoids and may reveal a currently unappreciated diversity in primary cilia composition. 509 510 511 512 513 514 ACKNOWLEDGEMENTS 515 We thank J. Goldstein and A. von der Malsburg for assistance in generating knockout IMCD3 cell 516 lines, E. Krause for fluorescence-activated cell sorting, K.V. Anderson, J.K. Chen, E. Ampofo, J.M. 517 Gerdes, C.G. Morrison, S. Mukhopadhyay, P.A. Beachy and M.P. Scott for reagents, P. Schuster for 518 experimental assistance, D.K. Breslow, S. Schuch and S.S. Taylor for helpful discussions, and M. von 519 Zastrow and M. Delling for comments on the manuscript. This work was supported by Deutsche 520 Forschungsgemeinschaft (DFG) funding to D.U.M. (SFB894/TPA-22) and NIH grants to S.P.G. 521 (GM67945) and M.V.N. (R01GM089933 and R21HD087126). This work was made possible, in part, 522 by NEI EY002162 - Core Grant for Vision Research and by the Research to Prevent Blindness 523 Unrestricted Grant (M.V.N.). 524 525 526 AUTHOR CONTRIBUTIONS 527 E.A.M. performed and analyzed most experiments. D.U.M. and M.V.N. conceptualized and wrote 528 the manuscript with support by all authors. I.G.A. conducted and analyzed the PKA-related 529 experiments. M.K. and D.U.M. established and performed the proximity labeling, TMT labeling and 530 mass spectrometry experiments and analyzed respective data together with S.P.G. 531 532 533 AUTHOR INFORMATION 534 The authors declare no competing financial interests. 535 536 537 538 539

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838 Wright, K.J., L.M. Baye, A. Olivier-Mason, S. Mukhopadhyay, L. Sang, M. Kwong, W. Wang, P.R. 839 Pretorius, V.C. Sheffield, P. Sengupta, D.C. Slusarski, and P.K. Jackson. 2011. An ARL3- 840 UNC119-RP2 GTPase cycle targets myristoylated NPHP3 to the primary cilium. Genes Dev. 841 25:2347–2360. doi:10.1101/gad.173443.111.

842 Xie, Y., Y. Zheng, H. Li, X. Luo, Z. He, S. Cao, Y. Shi, Q. Zhao, Y. Xue, Z. Zuo, and J. Ren. 2016. 843 GPS-Lipid: a robust tool for the prediction of multiple lipid modification sites. Scientific Reports. 844 6:28249. doi:10.1038/srep28249.

845 Yang, N., L. Li, T. Eguether, J.P. Sundberg, G.J. Pazour, and J. Chen. 2015. Intraflagellar transport 846 27 is essential for hedgehog signaling but dispensable for ciliogenesis during hair follicle 847 morphogenesis. Development (Cambridge, England). doi:10.1242/dev.115261.

848 Ye, F., A.R. Nager, and M.V. Nachury. 2018. BBSome trains remove activated GPCRs from cilia by 849 enabling passage through the transition zone. J. Cell Biol. 217:1847–1868. 850 doi:10.1083/jcb.201709041.

851 Ye, H., X. Wang, M.M. Constans, C.R. Sussman, F.T. Chebib, M.V. Irazabal, W.F. Young, P.C. 852 Harris, L.S. Kirschner, and V.E. Torres. 2017. The regulatory 1α subunit of protein kinase A 853 modulates renal cystogenesis. Am. J. Physiol. Renal Physiol. 313:F677–F686. 854 doi:10.1152/ajprenal.00119.2017.

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25 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 A SAG GLI2/3A B cilia-APEX Myr made available under aCC-BY-NC-ND 4.0 International license. 2 200 211 488 501 751 pEF1α target NPHP3 GFP APEX SHH PTCH1 SMO PKA GLI2/3 genes cilia-APEX2 Myr 2 200 211 488 501 751 CYC GPR161 GLI2/3R pCMV(Δ6) NPHP3 GFP APEX2 C GPR161ARL13B D GFP-APEX DNA GFP-APEX ARL13B GPR161 20

15 – Shh

10 GPR161 (RFU) parental 5 Ciliar y + Shh 0 Shh– + – + – +

– Shh parental cilia-APEX cilia-APEX2

cilia-APEX E F cilia-APEX

+ Shh cilia-APEX2 cilia-APEX2 parental cilia-APEX 6 kDa 5 100 – GFP 4 – Shh 3 100 – Ift88 2

cilia-APEX2 1

40 – e l. GFP signals (RFU)

actin r + Shh

G biotin ARL13B H I GFP-APEX DNA GFP-APEX ARL13B biotin 15 20

FU ) 15 (RFU )

parental 10 10 bioti n 5 5 Cilia ry GFP(R Ciliar y

cilia-APEX 0 0

cilia-APEX cilia-APEX cilia-APEX2 cilia-APEX2 cilia-APEX2

May et al., Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

857 Figure 1. An IMCD3 cell line expressing cilia-APEX2 at low levels efficiently labels 858 proteins of the primary cilium without disrupting ciliary localization of Hh signaling 859 components. 860 (A) Schematic overview of the key Sonic Hedgehog signaling components. Positive and negative 861 regulators are indicated in green and red boxes, respectively. Pharmacological agents used in this study 862 are displayed in ovals. Smoothened agonist (SAG) activates the pathway, while cyclopamine (CYC) 863 inhibits the pathway at the level of SMO. 864 (B) Diagram of the cilia-APEX and cilia-APEX2 transgenes. Both contain a cilia-targeting signal based 865 on the N-terminus of NPHP3, which is myristoylated at a glycine residue at position 2 (Myr), followed 866 by a GFP moiety. Numbers indicate amino acid positions. cilia-APEX is expressed from an EF1a 867 promoter (pEF1a), cilia-APEX2 from a truncated CMV promoter (pCMV(∆6)) for reduced expression. 868 (C) Immunofluorescence of parental IMCD3 cells and of stable clones expressing cilia-APEX or cilia- 869 APEX2. Cells were serum-starved for 24 h in the presence or absence of Sonic Hedgehog-conditioned 870 medium (Shh), fixed and stained for GPR161 (white) and ARL13B (red) using specific antibodies. Cilia- 871 APEX and cilia-APEX2 were visualized via the intrinsic fluorescence of GFP (green). DNA was stained 872 with DAPI (blue). Representative micrographs are shown for each condition. 873 (D) Box plots showing the relative GPR161 fluorescence normalized to ARL13B in the primary cilium 874 of parental, cilia-APEX and cilia-APEX2 cell lines after Shh treatment as in (A). 50 cilia were analyzed 875 for each condition (n = 50). In these and all subsequent box plots, crosses indicate mean values, 876 whiskers indicate values within 1.5x interquartile range and dots represent outliers. 877 (E) Cell lysates of parental, cilia-APEX and cilia-APEX2 cell lines were resolved by SDS-PAGE and 878 analyzed by quantitative immunoblotting using indicated antibodies. APEX fusion proteins were 879 detected using anti-GFP antibodies. 880 (F) Dot plot showing the ratio of total GFP protein detected in the cilia-APEX relative to the cilia- 881 APEX2 cell line (n = 3). 882 (G) Parental, cilia-APEX and cilia-APEX2 IMCD3 cells were subjected to APEX labeling, fixed and 883 stained for ARL13B (red) and biotin (white). Cilia-APEX and cilia-APEX2 were visualized via the 884 intrinsic fluorescence of GFP (green). DNA was stained with DAPI (blue). Representative micrographs 885 are shown. 886 (H and I) Box plots showing background-subtracted intensities of GFP (I) and biotin (J) signals in the 887 primary cilium from images as in (E). 30 cilia were analyzed for each condition (n = 30). 888 Scale bars = 2 µm in all panels. 889

26 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 A made available under aCC-BY-NC-NDB 4.0 International license. 3 sets control -/- control- Cep164-/- 0.96 cilia-APEX2 cilia-APEX2 2.77 APEX2 cilia-APEX2

2 4.58 Cep164 O APEX2

2 6.39 control- cilia-APEX2 Seed cilia-APEX2 –72 h 8.19 cells 21 3 31 2 – H 31 2 set no. 10.0 Arl13b Cep104 24.7 Inpp5e Armc9 39.4 Ift46 54.1 Rita1 Cep41 68.8 –24 h Serum starvation Ift81 Ift74 83.4 abundance (in %) protein rel. Ift57 Tmem2 Stradb Arl3 Ift88 Ttc30b –30 min + biotin tyramide Efcab7 Lgals3 Ckap2 Cys1 Ift52 Ttc30a1 cilia Ttc26 Nphp3 cluster 1 –2 min + H2O2 – H2O2 Snap29 Sri Cspp1 Rpgr Dync2h1 Kiaa1430 Lca5 Traf3ip1 0 Quenching + lysis Wdr35

Sample preparation (APEX labeling) Ccdc66 Cluap1 Stk11 Ift172 Gtl3 Cc2d2a Ick Streptavidin chromatography Ift43 Mdm1 Capg Plin3 Bbs7 Fam149a Evc Bbs9 Tryptic digest Ist1 Anks6 Iqce Tbc1d32 Fam179b Nphp1 Ift22 Kif3b Isobaric TMT labeling (10plex) Mapre1 Lasp1 Ppp1r2 Pdap1 Cdv3 Cd81 Oscp1 Hn1l Pool labeled peptides Cep290 Wbp2 Anxa7 Rnf25 Igbp1 Dpysl2 Zc2hc1a cilia Kifap3 cluster 2 High pH fractionation Mapre3 Sufu Bbs1 Dync2li1

Sample processing for mass-spectrometry SPS MS3 quantitation

C enrichment over control-APEX2 E -7 TIER 1 HITS 10 TIER 2 HITS 10-6 NOT IDENTIFED 10-5 Hh signaling dynein-1b 10-4 PTCH1 GLI1 kinesin-2 -3 DYNC2H1 WDR60 p value p 10 BBSome SMO GLI2 DYNC2LI1 WDR34 SUFU GLI3 KIF3A -2 TXTEX1D2 DYNLI1 10 BBS1 BBS7 GPR161 KIF7 KIF3B 10-1 BBS2 BBS8 PKA-RIα KIFAP3 p=0.05 BBS4 BBS9 EvC zone 0 10 BBS5 BBIP10 EVC IQCE EVC2 EFCAB7 -5-4-3-2-1 0 1 2 3 4 5 IFT-B average fold change log 2 IFT20 IFT56/Ttc26 Transition zone (cilia-APEX2 / control-APEX2) IFT22 IFT57 MKS1 NPHP1 IFT25/Hspb11IFT70A/Ttc30a RPGRIL1L NPHP3 D enrichment over Cep164-/- IFT27 IFT70B/Ttc30b MKS2/TMEM216 NPHP4 10-7 IFT38/Cluap1 IFT74 MKS3/TMEM67 IQCB1 IFT46 IFT80 -6 TMEM17 INVS 10 IFT52 IFT81 TMEM107 NEK8 IFT54/Traf3ip1 IFT88 10-5 TMEM218 ANKS6 IFT172 TMEM231 ANKS3 10-4 TMEM237 ATXN0 IFT-A -3 B9D1 TCTN1

p value p 10 IFT43 IFT139/TTC21B B9D2 TCTN2 10-2 IFT121/WDR35 IFT140 CEP290 TCTN3 IFT122 IFT144/WDR19 -1 CC2D2A DZIP1l 10 p=0.05 FAM92A 100 -2 -1 0 1 2 3 4 5

average fold change log2 -/- (cilia-APEX2 / cilia-APEX2 Cep164 ) May et al., Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

890 Figure 2. Cilia-APEX2-based proteomics reveals the proteome of primary cilia with high 891 sensitivity. 892 (A) Schematic representation of experimental workflow. Cells were seeded 72 h before the APEX 893 labeling reaction. Cells were first grown in serum-rich medium for 48 h and then switched to serum- 894 reduced medium to induce ciliation. APEX labeling was conducted by pre-incubating cells with biotin 895 tyramide for 30 min and the labeling reaction was then initiated by addition of hydrogen peroxide

896 (H2O2) for 2 min. The APEX reaction was then quenched with cyanide, ascorbate and the antioxidant

897 Trolox and cells put on ice. In one control, H2O2 was omitted from the experimental scheme. This last 898 control was conducted in singlet as all 10 TMT channels were then occupied. After labeling and 899 quenching, cells were lysed and biotinylated proteins isolated by streptavidin chromatography. Bound 900 material was extensively washed and tryptic peptides released via on-bead digest. Peptides of each 901 individual sample were labeled with a unique tandem mass tag (TMT), all samples were pooled, and 902 peptides were fractionated by offline high pH reversed phase chromatography to generate 12 fractions, 903 which were analyzed using a synchronous precursor selection (SPS) MS3 method for mass 904 spectrometric identification and quantitation. 905 (B) Hierarchical two-way cluster analysis of a cilia-APEX2/TMT experiment conducted according to 906 the scheme outlined in (A). Clustering of the relative abundances of each identified protein (rows) in 907 the individual samples (columns) was performed based on Ward’s minimum variance method. The 908 relative abundance of a given protein was calculated by dividing the TMT signal to noise ratio in one 909 sample by the sum of TMT signal to noise ratios in all samples. Legend depicts color scheme for relative 910 abundances (in %). The clusters containing cilia proteins are shown (see Fig. S1E for full cluster 911 analysis). 912 (C and D) Volcano plots showing protein enrichment in cilia-APEX2 compared to control-APEX2 913 samples (C) or in cilia-APEX2 WT vs. Cep164-/- samples (D). Average enrichments of cilia-APEX2 914 samples versus the respective controls were plotted against the calculated p values (statistical 915 significance of enrichment calculated from unpaired student’s t-tests). Proteins fulfilling all 4 916 significance and enrichment criteria are represented by blue dots, proteins that met 3 criteria are 917 represented by black dots and proteins that failed 2 or more significance criteria are shown in grey (see 918 text for details). Known cilia proteins outside of significance criteria or quantified from one peptide are 919 highlighted in purple and salmon, respectively. See Table S1. 920 (E) Schematic of a primary cilium with indicated hallmark protein complexes, structures or pathways. 921 Boxes contain proteins of the respective entities identified as Tier1 (black) or Tier2 (grey) hits of the 922 cilia-APEX2 proteome. Proteins not identified are indicated by ‘ghost’ lettering. Proteins names are 923 shown and Gene symbols added for cases, in which gene symbols in our datasets differ from

27 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

924 conventional protein names. Note that Ttc30a1 and Ttc30a1 have been grouped into 925 IFT70A/Ttc30a. 926

28 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 A made available under aCC-BY-NC-ND 4.0 International license. 2 sets controls control- cilia-APEX2 cilia-APEX2 cilia-APEX2 cilia-APEX2 cilia-APEX2 APEX2 Seed –72 h cells

Serum starvation

–24 h + Shh – Shh

–4 h + Shh

–1 h + Shh

–0.5 h + biotin tyramide

–2 min + H2O2 – H2O2

0 Quenching + lysis

Sample processing for mass spectrometry

B changes after 24 h Shh addition 10-4

10-3

-2

value 10

p Gpr161 Prkar1a Pald1 10-1 Ptch1 Smo

100 -3 -2 -1 0 1 2

fold change log2 (24h Shh / no Shh)

May et al., Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

927 Figure 3. Experimental outline of time-resolved cilia-APEX2 proteomics after Sonic 928 stimulation. 929 (A) Schematic of experimental workflow for time-resolved cilia-APEX2 proteomics. Cilia-APEX2 (and 930 control-APEX2) expressing IMCD3 cells were seeded 72 h before the APEX labeling reaction. 24 h 931 before labeling, cells were deprived of serum, and Shh-conditioned medium was added for 24 h, 4 h 932 or 1 h before labeling (as indicated). ‘–Shh’ indicates addition of conditioned medium without Shh. 933 APEX labeling and sample preparation were performed as in Fig. 2A. 934 (B) Volcano plot showing significance vs. enrichment in 24 h Shh-treated compared to no Shh 935 samples. Hh signaling components known to change their ciliary localization are shown in blue, 936 proteins with newly identified changes are shown in orange. 937

29 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 A made available under aCC-BY-NC-ND 4.0 International license. Housekeeping ciliary proteins

1.2 IFT88 1.2 BBS1 1.2 INPP5E 1.2 PKD2 1.0 1.0 1.0 1.0 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2

maximum signal l. to maximum 0.0 0.0 0.0 0.0

re 0 4 8 12162024 0 4 8 12162024 0 4 8 12162024 0 4 8 12162024 time after Shh addition (h)

B Ciliary Hedgehog pathway proteins 1.2 1.2 1.2 1.0 PTCH1 1.0 PKA-RIα 1.0 GPR161 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0.0 0.0 0.0 0 4 8 12162024 0 4 8 12162024 0 4 8 12162024 1.2 1.2 SMO PALD1 1.2 1.0 1.0 1.0 KIF7 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0.0 0.0 0.0 maximum signal l. to maximum

re 0 4 8 12162024 0 4 8 12162024 0 4 8 12 16 20 24 1.2 1.4 SUFU 1.2 GLI2 1.2 GLI3 1.0 1.0 0.8 1.0 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0.0 0.0 0.0 0 4 8 12162024 0 4 8 12162024 0 4 8 12162024 time after Shh addition (h)

C 1.0 GPR161 1.0 PKA-RIα 1.0 1.0 0.8 0.8 0.8 IFT88 0.8 0.6 0.6 0.6 0.6 SUFU 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 0.0 0.0 0.0 0.0 0 15 30 45 60 0 15 30 45 60 0 15 30 45 60 0 15 30 45 60

SMO PALD1 1.0 PTCH1 1.0 1.0 1.0 0.8 0.8 0.8 0.8 e.tomaximumsignal rel. 0.6 0.6 β-arrestin2 0.6 0.6 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 0.0 0.0 0.0 0.0 0 15 30 45 60 0 15 30 45 60 0 15 30 45 60 0 15 30 45 60 time after Shh addition (h)

May et al., Figure 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

938 Figure 4. Time-resolved cilia-APEX2 proteomics reveals the extent of ciliary proteome 939 dynamics in response to Sonic Hedgehog. 940 (A) Relative protein abundances in primary cilia assessed by mass-spectrometric TMT quantitation 941 were plotted over time. Data points represent averages of duplicate measurements, error bars depict 942 individual values. Error bars smaller than indicated datapoint symbols have been omitted. For each 943 individual protein, the background signal in the control-APEX2 sample was set to 0 and the maximum 944 average signal across all time points was set to 1. 0 h, represents –Shh as in (Fig. 3A). 945 (B) Hh signaling components change their abundance in cilia in response to Shh addition. Relative 946 abundances of indicated proteins in primary cilia are plotted over time as in (A). 947 (C) Relative abundances of indicated proteins in primary cilia were assessed by cilia-APEX2/TMT 948 proteomics at indicated timepoints after Shh addition (see Fig. S3). Normalized intensities (relative to 949 ARL13B) were plotted over time. Maximum signals were set to 1, TMT signals in control samples set 950 to 0. t= 0 corresponds to the ‘–Shh’ sample. 951

30 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this preprint A (which was notset certified #1 by peer review)set #2 is the author/funder, who has grantedB bioRxiv a license to setdisplay #1 the preprint inset perpetuity. #2 It is made available under aCC-BY-NC-ND 4.0 International license. 2 2 O O 2 2 –H –Shh 1h Shh 4h Shh 24h Shh control-APEX –Shh 1h Shh 4h Shh 24h Shh –H –Shh 1h Shh 4h Shh 24h Shh control-APEX –Shh 1h Shh 4h Shh 24h Shh Cdc37l1 Evc2 Nfkbia Evc Pkd1 Nphp3 Gli2 Fam179b Smo Invs Pald1 Ptch1 Gphn Prkar1a Trp53bp1 Gpr161 Nek9 Dsc2 Ccdc181 rel. protein abundance (in %) Sema4b 0,31 2,25 4,19 6,13 8,06 10,0 19,6 29,1 38,7 48,3 57,8

C NG D PKA-RIα ac-tub DNA ac-tub NGPKA-RIα 400

(RFU) 300

–Shh 200 PKA-RIα NG 100 ary Cili ary 0

+Shh 0 1 2 4 24 time after Shh addition (h)

E NG F GPR161 ac-tub DNA ac-tub GPR161NG 600 (RFU)

NG 400 –Shh

200 y GPR161 Cili ar 0

+Shh 0 1 2 4 24 time after Shh addition (h)

G 600 H siControl – Hh + Hh 400 early late

GLI3 GLI3 GLI3 200 P Arr SMO SMO (RFU) GPR161 C GPR161 P NG 0 C RIα 0 1 2 4 RIα 24 Gαi Gαi 800 Gαs Gαs

y GPR161 siPKA-RIα [cAMP] ↑ [cAMP] [cAMP] ↓

Cili ar 600

400 Arr P C 200 RIα

0 0 1 2 4 24 time after Shh addition (h) May et al., Figure 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

952 Figure 5. Hierarchical cluster analysis reveals ciliary exit of PKA-RIα together with 953 GPR161 in response to Hedgehog signal. 954 (A) and (B) Hierarchical cluster analysis of the two sets of time-resolved cilia-APEX2 proteomics 955 experiments conducted following the scheme in Fig. 3A. (A) Magnified view of the SMO mini-cluster 956 (green) and neighboring branches. (B) Magnified view of the GPR161 mini-cluster (red) and 957 neighboring branches. Prkar1a is the gene name for PKA-RIα. The complete clustering analysis is 958 shown in Fig. S2A. 959 (C) IMCD3 cells stably expressing mNeonGreen-tagged PKA-RIα (NGPKA-RIα) were serum-starved 960 for 24 h and treated with conditioned medium with or without Shh. Cells were fixed and stained for 961 acetylated tubulin (ac-tub, red) and DNA (blue). NGPKA-RIα was visualized by the intrinsic 962 fluorescence of mNeonGreen (green). 963 (D) Box plot showing background-corrected NGPKA-RIα fluorescence in cilia at indicated timepoints 964 after Shh addition. 60 cilia (n=60) were analyzed for each time point. 965 (E) IMCD3 cells expressing GPR161NG were treated and analyzed as in (C). GPR161NG was visualized 966 via the intrinsic fluorescence of mNeonGreen. 967 (F) Box plot showing background corrected ciliary GPR161NG signal at indicated timepoints after Shh 968 addition. 60 cilia (n=60) were analyzed for each time point. 969 (G) Box plots showing background corrected GPR161NG fluorescence signals in the primary cilium of 970 cells transfected with siRNA against Prkar1a or control siRNA at indicated times after Shh addition. 60 971 cilia were analyzed for each condition (n = 60) as in Fig. 4D. 972 All scale bars represent 2 µm. 973 (H) Model of the functional interaction between GPR161, PKA and SMO inside cilia. In unstimulated

974 cells (–Hh), GPR161 keeps [cAMP]cilia high via activation of Gαs. GPR161-bound PKA-RIα releases 975 the fully active catalytic PKA subunits (C) into the lumen of cilia. Early after pathway activation (+Hh,

976 early), SMO begins to accumulate in cilia and lowers [cAMP]cilia via Gαi activation. This leads to the 977 association of the PKA-C with PKA-RIα to form a partially active holoenzyme that locally 978 phosphorylates the GPR161 C-terminal tail. GPR161 phosphorylation results in the exit of GPR161 979 from cilia together with a bound PKA holoenzyme (+Hh, late). The removal of GPR161 from cilia

980 eliminates the source of tonic Gαs activation, which leads to a further reduction of [cAMP]cilia. ‘Arr’ 981 indicates β-arrestin2. 982

31 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license. A 1500 C PALD1 ac-tub DNA ac-tub PALD1

(RFU) 1000 –SAG PALD1 500 ary Cili ary

0

0 1 2 4 +SAG 24 time after Shh addition (h)

B 5000 D SMO ac-tub DNA ac-tub SMO 4000

3000 –SAG MO (RFU) 2000

ary S Cili ary 1000

0

0 1 2 4 +SAG 24 time after Shh addition (h)

E F PALD1 SMO ac-tub DNA ac-tub SMO PALD1 8 *

6 +CYC 4

2 Ciliary signals (RFU)

+SAG 0 CYC – + – + SMO PALD1 G H SSTR3NG PALD1 ac-tub DNA ac-tub SSTR3NG PALD1 * 10

–sst * 5 Ciliary signals (RFU)

+sst 0 sst – + – + SSTR3NG PALD1

May et al., Figure 6 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

983 Figure 6. PALD1 accumulates in primary cilia in response to ciliary Gαi activation. 984 (A and B) IMCD3 cells were serum-starved for 24 h and treated with Shh for indicated times before 985 fixation and staining for PALD1 (A) or SMO (B). Box plots display the background-corrected signals 986 of PALD1 and SMO in primary cilia. 59 cilia were analyzed for each condition (n = 59). 987 (C and D) IMCD3 cells were serum-starved and treated with or without SAG for 24 h and analyzed 988 by immunofluorescence microscopy using indicated antibodies. 989 (E and F) IMCD3 cells were serum-starved and treated with cyclopamine (+CYC) or SAG for 24 h 990 and analyzed as in (C and D). (E) Micrographs of representative images. (F) Box plots showing 991 background-corrected, relative ciliary fluorescence intensities of respective proteins normalized to 992 acetylated tubulin signals. 30 cilia were analyzed for each condition (n = 30). Data was analyzed using 993 two-way ANOVA with multiple comparisons (Tukey test) with a defined confidence of 95%. *, p < 994 0.05. 995 (G and H) IMCD3 cells stably expressing Sstr3NG were serum-starved for 24 h in the presence or 996 absence of 10 µM somatostatin and analyzed as in (E and F) (n = 30). Sstr3NG was detected by 997 mNeonGreen fluorescence. Data analyzed using two-way ANOVA with multiple comparisons (Sidak 998 test) with a defined confidence of 95%. *, p < 0.05; n.s., not significant. 999 All scale bars represent 2 µm. 1000

32 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

A -/- E

WT

HEK 293

IMCD3

MIN6

IMCD3 RPE Pald1 C2C12 3T3 Myr SAG – + – + – + – + – + – + – + 55 118 312 333 441 736 80 – PALD1 30 – N C kDa GAPDH Gln154ArgfsX68 859 1 2 3 4 5 6 7 8 9 10 11 12 13 14

6 Predicted protein tyrosine phosphatase domains Predicted catalytic motifs (CXXXXXR) 4

ALD 1 levels 2 F rel. P rel. IFT-B PALD1IFT25 SMO CFAP54Vash1/2OFD1 CEP19Phospho1Phospho2 0 H.sapiens SAG – + – + – + – + – + – + – +

-/- C.intestinalis

3T3

RPE C.elegans

WT

MIN6

IMCD3

IMCD3

Pald1 C2C12 D.melanogaster

HEK 293 N.vectensis YFP B PALD1 SMO M.brevicollis ac-tub DNA ac-tub SMOYFP PALD1 S.cerevisiae B.dendrobatidis D.discoideum

–SAG O.tauri Chlorella ?

3T3 C.reinhardtii

A.thaliana

P.patens +SAG C.merolae T.gondii C P.falciparum PALD1 D T.thermophila ac-tub DNA ac-tub PALD1 T.pseudonana 5 ? E.huxleyi

4 T.vaginalis –SAG 3 G.lamblia T.cruzi 2

C2C12 N.gruberi 1 Ciliary PALD1 (RFU)

+SAG 0 SAG – + May et al., Figure 7 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

1001 Figure 7. PALD1 accumulates in primary cilia of selected cell types upon Hh pathway 1002 activation. 1003 (A) Cell lysates of indicated cell lines were separated by SDS-PAGE and analyzed by quantitative 1004 Western blotting using anti-PALD1 antibody and anti-GAPDH as loading control. Dot plot indicates 1005 PALD1 protein levels relative to GAPDH in presence or absence of SAG as indicated (n = 2 except 1006 for for PALD1-/- where n = 1). Mean values are indicated by horizontal lines. 1007 (B) PALD1 does not detectably accumulate in primary cilia of 3T3 cells after Hh pathway activation 1008 whereas SMO does. 3T3 cells expressing YFPSMO (Rohatgi et al., 2009) were serum-starved and 1009 treated with or without SAG for 24 h and analyzed by immunofluorescence microscopy using indicated 1010 antibodies. SMO was detected by YFP fluorescence. Scale bars = 2 µm. 1011 (C and D) PALD1 is enriched in C2C12 myoblast primary cilia after Hh pathway activation. C2C12 1012 cells were treated and analyzed as in (B). Box plots show background-corrected, relative fluorescence 1013 normalized to acetylated tubulin signals. 30 cilia were analyzed for each condition (n = 30). 1014 (E) Schematic representation of PALD1 protein. Predicted protein domains and post-translational 1015 modifications. Numbers indicate amino acid positions in Mus musculus PALD1, Myr depicts 1016 myristoylation site at the N-terminus. Red arrow indicates location of missense mutation in PALD1-/- 1017 cells (see Fig. S4). 1018 (F) Phylogenetic analysis of PALD1 orthologs and co-conserved proteins (IFT25, CFAP54, Vash1/2, 1019 OFD1, CEP19, Phospho1/2) identified by Clustering by Inferred Models of Evolution (CLIME) (Li et 1020 al., 2014). The strongest co-conservation with PALD1 was observed for IFT25 (a mobile subunit of the 1021 IFT-B complex). Shown is a simplified taxonomic tree with crown eukaryotic groups in different colors 1022 (modified from (Carvalho-Santos et al., 2010)). Branch color code: purple, opisthokonts; blue, 1023 amebozoa; green, plants; yellow, alveolates and heterokonts; orange, haptophytes; and brown, 1024 excavates. When present in the respective organism, motile cilia are shown in green and primary cilia 1025 in blue. The presence of cilia in T. pseudomonas remains controversial. The presence of the 1026 corresponding proteins is indicated by black circles. Conservation of IFT-B complex subunits are 1027 depicted by circles with shades of grey that correspond to percentage of subunits, for which orthologs 1028 are found (black, 100%; dark grey <100%; light grey, <60%; white, <30%). The presence of orthologs 1029 was determined by CLIME, except for B. dendrobatidis, Chlorella, E. huxleyi, which were analyzed by 1030 BLASTp (blast.ncbi.nlm.nih.gov). Proteins with E-values ≤ E-25 were scored as hits. 1031

33 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

A IFT88 B C ac-tub DNA ac-tub IFT88 4 * -/- -/- WT Pald1 WT Pald1 n.s. kDa 3 kDa – + – + Shh 180 – – + – + Shh WT 100 – PALD1 GLI3FL

(rel. to control) 2 100 – IFT88 R 100 – R 40 – GLI3 /GLI3

-/- actin

FL 1 185 – 50 – PTCH1 GAPDH GLI3 Pald1 1 2 3 4 5 6 7 8 0 Shh – + – + WT Pald1-/- D E F GPR161 SMO ac-tub DNA ac-tub GPR161 SMO n.s. * * * * * 10 3 – SAG 8 WT 6 2

+ SAG 4 1 Ciliary SMO (RFU) 2 Ciliary GPR161 (RFU) – SAG

-/- 0 0 SAG – + – + SAG – + – + WT Pald1-/- WT Pald1-/- Pald1 + SAG

May et al., Figure 8 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

1032 Figure 8. PALD1 is required for efficient GLI3 repressor formation in IMCD3 cells. 1033 (A) Pald1-/- and parental IMCD3 cells were serum-starved for 24 h and analyzed by 1034 immunofluorescence microscopy using indicated antibodies. 1035 (B) Lysates of wild-type and Pald1-/- IMCD3 cells cultured in the presence or absence of Shh were 1036 separated by SDS-PAGE and analyzed by immunoblotting using indicated antibodies. GLI3 repressor 1037 (GLI3R) and full-length (GLI3FL) forms are indicated. 1038 (C) GLI3R and GLI3FL signals from 3 independent experiments as in (B) were quantified and 1039 GLI3FL/GLI3R ratios plotted. Horizontal lines depict means (n = 3). Each experiment was internally 1040 normalized to the GLI3FL/GLI3R ratio in WT in the absence of signal (WT –Shh GLI3FL/GLI3R ratio 1041 = 1). Data were analyzed using two-way ANOVA with multiple comparisons in a Tukey test with a 1042 defined confidence of 95%. *, p < 0.05; n.s., not significant. 1043 (D) WT and PALD1-deficient IMCD3 cells were serum-starved and treated with Smoothened agonist 1044 (SAG) for 24 h (as indicated) and analyzed as in (A). 1045 (E) and (F) Box plots showing background-corrected, relative fluorescence normalized to acetylated 1046 tubulin signals. (E) Two independent experiments were performed and 30 cilia were analyzed for each 1047 condition in each experiment (n = 60). (F) Three independent experiments were performed and 30 1048 cilia were analyzed for each condition in each experiment (n = 90). Data were analyzed using two-way 1049 ANOVA with multiple comparisons in a Tukey test with a defined confidence of 95%. *, p < 0.05; 1050 n.s., not significant. 1051 All scale bars represent 2 µm.

34 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

A B E F 7.5 -/- -/- 2 326 328 330 332 334 336 338 Cep164 2 O O 2 5.0 wild-type 2 Cep164 Cep164 APEX2 APEX2 – H – H control- control- cilia-APEX2 cilia-APEX2 cilia-APEX2 cilia-APEX2 2.5 -/- Cep164 213 312 312 set no. 213 312 312 set no. 326 328 330 332 334 336 338 cilia Hs6st1 0.96 Npc1 cluster 1 Rtel1

Total IFT88 (RFU) allele 0.0 2.77 Ctsa 4.58 Ntmt1 Mus musculus, Exon8 Spice1 cilia 6.39 Cdk5rap2 Ston1 cluster 2 8.19 Tgm2 Akr1b1 parental 10.0 Pcnt 24.7 Cyr61 cilia-APEX Rpf2 cilia-APEX2 39.4 Rbm8a Rap2c 54.1 Ncl Ap2a1 68.8 Cdc40 Asna1 83.4 abundance (in %) protein rel. C Sptbn1 Itsn1 GFP-APEX Hip1 Plec ninein DAPI GFP-APEX ninein Plat Anp32b Pa2g4 Snw1 Sptan1 Cltc Rpl35 Patz1 Slirp WT Ccdc61 Parp1 Kif4 Smchd1 Tmpo Fh Glud1 Phc3 Tfcp2l1 -/- Znf326 Rpa2 Glipr2 Cox4i1 Lmna Rpa3 Mcm3 Ppia Gpi Cep164 control Fech cluster Lmnb1 Aco2 Acadm Cpeb4 Etfb Idh2 D Mme Atad2 CEP164 γ-tub Rpa1 Lmnb2 Atrx GFP-APEX DAPI CEP164 γ-tub GFP-APEX Ap1b1 Bin1 Idh3a Cbx3 Sprr2b Snrpf Mapk14 Nasp Ankrd11

WT Cand2 Ppp2r5d Snrpd2 -/- Cep164

May et al., Figure S1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

1052 Figure S1. A CEP164-deficient cilia-APEX2 IMCD3 cell line serves as a specificity control 1053 for cilia-APEX2 proteomics 1054 (A) Dot plot showing total protein levels of IFT88 relative to actin in indicated cell lines as determined 1055 by quantitative immunoblotting (see Fig. 1E). Mean values are indicated by horizontal lines (n = 3). 1056 (B) Cep164-/- cDNA was sequenced and aligned with the Cep164 gene sequence from Mus musculus. A 1057 homozygous single base pair deletion in exon 8 leads to a frameshift mutation and protein truncation. 1058 DNA sequences were analyzed using Benchling. 1059 (C) and (D) Immunofluorescence micrographs of wild-type (WT) or CEP164-deficient (Cep164-/-) cell 1060 lines stably expressing cilia-APEX2. Cell lines were serum-starved for 24 h before fixation. cilia- 1061 APEX2 proteins were detected by GFP fluorescence. (C) Ninein marks centrioles and is visualized by 1062 antibody staining. (D) γ-tubulin and CEP164 are detected by specific antibodies. Representative 1063 micrographs are shown for each condition. Scale bars represent 2 µm. 1064 (E) High reproducibility of cilia-APEX2 proteomic setup. Hierarchical cluster analysis based on 1065 Ward’s minimum variance method (two-way clustering) of the relative abundances of each identified 1066 protein (rows) in the individual samples (columns). Relative scaled abundances were calculated by 1067 dividing the TMT signal to noise of an individual protein by the sum of TMT signal to noise ratios in 1068 all samples. Legend depicts color scheme for relative abundances (in %). Brackets indicate cilia clusters 1069 (red and purple, see Fig. 2B) and unrelated cluster (orange) without cilia proteins. 1070 (F) Zoom into hierarchical cluster analysis (E) at control cluster (orange) without cilia proteins. 1071 1072

35 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

A B D NGPKA-R1α GPR161NG NG NG 2 ac-tub DNA ac-tub PKA-R1α ac-tub DNA ac-tub GPR161 O 2 –H control-APEX2 –Shh #1 –Shh #2 1h Shh #2 4h Sh #2 24h Shh #2 1h Shh #1 4h Shh #1 24h Shh #1 +Shh +SAG 4h 2h 1h 0h 4h 2h 1h 0h

Smo mini-cluster

Gpr161 24h mini-cluster 24h

C 400 E 1.2 AKAP9 1.2 AKAP11 300 1.0 1.0 0.8 0.8 200

PKA-RIα (RFU) 0.6 0.6

NG 0.4 0.4 rel. protein abundance (in %) 100 0.2 0.2 maximum signal l. to maximum signal l. to maximum Ciliary re re

0,31 2,25 4,19 6,13 8,06 10,0 19,6 29,1 38,7 48,3 57,8 0 0.0 0.0 0 1 2 4 24 0 4 8 12 16 20 24 0 4 8 12 16 20 24 time after SAG addition (h) time after Shh addition (h) time after Shh addition (h)

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1073 Figure S2. PKA-RIα and GPR161 are removed from cilia in response to Smoothened 1074 agonist, while the A-kinase anchoring proteins (AKAPs) identified by cilia-APEX2 1075 remain unchanged. 1076 (A) Two-way hierarchical cluster analysis (Ward’s method) of the relative protein abundances (rows) 1077 in the individual samples (columns) shows high inter-set reproducibility. Legend depicts the color 1078 scheme of relative abundances (calculated as in Fig. S1E). SMO and GPR161 mini-clusters are 1079 indicated by green and red arrows, respectively (see Fig. 5A and B for magnified views of the mini- 1080 clusters). 1081 (B and C) NGPKA-RIα-expressing IMCD3 cells were serum-starved for 24 h and treated with or 1082 without SAG for indicated times. Cells were fixed and stained for ac-tub (red) and DNA (blue). NGPKA- 1083 RIα was visualized by mNeonGreen fluorescence. Representative micrographs are shown (B). (C) 1084 Box plot shows background-corrected NGPKA-RIα fluorescence in cilia at indicated timepoints after 1085 SAG addition. 50 cilia (n=50) were analyzed for each time point. 1086 (D) IMCD3 cells expressing GPR161NG were serum-starved for 24 h in the presence of Shh- 1087 conditioned medium for indicated times and analyzed as in (B). GPR161NG was detected by 1088 mNeonGreen fluorescence. For quantitative analysis, see Fig. 5F. 1089 (E) Relative AKAP abundances assessed by mass-spectrometric TMT quantitation were plotted over 1090 time. Data points represent averages of duplicate measurements, error bars depict individual values. 1091 Maximum average signal was set to 1, background signals as assessed from control-APEX2 labeled 1092 samples set to 0. ‘0 h’ represents –Shh. 1093 Scale bars represent 2 µm. 1094

36 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license. –72 h –24 h –60 min –45 min –30 min –15 min –10 min –5 min –2 min 0

Seed cells

control-APEX2 + Shh

cilia-APEX2 + Shh cilia-APEX2 + Shh 2

cilia-APEX2 O 2 + H + Shh + biotin tyramide Serum starvation cilia-APEX2 Quenching + lysis + Shh cilia-APEX2 Sample processing for mass-spectrometry + Shh cilia-APEX2 – Shh cilia-APEX2

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1095 Figure S3. Schematic of experimental workflow for high resolution time-resolved cilia- 1096 APEX2 profiling of the ciliary Hh response (see Figure 4C). 1097 Cilia-APEX2 (and control-APEX2) IMCD3 cells were seeded 72 h before the APEX labeling reaction. 1098 24 h before labeling, cells were starved of serum. 60 min, 45 min, 30 min, 15 min, 10 min and 5 min 1099 before APEX-labeling Shh-conditioned medium was added (as indicated). ‘–Shh’ indicates addition of 1100 conditioned medium without Shh 60 min prior to labeling. APEX labeling and sample preparation 1101 were performed as in Fig. 2A. In brief, biotin tyramide was added 30 min before the 2 min labeling

1102 reaction in the presence of hydrogen peroxides (H2O2). Samples were quenched and kept on ice for 1103 lysis, followed by streptavidin chromatography and on-bead tryptic digestion. Peptides of each sample 1104 were labeled with individual tandem-mass-tags (TMTs), pooled and fractionated offline via high pH 1105 reversed phase chromatography before mass-spectrometric analysis. 1106

37 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

148 150 152 154 156 158 160 162 Pald1 wild-type

Pald1-/- allele 1 148 150 152 154 156 158 160 162

Pald1-/- allele 2 148 150 152 154 156 158 160 162

Mus musculus, Exons 4 and 5

May et al., Figure S4 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

1107 Figure S4. Pald1-/- IMCD3 cells exhibit early missense mutation. 1108 Pald1-/- genomic DNA was sequenced and aligned with the PALD1 WT gene sequence from Mus 1109 musculus. Single base pair deletions in exon 4 lead to frameshift mutations in both alleles and protein 1110 truncation. The DNA sequences have been analyzed using Benchling. 1111 1112 1113 1114 1115 1116

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1117 SUPPLEMENTARY TABLES 1118 1119 Table S1. Cilia-APEX2/TMT proteomics reveals the proteome of primary cilia with high 1120 sensitivity. 1121 First tab ‘Legend’ shows the color scheme. Second tab ‘cilia-APEX2 TMT - Fig. 2’ enumerates the 1122 candidate cilia proteins identified by cilia-APEX2/TMT proteomics (see Figs. 2A-D). Proteins are 1123 listed by Gene symbols according to the Mus musculus proteome database (Uniprot 07/2014). Column 1124 B shows the numbers of quantified peptides. ‘TMT ratio’ in columns C and E is the average relative 1125 enrichment between three cilia-APEX2 samples and the indicated control samples. p values in columns 1126 D and F were calculated from multiple t-tests (statistical significance of enrichments of cilia-APEX2 1127 samples versus the respective controls determined by unpaired student’s t-tests).Third tab ‘known cilia 1128 proteins below cut’ lists known cilia proteins quantified by cilia-APEX2/TMT proteomics that did not 1129 meet the inclusion criteria. 1130 1131 Table S2. Cilia-APEX2 proteome list. 1132 First tab ‘Legend’ shows the color scheme. Second tab ‘cilia-APEX2 proteome’ lists the high 1133 confidence cilia proteins that have been scored as hits in three (Tier 1, detailed in third tab) or at least 1134 two out of three experiments (Tier 2, detailed in fourth tab). The numbers of quantified peptides and 1135 the average enrichment scores (TMT ratios) in the individual experiments are shown.

39 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

1136 MATERIALS AND METHODS 1137 Cell line generation, cultivation and manipulation 1138 C2C12 myoblasts, NIH 3T3 and HEK cells were cultured in DMEM medium, RPE1-hTERT 1139 (described as RPE in the text) and all IMCD3 cell lines were grown in DMEM/F12, all supplemented 1140 with 10% FBS. The pancreatic beta cell line MIN6 was cultured in DMEM medium, supplemented 1141 with 15 % FBS. Ciliation was induced by growth factor deprivation by reducing the growth media to 1142 0.2% FBS for 24 h. Transfections were carried out using XtremeGene9 (Roche) or FuGene6 (Promega) 1143 according to manufacturers’ guidelines. Cep164 and Pald1 genes were disrupted in IMCD3 FlpIn cells 1144 using CRISPR/Cas9-mediated genome editing with gRNAs targeting exons 8 and 4, respectively (Ran 1145 et al., 2013). Clones of each cell line were obtained by limited dilution (Cep164) or single cell sorting 1146 (Pald1). Clones with disrupted genes were screened by immunofluorescence microscopy and Western 1147 Blotting using protein-specific antibodies. Selected positive clones were further characterized by 1148 sequencing, confirming missense mutation leading to early termination of translation. 1149 IMCD3 cell lines stably expressing cilia-APEX2, control-APEX2, NGPKA-RIα were generated using 1150 the FlpIn system as described (Breslow and Nachury, 2015). Plasmids encoding cilia-APEX2 and 1151 control-APEX2 were created by site-directed mutagenesis of cilia-APEX and control-APEX plasmids, 1152 and confirmed by sequencing. A plasmid encoding NGPKA-RIα was generated using the Gateway 1153 cloning system (Life technologies) by LR clonase reaction of pEF5/FRT/NG-DEST with pENTR- 1154 PRKAR1A (obtained from Addgene, #23741). IMCD3 cells stably expressing GPR161NG have 1155 previously been described (Ye et al., 2018). 1156 To induce Hh signaling, growth media were supplemented with either 200 nM Smoothened agonist 1157 (SAG) or Shh-N conditioned medium (10-16% (v/v) depending on batch) produced with EcR-ShhN 1158 cells (gift from Phil Beachy). To block Hh signaling, cyclopamine (CYC) was added to the growth 1159 medium to a final concentration of 10 µM. 1160 1161 APEX labeling experiments 1162 Cells were incubated in the presence of 0.5 mM biotin tyramide for 30 min before the addition of

1163 hydrogen peroxide (H2O2) to a final concentration of 1 mM. For non-labeling samples water was added

1164 instead of H2O2. After 2 min of incubation at room temperature, the medium was aspirated quickly 1165 and cells were washed three times with quenching buffer (1x PBS supplemented with 10 mM sodium 1166 ascorbate, 10 mM sodium azide and 5 mM Trolox). For fluorescence microscopy, cells were 1167 immediately fixed. For proteomic and Western Blot analyses cells were lysed by scraping them off the 1168 growth surface in ice-cold lysis buffer (0.5 % (v/v) Triton X-100, 0.1 % (w/v) SDS, 10% (w/v) glycerol,

40 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

1169 300 mM NaCl, 100 mM Tris/HCl pH 7.5, protease inhibitors) supplemented with 10 mM sodium 1170 ascorbate, 10 mM sodium azide and 5 mM Trolox. After collecting the lysate in a reaction tube, the 1171 lysate was vortexed, incubated on ice for 15 min and cleared by centrifugation (16,000 x g for 20 min 1172 at 4°C). 1173 1174 Streptavidin chromatography 1175 After determining protein concentrations of lysates from APEX-labeling experiments they were 1176 adjusted to equal concentrations and volumes as starting material for chromatography, from which 1177 samples were taken as loading control for SDS-PAGE and Western Blot analysis. Samples were added 1178 onto washed and equilibrated Streptavidin-Sepharose beads (Thermo Scientific) and biotinylated 1179 proteins were allowed to bind for 1 h at room temperature. Unbound material was removed and 1180 samples taken for Western Blot analysis. Beads with bound proteins were washed extensively with lysis 1181 buffer, then with urea wash buffer (4 M urea 10 mM Tris/HCl, pH 7.5) and finally with urea wash 1182 buffer supplemented with 50 µM biotin. For mass-spectrometric analyses, bound proteins were 1183 alkylated and digested with endopeptidase Lys-C (Wako) for 3 hours and trypsin (Promega) on beads 1184 overnight at 37°C. 1185 1186 Mass spectrometry 1187 Tryptic digests were directly labelled in 200 mM HEPES pH 8.5 with tandem mass tag (TMT) 10- 1188 plex reagents (Thermo Fisher Scientific #90406). After efficient labeling was checked by MS, peptides 1189 were subjected to alkaline reversed phase fractionation as described (Paek et al., 2017). Pooled fractions 1190 were analyzed on a Fusion Lumos Orbitrap mass spectrometer coupled to a Proxeon EASY-nLC 1000 1191 liquid chromatography (LC) system (Thermo Fisher Scientific) using a synchronous precursor selection 1192 (SPS) MS3 method (McAllister and Gygi, 2013). Capillary columns had an inner diameter of 100 m 1193 and were packed with 2.6 m Accucore beads (Thermo Fisher Scientific). Peptides were analyzed on 1194 acidic acetonitrile gradients for 5 h with MS1 (Orbitrap, resolution 120k) scans, MS2 scans after 1195 collision-induced dissociation (CID, CE-35) in the ion trap and MS3 precursor fragmentation by high- 1196 energy collision-induced dissociation (HCD). Reporter ions were analyzed by MS3 in the orbitrap at a 1197 resolution of 50k. Further details on LC and MS parameters can be found in (Paek et al., 2017). 1198 1199 Mass spectrometry data analysis 1200 Mass spectra were processed and peptide-spectrum matches (PSM) were obtained by a SEQUEST 1201 (V.28, rev. 12) based software. Searches used a size-sorted forward and reverse database of the M. 1202 musculus proteome (Uniprot 07/2014) with a mass tolerance of 20 ppm for precursors and a fragment

41 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

1203 ion tolerance of 0.9 Da. Oxidized methionine residues were dynamically searched (+15.9949 Da). A 1204 false discovery rate of 1% was set for PSM following linear discriminant analysis and FDR for final 1205 collapsed proteins was 1% as well. 1206 Relative protein quantification used summed MS3 TMT signal / noise (s/n) per protein filtered for 1207 summed s/n >180 over all channels per peptide and an isolation specificity >70% for each peptide. 1208 Details of the TMT intensity quantification method can be found in (Paulo et al., 2016). 1209 1210 Immunofluorescence microscopy 1211 For microscopic analyses all cells were grown on round 12 mm #1.5 coverslips and fixed in 4% 1212 paraformaldehyde for 10 to 15 min at room temperature. After fixation cells were permeabilized in - 1213 20°C methanol for 5 min and rehydrated in 1x PBS at room temperature. After extensive washing in 1214 1x PBS, fixed cells were blocked in blocking buffer (3% bovine serum albumin, 5% serum in 1x PBS) 1215 for 30 min. After blocking, cells were incubated with primary antibody dilutions in blocking buffer for 1216 1 h at room temperature or 4°C overnight, washed three times with 1x PBS over 15 minutes, and 1217 incubated with AlexaFluor488-, Cy3- or Cy5-coupled secondary antibodies (Jackson 1218 Immunoresearch), or AlexaFluor647-conjugated streptavidin (Invitrogen) in blocking buffer for 30 1219 min. Finally, cells were washed five times in PBS and mounted on glass slides using Roti®-Mount 1220 FluorCare DAPI (Carl Roth; Figs. 1, 6C, 6D, 7 and 8) or DNA stained with Hoechst 33258 and 1221 mounted on glass slides using Fluoromount G (Electron Microscopy Sciences; Figs. 5, 6E, 6G, S1). 1222 APEX enzymes and YFPSMO were detected by GFP and YFP fluorescence, NGPKA-RIα and 1223 GPR161NG by mNeonGreen fluorescence. 1224 Prepared specimens were imaged on an AxioImager.M1 microscope (Carl Zeiss, Figs. 5C, 5E, 5G) or 1225 a Leica DMi8 with PlanApochromat oil objectives (63x, 1.4NA) using appropriate filters. Images were 1226 captured using a CoolSNAP HQ (Photometrics) or Leica DFC3000 G camera system, respectively. 1227 1228 SDS-PAGE and Western Blotting 1229 Standard techniques were used for SDS-PAGE and Western blotting. Cell lysates were generated after 1230 washing cells with 1x PBS and scraping cells of the growth surface in solubilization buffer (25 mM 1231 Tris/HCl, 300 mM NaCl, 1 mM EDTA, 10 % Glycerol, 1 % Triton X-100 (v/v), 0.1 % SDS (w/v), 1232 1 mM PMSF and proteinase inhibitors (Roche)). Lysates were cleared by centrifugation (20.000 g at 1233 4°C for 45 min), 25 µg of protein was separated on 10% Bis-Tris polyacrylamide gels and transferred 1234 onto nitrocellulose membranes. After blocking in 5% milk or Intercept® (TBS) Protein-Free Blocking 1235 Buffer (LI-COR) and specific antibody decoration, membranes were washed and primary antibodies 1236 visualized using IRDye800-conjugated and IRDye680-conjugated secondary antibodies on a LI-COR

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1237 Odyssey laser scanner. Quantitation of bands was performed using the Image Studio Lite software 1238 (version 5.2.5). 1239 1240 Statistical analyses 1241 Statistical analyses were performed with Graphpad Prism v8.3.1. For Western Blot analyses mean 1242 values from independent experiments (exact n stated in figure legend) were calculated and are shown 1243 with either SD or SEM as described in the figure legends. For GLI3 analysis, each experiment was 1244 internally normalized to the GLI3FL/GLI3R ratio in WT cells in the absence of Shh-N. For 1245 Immunofluorescence experiments at least 30 cilia were analyzed (exact n stated in figure legends). To 1246 compare biological replicates, relative fluorescence values were normalized to the average relative 1247 fluorescence signal in WT cells in the absence of inducing reagents. In all statistically analyzed 1248 experiments, significance was assessed by two-way ANOVA assuming normal distribution and 1249 multiplicity adjusted p-values were obtained by Holm-Sidak post-hoc or Tukey testing (p<0.05 was 1250 considered statistically significant). For volcano plot graphs, Student’s t-test were used and statistical 1251 analyses were performed using Prism 8 (Graphpad Software). Hierarchical cluster analyses were 1252 performed according to Ward’s minimum variance method using JMP software (from Statistical 1253 Analysis System, v15.1.0). 1254 1255 Antibodies and reagents 1256 Antibodies against the following proteins were used at indicated dilutions: anti-acTub (Sigma-Aldrich 1257 T7451, mouse 1:2,000), anti-Arl13b (Proteintech 17711-1-AP, rabbit 1:2,000), anti-IFT88 1258 (Proteintech 13967-1-AP, rabbit 1:200 in IF, 1:1,000 in WB), anti-GFP (raised against 6His-tagged 1259 eGFP, rabbit 1:1,000), anti-GPR161 (gift from S. Mukhopadhyay, rabbit 1:500), anti-GLI3 (R&D 1260 Systems nachuryAF3690, goat 1:1,000) anti-actin (self-made, rabbit 1:5,000), anti-GAPDH 1261 (Proteintech 60004-1-Ig, mouse 1:2,000), anti-PALD1 (Sigma-Aldrich HPA017343, rabbit, IF: 1:250, 1262 WB: 1:1,000), anti-SMO (Santa Cruz sc-166685, mouse IgG2a in IF 1:200; Abcam ab236465 for Figs. 1263 5B, D and E), anti-PTCH1 (Abcam ab53715, rabbit, WB 1:1,000), anti-ninein (gift from M. Bornens, 1264 rabbit 1:10,000), anti-CEP164 (gift from C. Morison, rabbit, IF 1:2,000), γ-tubulin (Proteintech 66320- 1265 1-AP, rabbit 1:1,000). Streptavidin-pHRP (Thermo Scientific #21140, 1:1,000), Streptavidin-AF647 1266 (Invitrogen S21374, 1:1,000), Smoothened agonist (SAG; Abcam ab142160), Shh (N-terminus of Shh, 1267 produced in cell line EcR-ShhN cells, gift from Phil Beachy), cyclopamine (CYC; Merck Millipore 1268 #239806), biotin tyramide (Iris Biotech GmbH), somatostatin (sst; Alfa Aesar J66168). 1269 1270

43 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.29.225797; this version posted July 29, 2020. The copyright holder for this 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 available under aCC-BY-NC-ND 4.0 International license.

1271 Cell lines used in this study Cell line Parental cell line reference Cep164-/- IMCD3 FlpIn This work Pald1-/- IMCD3 FlpIn This work Cilia-APEX2 IMCD3 FlpIn This work Control-APEX2 IMCD3 FlpIn This work Cep164-/- cilia-APEX2 IMCD3 FlpIn This work NGPKA-RIα IMCD3 FlpIn This work GPR161NG IMCD3 FlpIn (Nager et al., 2017) SSTR3NG IMCD3 FlpIn (Nager et al., 2017) YFPSMO 3T3 (Milenkovic et al., 2009) C2C12 - (Blau et al., 1985) RPE1-hTERT - (Bodnar et al., 1998) MIN6 - (Poitout et al., 1995) 1272

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