APEX2 Proximity Proteomics Resolves Flagellum Subdomains and Identifies Flagellum Tip-Specific

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bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 APEX2 proximity proteomics resolves flagellum subdomains and identifies flagellum tip-specific

2 proteins in Trypanosoma brucei

4 Daniel E. Vélez-Ramírez,a,b Michelle M. Shimogawa,a Sunayan Ray,a* Andrew Lopez,a Shima

5 Rayatpisheh,c* Gerasimos Langousis,a* Marcus Gallagher-Jones,d Samuel Dean,e James A. Wohlschlegel,c

6 Kent L. Hill,a,f,g#

8 aDepartment of Microbiology, Immunology, and Molecular Genetics, University of California Los

9 Angeles, Los Angeles, California, USA

10 bPosgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, Coyoacán, Ciudad de

11 México, México

12 cDepartment of Biological Chemistry, University of California Los Angeles, Los Angeles, California, USA

13 dDepartment of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Los

14 Angeles, California, USA

15 eWarwick Medical School, University of Warwick, Coventry, England, United Kingdom

16 fMolecular Biology Institute, University of California Los Angeles, Los Angeles, California, USA

17 gCalifornia NanoSystems Institute, University of California Los Angeles, Los Angeles, California, USA

19 Running title: APEX2 proteomics in Trypanosoma brucei

21 #Address correspondence to Kent L. Hill, [email protected].

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22 *Present address: Sunayan Ray: Invivoscribe, San Diego, California, USA. Shima Rayatpisheh: Genomics

23 Institute of the Novartis Research Foundation, San Diego, California, USA. Gerasimos Langousis:

24 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

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25 ABSTRACT

26 Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne

27 disease. T. brucei has a single flagellum that plays critical roles in parasite biology, transmission and

28 pathogenesis. An emerging concept in flagellum biology is that the organelle is organized into

29 subdomains, each having specialized composition and function. Overall flagellum proteome has been

30 well-studied, but a critical gap in knowledge is the protein composition of individual flagellum

31 subdomains. We have therefore used APEX-based proximity proteomics to examine protein composition

32 of T. brucei flagellum subdomains. To assess effectiveness of APEX-based proximity labeling, we fused

33 APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, an axonemal complex distributed

34 along the flagellum. We found that DRC1-APEX2 directs flagellum-specific biotinylation and purification

35 of biotinylated proteins yields a DRC1 “proximity proteome” showing good overlap with proteomes

36 obtained from purified axonemes. We next employed APEX2 fused to a flagellar membrane protein that

37 is restricted to the flagellum tip, adenylate cyclase 1 (AC1), or a flagellar membrane protein that is

38 excluded from the flagellum tip, FS179. Principal component analysis demonstrated the pools of

39 biotinylated proteins in AC1-APEX2 and FS179-APEX2 samples are distinguished from each other.

40 Comparing proteins in these two pools allowed us to identify an AC1 proximity proteome that is

41 enriched for flagellum tip proteins and includes several proteins involved in signal transduction. Our

42 combined results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can

43 be used to resolve proteome composition of flagellum subdomains that cannot themselves be readily

44 purified.

45 IMPORTANCE

46 Sleeping sickness is a neglected tropical disease, caused by the protozoan parasite Trypanosoma brucei.

47 The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility,

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48 transmission, and virulence depend on its flagellum (aka cilium), which consists of several different

49 specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is

50 need of approaches that enable proteomic analysis of individual subdomains. Our work establishes that

51 APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei, and

52 has allowed identification of proximity proteomes for different subdomains. This capacity opens the

53 possibility to study the composition and function of other compartments. We further expect that this

54 approach may be extended to other eukaryotic pathogens, and will enhance the utility of T. brucei as a

55 model organism to study ciliopathies, heritable human diseases in which cilia function is impaired.

56 INTRODUCTION

57 Trypanosoma brucei is a flagellated parasite that is transmitted between mammalian hosts by a

58 hematophagous vector, the tsetse fly, and is of medical relevance as the causative agent of sleeping

59 sickness in humans [1]. T. brucei also presents a substantial economic burden in endemic regions due to

60 infection of livestock, causing an estimated loss of over 1 billion USD/year [2]. As such, T. brucei is

61 considered both cause and consequence of poverty in some of the poorest regions in the world. T.

62 brucei also provides an excellent model system for understanding the cell and molecular biology of

63 related flagellates T. cruzi and Leishmania spp., which together present a tremendous health burden

64 across the globe.

65 T. brucei has a single flagellum that is essential for parasite viability, infection and transmission [3-5].

66 The flagellum drives parasite motility, which is necessary for infection of the mammalian host [4], and

67 for transmission by the tsetse fly [5]. In addition to its canonical function in motility, the flagellum plays

68 important roles in cell division and morphogenesis [6-8] and mediates direct interaction with host

69 tissues [9]. Moreover, recent work has demonstrated that the trypanosome flagellum is the site of

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70 signaling pathways that control the parasite’s response to external signals and are required for

71 transmission and virulence [4, 10-15].

72 The trypanosome flagellum is built on a canonical “9 + 2” axoneme that originates at the basal body in

73 the cytoplasm near the posterior end of the cell [3]. Triplet microtubules of the basal body extend to

74 become doublets in the transition zone, which marks the boundary between the basal body and 9 + 2

75 axoneme [16]. The axoneme exits the cytoplasm through a specialized invagination of the plasma

76 membrane, termed the “flagellar pocket” (FP) [17]. As it emerges from the flagellar pocket, the

77 axoneme is attached to an additional filament, termed the “paraflagellar rod” (PFR) [18], that extends

78 alongside the axoneme to the anterior end of the cell. The axoneme and PFR remain surrounded by cell

79 membrane and this entire structure is laterally attached to the cell body along its length, except for a

80 small region at the distal tip that extends beyond the cell’s anterior end [19]. Lateral flagellum

81 attachment is mediated by proteins in the flagellum and cell body that hold the flagellum and plasma

82 membranes in tight apposition, constituting a specialized “flagellar attachment zone” (FAZ) that extends

83 from the flagellar pocket to the anterior end of the cell [19, 20].

84 As in other flagellated eukaryotes, the trypanosome flagellar apparatus (Fig. 1) can be subdivided into

85 multiple subdomains, each having specialized function and protein composition. The FP, for example,

86 demarcates the boundary between the flagellar membrane and cell membrane. In T. brucei, the FP is

87 the sole site for endocytosis and secretion, thus presenting a critical portal for host-parasite interaction

88 [17]. The basal body functions in flagellum duplication, segregation and axoneme assembly [21]. The

89 region encompassing the transition zone lies between the cytoplasm and flagellar compartment and

90 includes proteins that control access into and out of the flagellum [13, 16]. The 9 + 2 axoneme is the

91 engine of motility [7], while the PFR in trypanosomes is considered to physically influence flagellum

92 beating and to serve as a scaffold for assembly of signaling and regulatory proteins [22-25]. The FAZ is a

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93 trypanosome-specific structure and is critical for parasite motility and cell morphogenesis [26]. The

94 flagellum tip marks the site of cleavage furrow initiation during cytokinesis [27] and mediates

95 attachment to the tsetse fly salivary gland epithelium, which is crucial for development into human

96 infectious parasites [28]. The flagellum tip is also the site of signaling proteins that function in cell-cell

97 communication and signaling [11, 29].

98 Given the essential and multifunctional roles of the trypanosome flagellum, much effort has been made

99 to define the protein composition of the organelle. However, although we know a lot about protein

100 composition of the flagellum as a whole, a critical knowledge gap is the protein composition of

101 individual flagellum subdomains. Classical proteomics approaches typically require purification of the

102 flagellum, or subfractions of the flagellum [16, 30-34]. This approach is very useful and has been used to

103 determine proteomes of the transition zone [16] and axoneme tip [31], but it can be cumbersome, is

104 dependent on quality of the purified fraction and cannot resolve subdomains that are not part of a

105 specific structure that can be purified.

106 To overcome limitations of conventional flagellum proteomics approaches, we have adapted APEX2

107 proximity labeling [35] for use in T. brucei. APEX2 is an engineered monomeric ascorbate peroxidase

108 that converts biotin-phenol into a short-lived biotin radical that is highly reactive. The biotin-phenol

109 radical interacts with nearby proteins, resulting in covalent attachment of a biotin tag. Biotinylated

110 proteins can be affinity-purified with streptavidin and identified by shotgun proteomics, allowing for

111 facile identification of proteins within a specific subcellular location from a complex and largely

112 unfractionated sample [35, 36]. Here, we report successful implementation of APEX2 proximity labeling

113 in T. brucei, to define the proximity proteome of flagellar proteins that are either distributed along the

114 axoneme or restricted to the tip of the flagellum membrane. Our results establish APEX-based proximity

115 proteomics as a powerful tool for T. brucei, demonstrate the approach can resolve flagellum

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116 subdomains that are not separated by a physical boundary and support the idea that the flagellum tip

117 subdomain is specialized for cell signaling.

118 RESULTS

119 To evaluate APEX2 labeling in T. brucei, we selected an axonemal protein as bait, because the axoneme

120 is a well-defined cellular component whose protein composition in T. brucei has been examined in prior

121 studies [16, 30, 31, 34]. We selected the DRC1 subunit of the nexin-dynein regulatory complex (NDRC)

122 [37], because this protein has a defined localization along the axoneme and its position relative to major

123 axonemal substructures, e.g. microtubule doublets, radial spokes and dynein arms, is known [38].

124 Having selected DRC1 as bait, we used in situ gene tagging [39] to generate cell lines expressing DRC1

125 fused to a C-terminal APEX2 tag that includes an HA-tag following the APEX2 tag, referred to as “DRC1-

126 APEX2”. Expression of DRC1-APEX2 was demonstrated in Western blots of whole cell lysates (Fig. 2A).

127 Extraction with non-ionic detergent leaves the axoneme intact in a detergent-insoluble cytoskeleton

128 fraction that can be isolated from detergent-soluble proteins by centrifugation [40]. We found that

129 DRC1-APEX2 fractionates almost completely with the detergent-insoluble cytoskeleton as expected for

130 an NDRC protein (Fig. 2A). Growth curves demonstrated that expression of DRC1-APEX2 does not affect

131 growth of T. brucei in vitro (Supplemental Fig. S1).

132 We next asked if APEX2 is functional within the biochemical environment of the T. brucei cell. Cells

133 expressing DRC1-APEX2 were incubated with biotin-phenol, which was then activated with brief H2O2

134 treatment followed by quenching with trolox and L-ascorbate. Cells were then probed with streptavidin-

135 Alexa 594 and subjected to fluorescence microscopy to assess biotinylation. As shown in Figure 2C, we

136 observed APEX2-dependent biotinylation and this was highly enriched in the flagellum. There was some

137 background staining in the cytoplasm, as revealed by parallel analysis of parental cells lacking the

138 APEX2-tagged protein (Fig. 2C), but flagellum staining was only observed in cells expressing DRC1-

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139 APEX2. In the proximal region of the flagellum, the streptavidin signal extended further than the PFR

140 (Fig. 2C), indicating streptavidin labeling is on the axoneme. Therefore, DRC1-APEX2 directs specific

141 biotinylation in the flagellum.

142 Having established that DRC1-APEX2 directs flagellum-specific biotinylation, we used shotgun

143 proteomics to identify biotinylated proteins. Samples were extracted with non-ionic detergent and

144 separated into detergent-soluble supernatant and detergent-insoluble pellet fractions. Biotinylated

145 proteins in each fraction were then isolated using streptavidin purification and subjected to shotgun

146 proteomics for protein identification. WT and DRC1-APEX2 cells were processed in parallel (Fig. 3A). Our

147 focus was the detergent-insoluble pellet because this fraction includes the axoneme and PFR, and the

148 protein composition of these structures has been characterized [19, 25, 30, 34, 41]. The pellet fraction

149 also includes non-axonemal structures such as the basal body, tripartite attachment complex,

150 cytoplasmic FAZ filament and subpellicular cytoskeleton, thus enabling us to test for enrichment of

151 axonemal proteins. The analysis was done using three independent biological replicates. In one case, the

152 sample was split into two aliquots and shotgun proteomics was done on both in parallel, giving a total of

153 four replicates each for DRC1-APEX2 and WT pellet samples.

154 Principal component analysis demonstrated that the biotinylated protein profile of the DRC1-APEX2

155 pellet samples (DRC1p) was distinct from that of WT samples (WTp) processed in parallel (Fig. 3B). We

156 detected bona fide axonemal proteins, including DRC1 and axonemal dynein subunits in some WT

157 replicates, but these were enriched in DRC1-APEX2 samples relative to WT. We therefore assembled a

158 “DRC1p proximity proteome” that included only proteins meeting the following three criteria: i)

159 detected in all four replicates of the DRC1p sample, ii) had a normalized spectrum count of two or more,

160 and iii) were enriched in the DRC1p versus WTp sample. This yielded a DRC1p proximity proteome of

161 697 proteins (Supplemental Table S1). Human homologues were identified for 372 proteins in the

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162 DRC1p proximity proteome and among these, 38 are linked to human diseases that have been

163 connected to cilium defects (Table 1).

164 To evaluate whether APEX proximity labeling was effective in identifying flagellar proteins, we used

165 Gene Ontology (GO) analysis [42], comparison to prior T. brucei flagellar proteomes [30, 32, 34] and

166 independent tests of localization [43]. GO analysis demonstrated significant enrichment of flagellar

167 proteins in the DRC1p proximity proteome compared to the genome as a whole (Fig. 3C and D). As

168 discussed above, our efforts were focused on the pellet fraction. We did however, complete GO analysis

169 on the detergent-soluble “DRC1s proximity proteome”, which also showed significant enrichment of

170 flagellar proteins, as well as signaling proteins (Supplemental Fig. S2).

171 When compared with prior proteomic analyses of T. brucei flagella, the DRC1p proximity proteome

172 encompassed a larger fraction (45%) of the flagellum skeleton proteome [30] than of the intact

173 flagellum proteome (36%) [34], perhaps due to the fact that the latter includes detergent-soluble

174 proteins, which are not expected in the DRC1p proximity proteome. As anticipated, minimal overlap was

175 observed with the flagellum surface plus matrix proteome [32], which includes only detergent-soluble

176 proteins.

177 TrypTag localization data [43] were available for 677 proteins in the DRC1p proximity proteome and 509

178 of these (75%) are annotated as having TrypTag localization that includes one or more flagellum

179 structures. The DRC1p proximity proteome includes 350 proteins that were not identified in prior

180 proteomic analyses of the T. brucei flagellum or axoneme fragments [16, 25, 30-32, 34] (Supplemental

181 Table S1A). TrypTag localization data were available for 337 of these and 241 (71%) are annotated as

182 having a TrypTag localization to one or more flagellum structures. In some cases, localization was

183 specific to flagellum structures, while in others the protein showed multiple locations. This finding

184 supports the idea that many of these 350 proteins are bona fide flagellar proteins despite going

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185 undetected in earlier flagellum proteome studies. The combined results demonstrate that APEX2

186 proximity labeling is functional in T. brucei and enables identification of flagellar proteins without the

187 need to purify the flagellum. The data also indicate protein composition of the T. brucei flagellum is

188 more complex than indicated by earlier studies alone.

189 APEX labeling readily distinguishes proteins in close proximity but separated by a membrane [36] and

190 this is evidenced in our data when considering protein components of the FAZ [19]. Proteins on the

191 flagellar side of the FAZ are substantially enriched in the DRC1-APEX2 sample, whereas proteins on the

192 cell body side of the FAZ are not (Supplemental Fig. S3). Furthermore, the short half-life of the biotin-

193 phenol radical [35] means that APEX labeling can resolve proteins separated by distance even in the

194 absence of a membrane boundary. As discussed above and shown previously [44], APEX resolves

195 flagellar versus cytoplasmic proteins despite these two compartments being contiguous. Within the

196 DRC1p proximity proteome, we noted that proteins distributed similarly to DRC1 along the axoneme

197 were well-represented, while proteins restricted to the distal or proximal end of the flagellum were less

198 represented (Fig. 4 and Supplemental Table S2). While total abundance may contribute to this result, it

199 nonetheless suggested that beyond flagellum versus cytoplasm, APEX labeling might also be able to

200 resolve proteins from different subdomains within the flagellum. We were particularly interested in the

201 flagellum tip because of its importance in trypanosomes and other organisms for signal transduction

202 [10, 11, 45, 46], flagellum length regulation [47-51] and interaction with host tissues [9].

203 We generated APEX2-tagged versions of flagellar proteins that are tip-specific, AC1 [29], or tip-excluded,

204 FS179 [32]. Expression of either AC1-APEX2 or FS179-APEX2 did not affect T. brucei doubling time

205 (Supplemental Fig. S1) and both tagged proteins fractionated in the detergent-soluble fraction as

206 expected. To assess biotinylation, AC1-APEX2 and FS179-APEX2-expressing cells were incubated with

207 biotin-phenol, activated with H2O2, then quenched, probed with streptavidin-Alexa 594 and examined

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208 by fluorescence microscopy (Fig. 5A-D). The signal in AC1-APEX2 expressors was enriched at the

209 flagellum tip, while the signal in FS179-APEX2 expressors was distributed along the flagellum but lacking

210 or diminished at the flagellum tip (Fig. 5A-D). Samples were solubilized with detergent and centrifuged

211 to remove insoluble material. Biotinylated proteins were isolated from the soluble fraction by

212 streptavidin affinity purification and then identified by shotgun proteomics. As negative controls,

213 samples from WT cells without an APEX2 tag and cells expressing AC145-APEX2, an AC1 truncation that

214 lacks the C-terminal 45aa and is localized to the cytoplasm instead of the flagellum [29], were processed

215 in parallel (Fig. 5E).

216 Principal component analysis demonstrated that the biotinylated protein profile of AC1-APEX2

217 detergent-soluble (AC1s) samples was readily distinguished from that of FS179-APEX2 (FS179s) and WT

218 (2913s) controls (Fig. 6A). Therefore, APEX2 proximity proteomics was able to distinguish protein

219 composition of the tip versus FAZ subdomains within the flagellum, even though they have no physical

220 barrier between them.

221 To define an “AC1s proximity proteome”, we compared the biotinylated protein profile of the AC1s

222 sample to that of 2913s and AC145s samples processed in parallel. We used known flagellum tip

223 proteins (Supplemental Table S3) to set the enrichment threshold for inclusion in the AC1s proximity

224 proteome. Finally, we included only those proteins that were enriched in AC1s versus FS179s and DRC1s.

225 This yielded a final AC1s proximity proteome of 48 proteins, including 27 adenylate cyclases and 21

226 additional proteins (Table 2). Extensive sequence identity among adenylate cyclases poses challenges for

227 distinguishing between some isoforms, so the number of 27 might be an overestimate. Nonetheless,

228 adenylate cyclases represent the majority of proteins identified. We performed a parallel analysis to

229 define an “FS179s proximity proteome”, in this case comparing to 2913s and AC145s samples as

230 negative controls and using intraflagellar transport (IFT) proteins [52] to set the enrichment threshold

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231 for inclusion. Gene ontology analysis showed enrichment of flagellar proteins and signaling proteins in

232 the AC1s proximity proteome, while the FS179s proximity proteome is enriched for flagellar proteins,

233 but not signaling proteins (Fig. 6B and C).

234 Prevalence of adenylate cyclases within the AC1s proximity proteome supports the idea that the dataset

235 is enriched for tip proteins, because all T. brucei adenylate cyclases studied to date are flagellar and, in

236 procyclics, many are enriched at the flagellum tip [29]. AC2 is localized all along the flagellum [29], yet it

237 is found in the AC1s proximity proteome, perhaps due to the fact that AC2 and AC1 dimerize and share

238 ~90% amino acid sequence identity [29].

239 Among the 21 non-AC proteins in the AC1s proximity proteome, 20 have independent data on

240 localization [43]. Four of these have previously been published as being flagellum tip-specific (FLAM8

241 and CALP1.3), flagellum-specific and tip-enriched (KIN-E), or located throughout the cell but also found

242 in the flagellum tip (CALP7.2) [34, 53, 54]. For the remaining 16 proteins we assessed localization by

243 referencing the TrypTag database [43] and/or epitope tagging directly. We find that half of these 16

244 proteins are either tip-specific or enriched at the flagellum tip while also being located elsewhere in the

245 cell (Fig. 7 and Table 2). Notably, most of the proteins that did not exhibit tip localization were among

246 the least enriched in the AC1s versus FS179s samples (Table 2). Among 11 proteins enriched >2-fold in

247 AC1s versus FS179s and having localization data, 9 are enriched in the flagellum tip. Therefore, the AC1s

248 proximity proteome is enriched for flagellum tip proteins.

249 DISCUSSION

250 The protein composition of flagellum subdomains in T. brucei is a knowledge gap in understanding the

251 biology of these pathogens. To overcome this, we implemented APEX2 proximity proteomics. Our

252 results demonstrate that APEX-based proximity labeling is effective in T. brucei and is capable of

253 resolving flagellum subdomains even if they are not separated by physical barriers. Use of the APEX

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254 system has allowed us to define a soluble flagellum tip proteome that indicates the tip is enriched for

255 signaling proteins. While this tip proteome is likely incomplete, our work represents an important step in

256 defining protein composition of flagellum subdomains and other trypanosome cellular compartments

257 that cannot themselves be purified.

258 One major advantage of proximity labeling-based proteomics versus other proteomic approaches to

259 define organelle protein composition is that it allows for isolation of proteins of interest from crude cell

260 lysates in a simple, one-step purification, without the need to purify the organelle. Prior proteomic

261 analyses of the T. brucei flagellum have required purification of the flagellum away from the cell body

262 [34]. This is problematic, because the flagellum in T. brucei is laterally connected to the cell body along

263 most of its length. Therefore, purification approaches have employed genetic manipulation to remove

264 lateral connections, followed by sonication or shearing to detach the flagellum at its base [32, 34] or

265 have employed detergent extraction followed by selective depolymerization of subpellicular

266 microtubules while leaving axoneme microtubules intact [30]. The latter approach does not allow for

267 identification of flagellum matrix or membrane proteins that are detergent-soluble. In both cases,

268 flagellum detachment is followed by centrifugation to separate flagellum fractions from cell bodies and

269 solubilized material, then electron microscopy to evaluate sample quality. The APEX approach eliminates

270 the need for subcellular fractionation and evaluation of the purified flagellum sample, although it can be

271 coupled with fractionation to distinguish detergent-soluble from insoluble proteins, as done in our case.

272 APEX labeling also enables easy detection of detergent-soluble and insoluble proteins from the same

273 cells. A disadvantage of proximity labeling-based proteomics is that owing to the requirement for

274 proteins to be close to the APEX-tagged bait, one might miss proteins that are part of the organelle in

275 question, but distant from the bait protein. For this reason, efforts to define a comprehensive, whole-

276 organelle proteome should employ multiple independent approaches.

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277 Our ability to distinguish protein profiles of AC1s and FS179s samples (Fig 6A) illustrates the capacity of

278 APEX to distinguish between subcellular regions that are not separated by a physical barrier. Although

279 this capacity was shown previously in mammalian cells for the cilium compartment versus the cytoplasm

280 [44] and for distinct locations in the cytoplasm [55], our studies now demonstrate this is also possible

281 within the spatially-restricted volume of the flagellum. The eukaryotic flagellum is comprised of specific

282 subdomains, each with specialized functions and protein composition [29]. The distal tip of the flagellum

283 in many organisms, for example, is important for transduction of extracellular signals, flagellum length

284 regulation and cell-cell adhesion [10, 11, 45-51]. The transition zone at the flagellum’s proximal end has

285 specialized functions controlling access into and out of the flagellar compartment [16, 56]. Even dyneins

286 are distributed differentially from proximal to distal ends of the axoneme [57]. APEX labeling has

287 previously been used to identify proteins throughout the cilium in mammalian cells [44]. To our

288 knowledge however, our studies are the first to extend this system to distinguishing protein composition

289 of specific flagellum subdomains, thus providing a powerful addition to tools available for dissecting

290 flagellum function and mechanisms of ciliary compartmentation.

291 Prior work has provided important advances by determining protein composition of two specific

292 subdomains within the T. brucei flagellum, the transition zone [16] and tip [58] of the detergent-

293 insoluble axoneme, the latter including an “axonemal capping structure” (ACS) and the T. brucei-specific

294 flagellar connector (FC). Those studies developed a novel method termed “structure

295 immunoprecipitation” (SIP), in which detergent-insoluble axonemes are prepared, then fragmented into

296 small pieces and immunoprecipitated using antibody to a marker protein localized to the domain of

297 interest. Independent localization, biochemical and functional analysis were then used to define a

298 corresponding transition zone, ACS and FC proteomes. Given that the FC and ACS are at the tip of the

299 flagellum, we compared their proteomes to the AC1s proximity proteome. We did not find any overlap,

300 as might be expected because the FC and ACS analyses were restricted to detergent-insoluble

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301 components, while the AC1s proximity proteome uniquely identifies detergent-soluble proteins. Our

302 work therefore complements and extends earlier proteome analyses of T. brucei flagellum sub-domains

303 and expands our knowledge of proteins that mediate flagellum tip-specific functions.

304 The AC1s proximity proteome is enriched for known and previously unknown flagellum tip proteins (Fig.

305 7 and Table 2). Function for many of these proteins remains to be determined but features and

306 properties in some cases indicate a role in signal transduction. One, previously identified as “cAMP

307 response protein 3” (CARP3), is a candidate cAMP effector [59] and over half of the proteins are

308 adenylate cyclases. A role for the flagellum tip in cAMP signaling has been previously demonstrated [10,

309 11] and flagellar cAMP signaling is required for the T. brucei transmission cycle in the tsetse fly [12].

310 Another protein group well-represented in the AC1s proximity proteome is calpain-like proteins.

311 Calpains function in Ca++ signal transduction, although the calpain-like proteins in the AC1s proximity

312 proteome possess only a subset of the domains typically seen in more classical calpains. Interestingly,

313 the flagellar tip kinesin KIN-E [53] contains a domain III-like domain typically found in calpains and

314 thought to function in lipid binding [60]. The AC1s proximity proteome includes four proteins annotated

315 as having homology to a serine-rich adhesin protein from bacteria [61]. Whether these function as

316 adhesins in the flagellum is unknown, but adhesion functions could contribute to attachment of the

317 axoneme to the flagellar membrane or in attachment functions of the FC [58].

318 The DRC1p proximity proteome showed substantial overlap with earlier proteome analyses of purified

319 flagellum skeletons [16, 30, 31, 34], while also including 350 proteins not found in those prior studies.

320 Independent data on 241 of these 350 proteins support flagellum association for 71%, indicating protein

321 composition of the T. brucei flagellum is more complex than suggested from earlier studies. Many

322 proteins uncovered in prior flagellum proteome analyses were not found in the DRC1p proximity

323 proteome. There are several potential explanations for this. First, some earlier studies include

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324 detergent-soluble matrix and membrane proteins [32, 34], which are not expected in the DRC1p

325 proximity proteome. Second, the threshold for inclusion in the DRC1p dataset is high, as proteins must

326 be present in all four DRC1p replicates and enriched over negative controls, while some of the earlier

327 studies examined a limited number of replicates [30], or lacked extensive negative controls [32]. Third,

328 each approach will contain false positives and false negatives. Lastly, proteins that are far away from

329 DRC1 might not be biotinylated in our analysis. We recognize that several bona fide axonemal proteins

330 were identified in WT, negative control samples processed in parallel to DRC1p samples. We do not

331 presently know the reason for this, but the problem was less evident in the analysis of detergent-soluble

332 AC1s samples, so may relate to residual insolubility of the axoneme, or high abundance of axoneme

333 proteins, which may be present at thousands of copies per axoneme.

334 To our knowledge, this is the first work to demonstrate APEX2 proximity proteomics in a pathogenic

335 eukaryote. This is an important point, because presence of systems for removing reactive oxygen

336 species in any given organism may limit suitability of APEX-based labeling [36]. For example,

337 Plasmodium spp. rely on a series of redundant glutathione- and thioredoxin-dependent reactions to

338 remove H2O2 and maintain redox equilibrium [62]. BioID [63] is an alternative proximity-labeling method

339 that has been used widely, including several applications in T. brucei [64] and other parasites [65]. BioID

340 and APEX methods are complementary, with APEX offering the added advantage of capacity for doing

341 time course analyses. Another advantage is that APEX catalyzes oxidation of 3,3’-diaminobenzidine

342 (DAB), which polymerizes and can be visualized by electron microscopy to determine precise subcellular

343 location of the APEX-tagged protein [66]. Therefore, our studies expand the capacity in T. brucei for

344 proximity-labeling, which is an increasingly important tool in post-genomic efforts to define protein

345 location and function in pathogenic organisms [67].

346 MATERIALS AND METHODS

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347 Trypanosoma brucei culture

348 Procyclic Trypanosoma brucei brucei (strain 29-13) [68] was used as control and to generate all the

349 APEX2-tagged cell lines. Cells were cultivated in SM media supplemented with 10% heat inactivated fetal

350 bovine serum and incubated at 28°C with 5% CO2. Selection for transformants was done using 1 μg/mL

351 of puromycin.

352 In situ Tagging

353 APEX2-tagged cell lines were generated by in situ tagging [69]. In each case, cells were transfected with

354 a cassette containing the APEX2-NES tag [35] followed by a puromycin resistance marker and flanked on

355 the 5’ end by the 3’ end of the target gene ORF and on the 3’ end by the target gene 3’ UTR. For AC1

356 (Tb927.11.17040) and FS179 (Tb927.10.2880), the AC1-HA [32] and FS179-HA [29] in situ tagging vectors

357 were modified by inserting the APEX2-NES tag in-frame between the target gene ORF and HAx3 tag to

358 generate the final APEX2 tagging cassettes. For DRC1 (Tb927.10.7880), the last 592 bp of the ORF were

359 PCR-amplified from genomic DNA and cloned upstream of the HAx3 tag in the pMOTag2H [69] vector

360 backbone. Similarly, the first 404 bp of the DRC1 3’-UTR were PCR-amplified from genomic DNA and

361 cloned downstream of the puromycin resistance marker. The APEX2-NES tag was then inserted between

362 the target gene ORF and HAx3 tag as described above. All sequences were verified by DNA sequencing.

363 Tagging cassettes were excised from the pMOTag backbone and gel purified. Trypanosome cells were

364 transfected by electroporation and selected with puromycin as described [39].

365 Biotinylation

366 Biotinylation was done using a modified version of [36]. Briefly, cells were harvested by centrifugation

367 for 10 min at 1,200 x g, then resuspended at 2 × 107 cells/mL in growth medium supplemented with 5

368 mM biotin-phenol (biotin tyramide, Acros Organics). After a one-hour incubation, cells were treated

Page 17 of 33

369 with 1 mM H2O2 for one minute. To quench unreacted hydrogen peroxide, an equal volume of 2x

370 quenching buffer (10 mM Trolox and 20 mM L-Ascorbic Acid Sodium Salt in PBS, pH 7.2) was added, cells

371 were harvested by centrifugation and two additional washes were made with 1x quenching buffer.

372 Immunofluorescence

373 Cells were washed once in PBS and fixed by addition of paraformaldehyde to 0.1% for 5 min on ice.

374 Fixed cells were washed once in PBS and air-dried onto coverslips. The coverslips were incubated for 10

375 min in -20°C methanol followed by 10 min in -20°C acetone, and then air-dried. Cells were re-hydrated

376 for 15 min in PBS and blocked overnight in blocking solution (PBS + 5% bovine serum albumin [BSA] + 5%

377 normal donkey serum). Coverslips were incubated with Streptavidin coupled to Alexa 594 (Life

378 Technologies) and anti-PFR [29] diluted in blocking solution for 1.5 h. After three washes in PBS + 0.05%

379 Tween-20 for 10 min each, coverslips were incubated with donkey anti-rabbit IgG coupled to Alexa 488

380 (Invitrogen) in blocking solution for 1.5 h. After three washes in PBS + 0.05% Tween-20 for 10 min each,

381 cells were fixed with 4% paraformaldehyde for 5 min. Coverslips were washed three times in PBS +

382 0.05% Tween-20 and one time in PBS for 10 min each. Cells were mounted with Vectashield containing

383 DAPI (Vector). Images were acquired using a Zeiss Axioskop II compound microscope and processed

384 using Axiovision (Zeiss, Inc., Jena, Germany) and Adobe Photoshop (Adobe Systems, Inc., Mountain

385 View, CA).

386 Fractionation of Whole Cells and Purification of Biotinylated Proteins

387 For purification of biotinylated proteins, 6 × 108 cells were washed once in PBS and lysed in lysis buffer:

388 PEME buffer (100 mM PIPES • 1.5 Na, 2 mM EGTA, 1 mM MgSO4 • 7 H2O, and 0.1 mM EDTA-Na2 • 2

389 H2O) + 0.5% Nonidet P-40 (NP40) + EDTA free protease inhibitors (Sigma), for 10 min on ice. Lysates

390 were centrifuged for 8 min at 2,500 x g at room temperature (RT) to separate the NP40-soluble

Page 18 of 33

391 supernatant and the insoluble pellet. The pellet was boiled for 5 min in lysis buffer + 1% SDS, centrifuged

392 for 3 min at 21,000 x g at RT to remove insoluble debris and SDS supernatant collected.

393 Capture of biotinylated proteins from the NP40-soluble and SDS supernatants on streptavidin beads was

394 done essentially as described [32]. Cell fractions were incubated with 120 µL streptavidin beads (GE

395 Healthcare) overnight at 4°C with gentle agitation. Biotinylated proteins bound to Streptavidin beads

396 were separated from the unbound molecules by centrifugation. Beads were washed once with lysis

397 buffer at 4°C, whereas the rest of the washes were made at RT as follows: once in buffer A (8 M urea,

398 200 mM NaCl, 2% SDS, and 100 mM Tris), once in buffer B (8 M urea, 1.2 M NaCl, 0.2% SDS, 100 mM

399 Tris, 10% ethanol, and 10% isopropanol), once in buffer C (8 M urea, 200 mM NaCl, 0.2% SDS, 100 mM

400 Tris, 10% ethanol, and 10% isopropanol), and 5 times in buffer D (8 M urea, and 100 mM Tris); pH of all

401 wash buffers was 8.

402 Shotgun Proteomics

403 Shotgun proteomics was done based on [32]. Streptavidin-bound proteins were digested on beads by

404 the sequential addition of lys-C and trypsin protease. Peptide samples were fractionated online using

405 reversed phase chromatography followed by tandem mass spectrometry analysis on a Thermofisher Q-

406 Exactive™ mass spectrometer (ThermoFisher). Data analysis was performed using the IP2 suite of

407 algorithms (Integrated Proteomics Applications). Briefly, RawXtract (version 1.8) was used to extract

408 peaklist information from Xcalibur-generated RAW files. Database searching of the MS/MS spectra was

409 performed using the ProLuCID algorithm (version 1.0) and a user assembled database consisting of all

410 protein entries from the TriTrypDB for T. brucei strain 927 (version 7.0). Other database search

411 parameters included: 1) precursor ion mass tolerance of 10 ppm, 2) fragment ion mass tolerance of

412 10ppm, 3) only peptides with fully tryptic ends were considered candidate peptides in the search with

413 no consideration of missed cleavages, and 4) static modification of 57.02146 on cysteine residues.

Page 19 of 33

414 Peptide identifications were organized and filtered using the DTASelect algorithm which uses a linear

415 discriminate analysis to identify peptide scoring thresholds that yield a peptide-level false discovery rate

416 of less than <1.8% as estimated using a decoy database approach. Proteins were considered present in

417 the analysis if they were identified by two or more peptides using the <1.8% peptide-level false

418 discovery rate.

419 For principal component analysis (PCA) and comparison of proteins identified in each sample,

420 proteomics data were parsed using the IP2 Integrated Proteomics Pipeline Ver. 5.1.2. The output from

421 the Protein Identification STAT Compare tool (IDSTAT_COMPARE) in IP2 was processed using a custom

422 R-based web app (https://uclaproteomics.shinyapps.io/iscviewer/) to generate the PCA graphs. For

423 proteins identified in different samples, the ID_COMPARE output was exported to Excel to generate

424 mass spectrometry data analysis tables (Supplemental Tables S4 and S5).

425 For the DRC1p proximity proteome, data were from three independent experiments each for DRC1-

426 APEX2 and 29-13 (WT) pellet samples. In one case each, the sample was split into two aliquots and

427 shotgun proteomics was done on both in parallel, giving a total of four replicates each for DRC1-APEX2

428 and WT samples. Ratios of average normalized spectra were used to determine inclusion in the DRC1p

429 proximity proteome as described in the text. Of 739 proteins identified, 42 redundant GeneIDs were

430 removed for a total proximity proteome of 697 (Table S1 and S4). For the AC1s proximity proteome,

431 data were from four independent experiments for 29-13 samples (0313, 0410, 0629-A and 0629-B) and

432 two independent experiments each for AC1 (0313, 0410) and FS179 (0629-A and O629-B). For the 0313

433 and 0410 experiments, each sample was split into two aliquots and shotgun proteomics done in parallel

434 on each, for a total of six replicates for WT and four replicates for AC1. Ratios of average normalized

435 spectra were used to determine inclusion in the AC1s proximity proteome as described in the text.

436 Bioinformatics

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437 To identify human homologues, we developed an algorithm to automatically return reciprocal best blast

438 hits from the large lists of proteins produced by shotgun proteomics. The algorithm was implemented in

439 python using the biopython library

440 (https://academic.oup.com/bioinformatics/article/25/11/1422/330687) and works as follows. Individual

441 sequences were parsed sequentially and used as a query for BLASTp to find a list of similar sequences

442 with an e-value threshold of 0.1 from a database of human protein sequences retrieved from NCBI. The

443 top three most similar sequences were then used as query sequences for a subsequent BLASTp call

444 against a database of T. brucei protein sequences. If the original sequence was found in the top three

445 from this call then the query was then returned as a homologue. The python code for performing this

446 can be found at the following URL: https://github.com/marcusgj13/Reciprocal-BB. Comparison to

447 published T. brucei flagellar proteomes [30, 32, 34] was done using the search tools in the TriTryp

448 Genome Database (https://tritrypdb.org/tritrypdb/) [61]. Word clouds (Figures 3, 6 and S2) were

449 obtained using the GO Enrichment tool of TriTrypDB, using T. brucei brucei TREU927 and a 0.05 P-Value

450 cutoff.

451 To assess protein localization as determined by TrypTag [43], proteins in the DRC1p proximity proteome

452 dataset were cross-referenced with TrypTag to look at matches vs mis-matches. The GO terms searched

453 for were: flagellum, transition zone, flagellar cytoplasm, pro-basal body, basal body, flagellar membrane,

454 flagella connector, paraflagellar rod, flagellum attachment zone, intraflagellar transport particle, hook

455 complex, ﬂagellar tip, and axoneme. For the full DRC1p proximity proteome, the number of genes that

456 were searched for was 739. The number of genes for which there is TrypTag localization data for at least

457 one terminus was 677. The number of TrypTag-tagged genes that matched a flagellum GO term were

458 509. The number of TrypTag-tagged genes that DID NOT match a flagellum GO term was 168. Therefore

459 of 677 proteins with TrypTag localization data, 75% have a TrypTag localization that matches the query

460 GO terms. For DRC1p proximity proteome proteins not found in earlier proteome analyses, the number

Page 21 of 33

461 of query genes searched for was 350. The number of query genes for which there is TrypTag localization

462 data for at least one terminus was 337. The number of query genes where TrypTag localization

463 MATCHES the query GO term was 241. The number of query genes where TrypTag localization MIS-

464 MATCHES the query GO term was 96. Therefore, of 337 proteins with TrypTag localization data, 71%

465 have a TrypTag localization that MATCHES the query GO terms.

466 Data availability

467 The python code used to identify human homologues was deposited in GitHub, and it can be found at

468 the following URL: https://github.com/marcusgj13/Reciprocal-BB. The code, explanation of it, as well as

469 instructions to install and run the program can be found there.

470 ACKNOWLEDGMENTS

471 We thank Vincent Tran for help with molecular cloning. Funding was provided by National Institutes of

472 Health grants AI052348 and AI142544 to K.H., and GM089778 to J.A.W. The authors would like to thank

473 the TrypTag consortium, which is supported by the Wellcome Trust [108445/Z/15/Z], for providing data

474 which enabled part of this work. D.E.V.R. was recipient of a Fulbright scholarship trough the United

475 States-Mexico Commission for Educational and Cultural Exchange (COMEXUS), a postdoctoral fellowship

476 of the Mexican National Council of Science and Technology (CONACyT) (CVU #325790), and a

477 postdoctoral fellowship of the University of California Institute for Mexico and the United States (UC

478 MEXUS), in conjunction with CONACyT. The funders did not have any role on the study design, data

479 collection and interpretation, nor the decision to submit the work for publication.

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632 2004. 117(Pt 9): p. 1641-51.

633 59. Gould, M.K., S. Bachmaier, J.A. Ali, S. Alsford, D.N. Tagoe, J.C. Munday, A.C. Schnaufer, D. Horn,

634 M. Boshart, and H.P. de Koning, Cyclic AMP effectors in African trypanosomes revealed by

635 genome-scale RNA interference library screening for resistance to the phosphodiesterase

636 inhibitor CpdA. Antimicrob Agents Chemother, 2013. 57(10): p. 4882-93.

637 60. Tompa, P., Y. Emori, H. Sorimachi, K. Suzuki, and P. Friedrich, Domain III of calpain is a ca2+-

638 regulated phospholipid-binding domain. Biochem Biophys Res Commun, 2001. 280(5): p. 1333-9.

639 61. Aslett, M., C. Aurrecoechea, M. Berriman, J. Brestelli, B.P. Brunk, M. Carrington, D.P. Depledge,

640 S. Fischer, B. Gajria, X. Gao, M.J. Gardner, A. Gingle, G. Grant, O.S. Harb, M. Heiges, et al.,

641 TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res, 2010.

642 38(Database issue): p. D457-62.

643 62. Jortzik, E. and K. Becker, Thioredoxin and glutathione systems in Plasmodium falciparum. Int J

644 Med Microbiol, 2012. 302(4-5): p. 187-94.

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645 63. Varnaite, R. and S.A. MacNeill, Meet the neighbors: Mapping local protein interactomes by

646 proximity-dependent labeling with BioID. Proteomics, 2016. 16(19): p. 2503-2518.

647 64. Morriswood, B., K. Havlicek, L. Demmel, S. Yavuz, M. Sealey-Cardona, K. Vidilaseris, D. Anrather,

648 J. Kostan, K. Djinovic-Carugo, K.J. Roux, and G. Warren, Novel bilobe components in

649 Trypanosoma brucei identified using proximity-dependent biotinylation. Eukaryot Cell, 2013.

650 12(2): p. 356-67.

651 65. Chen, A.L., E.W. Kim, J.Y. Toh, A.A. Vashisht, A.Q. Rashoff, C. Van, A.S. Huang, A.S. Moon, H.N.

652 Bell, L.A. Bentolila, J.A. Wohlschlegel, and P.J. Bradley, Novel components of the Toxoplasma

653 inner membrane complex revealed by BioID. MBio, 2015. 6(1): p. e02357-14.

654 66. Martell, J.D., T.J. Deerinck, S.S. Lam, M.H. Ellisman, and A.Y. Ting, Electron microscopy using the

655 genetically encoded APEX2 tag in cultured mammalian cells. Nat Protoc, 2017. 12(9): p. 1792-

656 1816.

657 67. Rees, J.S., X.W. Li, S. Perrett, K.S. Lilley, and A.P. Jackson, Protein Neighbors and Proximity

658 Proteomics. Mol Cell Proteomics, 2015. 14(11): p. 2848-56.

659 68. Wirtz, E., S. Leal, C. Ochatt, and G.A. Cross, A tightly regulated inducible expression system for

660 conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol

661 Biochem Parasitol, 1999. 99(1): p. 89-101.

662 69. Oberholzer, M., S. Morand, S. Kunz, and T. Seebeck, A vector series for rapid PCR-mediated C-

663 terminal in situ tagging of Trypanosoma brucei genes. Mol Biochem Parasitol, 2006. 145(1): p.

664 117-20.

665

666 Legends

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667 Figure 1. Schematic diagram of major flagellum substructures. Schematic depicts emergence of the

668 flagellum from the basal body near the cell’s posterior end (left), extending to the cell’s anterior end

669 (right). Major substructures are labeled.

670

671 Figure 2. APEX2 directs organelle-specific biotinylation in T. brucei.

672 Panel A. Western blot of whole cell lysate (W), NP40-extracted supernatant (S) and pellet (P) samples

673 from WT and DRC1-APEX2-expressing cells. Samples were probed with anti-HA antibody.

674 Panel B. Samples as in A were stained with SYPRO Ruby to assess loading.

675 Panel C. WT and DRC1-APEX2 cells were examined by immunofluorescence with anti-PFR antibody

676 (Alexa 488, green), Streptavidin (Alexa 594, red) and DAPI.

677

678 Figure 3. DRC1-APEX2 proximity proteome is enrichened for flagellar proteins.

679 Panel A. Scheme used to identify biotinylated proteins from WT and DRC1-APEX2 expressing T. brucei

680 cells.

681 Panel B. Principal Component Analysis of proteins identified in pellet fractions from WT cells (WTp) and

682 DRC1-APEX2 cells (DRC1p). The experiment was done using three independent biological replicates

683 (0313, 0623-A and 0623-B), and the 0313 protein sample was split into two aliquots and shotgun

684 proteomics done on each in parallel (0313-A1 and 0313-A2).

685 Panels C and D. Word clouds showing GO analysis of the DRC1p proximity proteome. Size and shading of

686 text reflects the p-value according to the scale shown.

687

688 Figure 4. Spatial distribution of proteins identified in the DRC1p proximity proteome.

689 Bars in the histogram indicate the percentage of proteins from each of the indicated complexes that

690 were identified in the DRC1p proximity proteome. Schematic below the histogram illustrates the relative

Page 31 of 33

691 distribution of the complexes indicated in the histogram, with the mitochondrion and kinetoplast

692 indicated in black at left.

693

694 Figure 5. APEX2-labeling resolves flagellum sub-domains.

695 Panels A, B. AC1-APEX2 cells were fixed and examined by fluorescence microscopy after staining with

696 Streptavidin (red) and DAPI (blue).

697 Panels C, D. FS179-APEX2 cells were examined by immunofluorescence with anti-PFR antibody (Alexa

698 488, green), Streptavidin (Alexa 594, red) and DAPI (blue). White brackets indicate the distal region of

699 the flagellum that is not labelled by streptavidin. Box shows zoomed in version of the cell in the middle

700 of the field.

701 Panel E. Scheme used to identify biotinylated proteins from the indicated cell lines (WT, AC-APEX2 and

702 FS179-APEX2).

703

704 Figure 6. APEX2 proximity proteomics differentiates protein composition of distinct flagellum

705 subdomains.

706 Panel A. Principal Component Analysis of proteins identified in supernatant fractions from WT (2913s),

707 AC1-APEX2 (AC1s) and FS179-APEX2 (FS179s) cells. For 2913 controls, three independent experiments

708 are shown (0410, 0629-A and 0629-B) and for one experiment the sample was split into two aliquots

709 (0410-A1 and 0410-A2) that were subjected to shotgun proteomics in parallel. For FS179s, two

710 independent experiments are shown (0629-A, 0629-B). For AC1s, one sample was split into two aliquots

711 that were subjected to shotgun proteomics in parallel (0410-A1, 0410-A2).

712 Panel B. Word cloud representing GO analysis for Cellular Component and Biological Process of the

713 proteins identified in the AC1 proximity proteome.

Page 32 of 33

714 Panel C. Word cloud representing GO analysis for Cellular Component and Biological Process of the

715 proteins identified in the FS179 proximity proteome.

716

717 Figure 7. AC1s proximity proteome identifies tip proteins.

718 Fluorescence microscopy of trypanosomes expressing the indicated protein tagged with neon green

719 (Green). Samples are stained with DAPI (Blue). Top panel shows fluorescence plus phase contrast

720 merged images. Bottom panel shows fluorescence image.

721

722 Supplemental Figure S1. Growth curves of APEX2-tagged cell lines.

723 Comparison of doubling times of 29-13 (parental cell line), AC1-APEX2, DRC1- APEX2, and FS179- APEX2

724 cell lines in suspension culture over the course of 8 days. Cell densities were adjusted daily to 1e6

725 cells/mL in order to insure logarithmic growth.

726

727 Supplemental Figure S2. Word cloud representing GO analysis for Cellular Component (A) and

728 Biological Process (B) of the proteins identified in the DRC1s proximity proteome.

729

730 Supplemental Figure S3. FAZ proteins identified on the DRC1p proximity proteome.

731 Panel A is a schematic of the FAZ. Panel B indicates the FAZ zone to which each protein has been

732 localized, as well as the ratio of spectra identified in the DRC1p versus 2913p sample. FAZ schematic and

733 zone locations are based on Sunter, J.D. and K. Gull, The Flagellum Attachment Zone: 'The Cellular Ruler'

734 of Trypanosome Morphology. Trends Parasitol, 2016. 32(4): p. 309-324.

735

Page 33 of 33 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 1. Human homologues with disease association T. brucei gene Human Human gene product Human disease phenotype e value homologue description Tb927.3.4020 XP_016884319.1 PI4KA phosphatidylinositol 4- Polymicrogyria, perisylvian, with cerebellar hypoplasia 2.00411e-94 kinase alpha isoform X4 and arthrogryposis Tb927.5.3650 EAW71112.1 MLPH melanophilin, isoform Griscelli syndrome type 3, identified in cancer 0.00600032 CRA_a susceptibility loci NP_055710.2 CEP162 centrosomal protein Associated with diabetic retinopathy 2.69684e-17 of 162 kDa isoform a Tb927.10.1510 AAH00779.2 CNOT1 protein, partial Associated with QT interval duration, PR interval, and 2.47459e-60 QRS duration Tb927.10.5380 XP_005247666.1 IFT122 intraflagellar transport Craneoectodermal dysplasia 1 0.0 protein 122 homolog isoform X13 EAW91274.1 asp (abnormal spindle)-like, Primary autosomal recessive microcephaly 5, lupus 0.000116493 microcephaly associated nephritis susceptibility in women Tb927.10.970 EAW91271.1 asp (abnormal spindle)-like, Primary autosomal recessive microcephaly 5, lupus 4.26269e-12 microcephaly associated, nephritis susceptibility in women, locus associated with isoform CRA_b ischemic stroke Tb927.8.6870 BAG06714.1 MYO5B variant protein Congenital microvillous atrophy 9.0414e-05 Tb927.7.7260 XP_011512661.1 kinesin-like protein KIF6 ADHD, antibody response to smallpox vaccine, dental 4.63801e- isoform X4 caries 135 Tb927.5.950 CAA09375.1 thioredoxin-like protein Susceptibility to HIV-1 1.87138e-32 Tb927.10.13000 EAW93986.1 PDE1C phosphodiesterase 1C, Endometriosis 8.12662e-66 calmodulin-dependent 70kDa, isoform CRA_b AAD50326.1 truncated RAD50 protein Nijmegen breakage syndrome-like disorder 5.70368e-59 Tb927.9.9580 AAF23275.2 KIAA0998, partial (tubulin Cone-rod dystrophy 19, susceptibility to ischemic 7.80409e-56 tyrosine ligase like 5) stroke and coronary heart disease EAW97654.1 UTP20, small subunit (SSU) Influences neurodegeneration in Alzheimer’s disease, 2.98428e-20 processome component, associated with subclinical atherosclerosis isoform CRA_c Tb927.10.9380 BAC04112.1 UBXN11 UBX domain protein Pathophysiology of childhood obesity 1.17265e-23 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

11 Tb927.8.5290 BAG64996.1 ITCH itchy E3 ubiquitin protein Autoimmune disease 2.61032e-17 ligase Tb927.11.3140 AAH31244.1 Dual-specificity tyrosine-(Y)- Allergic rhinitis 5.35331e- phosphorylation regulated 119 kinase 4 Tb927.5.1920 EAW64669.1 CCDC13 coiled-coil domain Clozapine-induced agranulocytosis, chronic 6.40896e-13 containing 13, isoform CRA_a periodontitis, pathophysiology of childhood obesity Tb927.8.5970 XP_011529701.1 CUL4B cullin-4B isoform X3 Syndromic X-linked mental retardation, Cabezas type 1.67244e-50 Tb927.3.4510 BAH13910.1 TRAF3 TNF receptor Schizophrenia, susceptibility to herpes simplex 0.0509298 associated factor 3 encephalitis 3 Tb927.8.4510 AAH36816.1 Thioredoxin domain Primary ciliary dyskinesia 6, risk of fracture, 2.4852e-06 containing 3 susceptibility for Alzheimer’s disease BAG37189.1 CCDC65 coiled-coil domain Primary ciliary dyskinesia 27 1.04609e-36 containing 65 Tb927.8.1890 AAA35730.1 cytochrome c1, partial Nuclear type 6 mitochondrial complex III deficiency 2.43602e-49 Tb927.8.5020 BAA25448.1 KIAA0522 protein, partial X-linked 1 mental retardation, triplosensitivity, 0.0592341 haploinsufficeincy CAD38878.1 VWA8 von Willebrand factor A Emphysema, TB risk 1.15659e- domain containing 8 116 Tb927.5.4110 AAH50721.1 CEP104 centrosomal protein Joubert syndrome 25 7.07883e-14 104 Tb927.3.1190 EAW64508.1 DLEC1 deleted in lung and Lung cancer, malignant tumor of esophagus 0.00498254 esophageal cancer 1, isoform CRA_a Tb927.6.4750 BAG56787.1 BUB3, mitotic checkpoint Age of menarche and natural menopause 0.000868412 protein Tb927.11.6350 XP_006712094.1 ATAD 2B ATPase family AAA Pathophysiology of childhood obesity 4.90261e- domain-containing protein 2B 102 isoform X10 Tb927.11.4210 AAC18038.1 antigen NY-CO-7 Autosomal recessive 16 spinocerebellar ataxia 0.00394461 Tb927.10.2900 BAG37316.1 KPNB1 karyopherin subunit Circulating levels of very long-chain saturated fatty 5.10086e-78 beta 1 acids, multiple sclerosis susceptibility Tb927.10.8390 AAH40929.1 HERC1 protein, partial Macrocephaly, dysmorphic facies, psychomotor 3.352e-64 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

retardation, neuronal pattern development Tb927.4.3810 BAF82512.1 POLR2B RNA polymerase II Age-related macular degeneration, adult height 0.0 subunit B Tb927.11.12430 AAT38107.1 CDC14A cell division cycle 14 Autosomal recessive 32 deafness 4.10273e-46 homolog A Tb927.7.1560 AAI03916.1 TTC6 protein Allergy-specific susceptibility 1.23027e-09 Tb927.7.290 XP_005250264.1 FBXL13 F-box/LRR-repeat Blood pressure response to interventions 1.17865e-09 protein 13 isoform X5 Tb927.11.14680 NP_001341508.1 serine/threonine-protein Familial cutaneous telangiectasia and cancer 1.05991e- kinase ATR isoform 2 syndrome, Seckel syndrome 1 113 Tb927.11.6430 XP_011524505.1 CCDC68 coiled-coil domain- Schizophrenia, severe diabetic retinopathy, psychiatric 0.0250287 containing protein 68 isoform disorders X3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Table 2. AC1s proximity proteome.

Source for location AC1s/FS179s TriTryp GeneID TriTryp Annotation Localization* data avg ratio&

Tb927.2.5860 Hypothetical Tip TrypTag1 15.88 Tb927.2.5870 Hypothetical Tip TrypTag 1 + this work 10.71 Tb927.4.1500 RNA editing associated helicase 2 Not Flagellar TrypTag1 7.45 Tb927.4.4400 Hypothetical Flagellum, distal points + Endocytic TrypTag1 5.18 Tb927.2.5760 Flagellar Member 8 Tip 2 5.03 Tb927.11.14410 Ankyrin repeats Not Flagellar TrypTag1 3.89 Tb927.1.2120 Calpain-like protein CALP1.3 Tip 3 3.60 Tb927.11.17040 AC1# Tip 4 3.37 Tb927.7.4060 cysteine peptidase, Clan CA, family C2 Flagellum, Tip-enriched + Cytoplasm TrypTag1 + 3 3.02 Tb927.9.7540 cysteine peptidase, Clan CA, family C2, putative Flagellum, Tip-enriched TrypTag 1 + this work 2.98 Tb927.7.4070 cysteine peptidase, Clan CA, family C2 Tip + Cytoplasm 3 2.86 Tb927.7.5340 cAMP response protein 3 Flagellum, Tip-enriched + Cytoplasm TrypTag + this Work 2.41 Tb927.10.15700 Hypothetical NA 2.11 Tb927.4.4220 small GTP-binding rab protein NA 2.11 1 Tb927.10.9380 SEP domain/UBX domain containing protein New Flagellum-distal points TrypTag 1.86 Tb927.5.2090 kinesin Flagellum + Cytoplasm TrypTag1 1.74 Tb927.6.4710 calmodulin Flagellum TrypTag1 1.68 Tb927.3.4640 VIT family Not Flagellar TrypTag1 1.50 Tb927.7.7260 kinesin Flagellum-puncta 5 1.32 Tb927.9.9690 Hypothetical Flagellum, Tip-enriched TrypTag1 1.22 Tb927.7.3090 Galactose Oxidase-central domain =-containing Not Flagellar TrypTag1 1.17 Tb927.5.2410 kinesin Flagellum, Tip-enriched TrypTag1 + 6 1.16 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

*Localization defined as: Tip = the prominent or sole location is flagellum tip. Flagellum, Tip-enriched = in the flagellum and enriched at tip. Tip- enriched = enriched at the flagellum tip, but also present outside the flagellum. 1Dean, S., J.D. Sunter, and R.J. Wheeler, TrypTag.org: A Trypanosome Genome-wide Protein Localisation Resource. Trends Parasitol, 2017. 33(2): p. 80-82. 2Subota, I., et al., Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub- localization and dynamics. Mol Cell Proteomics, 2014. 13(7): p. 1769-86. 3Liu, W., et al., Expression and cellular localisation of calpain-like proteins in Trypanosoma brucei. Mol Biochem Parasitol, 2010. 169(1): p. 20-6. 4Saada, E.A., et al., Insect stage-specific receptor adenylate cyclases are localized to distinct subdomains of the Trypanosoma brucei Flagellar membrane. Eukaryot Cell, 2014. 13(8): p. 1064-76. 5Demonchy, R., et al., Kinesin 9 family members perform separate functions in the trypanosome flagellum. J Cell Biol, 2009. 187(5): p. 615-22. 6An, T. and Z. Li, An orphan kinesin controls trypanosome morphology transitions by targeting FLAM3 to the flagellum. PLoS Pathog, 2018. 14(5): p. e1007101. #AC1 represents a total of 39 ACs identified in the AC1s proximity proteome (Supplementary Table S4). &Normalized spectral count average of all experiments and replicates of AC1s, over normalized spectral count average of all experiments and replicates of FS179s. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.984815; this version posted March 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.