2 proteins in Trypanosoma brucei · Page 3 of 33 25 ABSTRACT 26 Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne 27 disease. T

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

proteins in Trypanosoma brucei 2

3

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

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

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

7

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

Angeles, Los Angeles, California, USA 9

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

México, México 11

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

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

Angeles, California, USA 14

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

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

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

18

Running title: APEX2 proteomics in Trypanosoma brucei 19

20

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

(which was not certified by peer review) is the author/funder.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

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

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

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


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ABSTRACT 25

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

purified. 44

IMPORTANCE 45

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

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


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

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

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

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

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

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

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

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

INTRODUCTION 56

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

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

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

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

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

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

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

across the globe. 64

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

communication and signaling [11, 29]. 97

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

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

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

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

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

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

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

specific structure that can be purified. 105

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

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

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

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

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

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

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

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

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

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


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

subdomain is specialized for cell signaling. 117

RESULTS 118

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

biotinylation in the flagellum. 141

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

connected to cilium defects (Table 1). 163

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

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

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

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

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

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

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

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

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

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

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

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

proteins. 176

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

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

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

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

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

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

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

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


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

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

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

more complex than indicated by earlier studies alone. 188

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

samples from WT cells without an APEX2 tag and cells expressing AC145-APEX2, an AC1 truncation that 213

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

in parallel (Fig. 5E). 215

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

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

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

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

barrier between them. 220

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

sample to that of 2913s and AC145s samples processed in parallel. We used known flagellum tip 222

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

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

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

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

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

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

define an “FS179s proximity proteome”, in this case comparing to 2913s and AC145s samples as 229

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

proximity proteome is enriched for flagellum tip proteins. 248

DISCUSSION 249

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

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

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

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


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

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

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

that cannot themselves be purified. 257

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

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

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

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

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

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

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

have employed detergent extraction followed by selective depolymerization of subpellicular 265

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

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

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

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

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

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

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

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

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

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

organelle proteome should employ multiple independent approaches. 276


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

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

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

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

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

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

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

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

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

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

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

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

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

flagellum function and mechanisms of ciliary compartmentation. 290

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

location and function in pathogenic organisms [67]. 345

MATERIALS AND METHODS 346


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Trypanosoma brucei culture 347

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

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

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

of puromycin. 351

In situ Tagging 352

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

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

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 355

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

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

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

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

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

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

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

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

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

Biotinylation 365

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

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

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


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

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

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

Immunofluorescence 372

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

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

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

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

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

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

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

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

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

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

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

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

View, CA). 385

Fractionation of Whole Cells and Purification of Biotinylated Proteins 386

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

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

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

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


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

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

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

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

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

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

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

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 398

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

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

wash buffers was 8. 401

Shotgun Proteomics 402

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

discovery rate. 418

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Bioinformatics 436


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

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

python using the biopython library 439

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

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

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

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

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

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

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

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

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

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

cutoff. 450

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

Data availability 466

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

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

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

ACKNOWLEDGMENTS 470

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

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

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

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

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

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

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

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

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

REFERENCES 480


https://doi.org/10.1101/2020.03.09.984815

Page 23 of 33

1. Stuart, K., R. Brun, S. Croft, A. Fairlamb, R.E. Gurtler, J. McKerrow, S. Reed, and R. Tarleton, 481

Kinetoplastids: related protozoan pathogens, different diseases. J Clin Invest, 2008. 118(4): p. 482

1301-10. 483

2. FAO, Controling tsetse and trypanosomosis to protect African livestock keepers, public health 484

and farmers' livelihoods. 2019. 485

3. Langousis, G. and K.L. Hill, Motility and more: the flagellum of Trypanosoma brucei. Nat Rev 486

Microbiol, 2014. 12(7): p. 505-18. 487

4. Shimogawa, M.M., S.S. Ray, N. Kisalu, Y. Zhang, Q. Geng, A. Ozcan, and K.L. Hill, Parasite motility 488

is critical for virulence of African trypanosomes. Sci Rep, 2018. 8(1): p. 9122. 489

5. Rotureau, B., C.P. Ooi, D. Huet, S. Perrot, and P. Bastin, Forward motility is essential for 490

trypanosome infection in the tsetse fly. Cell Microbiol, 2014. 16(3): p. 425-33. 491

6. Ralston, K.S., A.G. Lerner, D.R. Diener, and K.L. Hill, Flagellar motility contributes to cytokinesis in 492

Trypanosoma brucei and is modulated by an evolutionarily conserved dynein regulatory system. 493

Eukaryot Cell, 2006. 5(4): p. 696-711. 494

7. Branche, C., L. Kohl, G. Toutirais, J. Buisson, J. Cosson, and P. Bastin, Conserved and specific 495

functions of axoneme components in trypanosome motility. J Cell Sci, 2006. 119(Pt 16): p. 3443-496

55. 497

8. Moreira-Leite, F.F., T. Sherwin, L. Kohl, and K. Gull, A trypanosome structure involved in 498

transmitting cytoplasmic information during cell division. Science, 2001. 294(5542): p. 610-2. 499

9. Vickerman, K., On the surface coat and flagellar adhesion in trypanosomes. J Cell Sci, 1969. 5(1): 500

p. 163-93. 501

10. Oberholzer, M., E.A. Saada, and K.L. Hill, Cyclic AMP Regulates Social Behavior in African 502

Trypanosomes. MBio, 2015. 6(3): p. e01954-14. 503


https://doi.org/10.1101/2020.03.09.984815

Page 24 of 33

11. Lopez, M.A., E.A. Saada, and K.L. Hill, Insect stage-specific adenylate cyclases regulate social 504

motility in African trypanosomes. Eukaryot Cell, 2015. 14(1): p. 104-12. 505

12. Shaw, S., S.F. DeMarco, R. Rehmann, T. Wenzler, F. Florini, I. Roditi, and K.L. Hill, Flagellar cAMP 506

signaling controls trypanosome progression through host tissues. Nat Commun, 2019. 10(1): p. 507

803. 508

13. Langousis, G., M.M. Shimogawa, E.A. Saada, A.A. Vashisht, R. Spreafico, A.R. Nager, W.D. 509

Barshop, M.V. Nachury, J.A. Wohlschlegel, and K.L. Hill, Loss of the BBSome perturbs endocytic 510

trafficking and disrupts virulence of Trypanosoma brucei. Proc Natl Acad Sci U S A, 2016. 113(3): 511

p. 632-7. 512

14. Salmon, D., G. Vanwalleghem, Y. Morias, J. Denoeud, C. Krumbholz, F. Lhomme, S. Bachmaier, 513

M. Kador, J. Gossmann, F.B. Dias, G. De Muylder, P. Uzureau, S. Magez, M. Moser, P. De 514

Baetselier, et al., Adenylate cyclases of Trypanosoma brucei inhibit the innate immune response 515

of the host. Science, 2012. 337(6093): p. 463-6. 516

15. Gould, M.K. and H.P. de Koning, Cyclic-nucleotide signalling in protozoa. FEMS Microbiol Rev, 517

2011. 35(3): p. 515-41. 518

16. Dean, S., F. Moreira-Leite, V. Varga, and K. Gull, Cilium transition zone proteome reveals 519

compartmentalization and differential dynamics of ciliopathy complexes. Proc Natl Acad Sci U S 520

A, 2016. 113(35): p. E5135-43. 521

17. Field, M.C. and M. Carrington, The trypanosome flagellar pocket. Nat Rev Microbiol, 2009. 7(11): 522

p. 775-86. 523

18. Cachon, J., M. Cachon, M.-P. Cosson, and J. Cosson, The paraflagellar rod: a structure in search 524

of a function. Biology of the Cell, 1988. 63(2): p. 169-181. 525

19. Sunter, J.D. and K. Gull, The Flagellum Attachment Zone: 'The Cellular Ruler' of Trypanosome 526

Morphology. Trends Parasitol, 2016. 32(4): p. 309-324. 527


https://doi.org/10.1101/2020.03.09.984815

Page 25 of 33

20. Sun, S.Y., C. Wang, Y.A. Yuan, and C.Y. He, An intracellular membrane junction consisting of 528

flagellum adhesion glycoproteins links flagellum biogenesis to cell morphogenesis in 529

Trypanosoma brucei. J Cell Sci, 2013. 126(Pt 2): p. 520-31. 530

21. Hoog, J.L., C. Bouchet-Marquis, J.R. McIntosh, A. Hoenger, and K. Gull, Cryo-electron 531

tomography and 3-D analysis of the intact flagellum in Trypanosoma brucei. J Struct Biol, 2012. 532

178(2): p. 189-98. 533

22. Hughes, L.C., K.S. Ralston, K.L. Hill, and Z.H. Zhou, Three-dimensional structure of the 534

Trypanosome flagellum suggests that the paraflagellar rod functions as a biomechanical spring. 535

PLoS One, 2012. 7(1): p. e25700. 536

23. Koyfman, A.Y., M.F. Schmid, L. Gheiratmand, C.J. Fu, H.A. Khant, D. Huang, C.Y. He, and W. Chiu, 537

Structure of Trypanosoma brucei flagellum accounts for its bihelical motion. Proc Natl Acad Sci U 538

S A, 2011. 108(27): p. 11105-8. 539

24. Oberholzer, M., P. Bregy, G. Marti, M. Minca, M. Peier, and T. Seebeck, Trypanosomes and 540

mammalian sperm: one of a kind? Trends Parasitol, 2007. 23(2): p. 71-7. 541

25. Portman, N., S. Lacomble, B. Thomas, P.G. McKean, and K. Gull, Combining RNA interference 542

mutants and comparative proteomics to identify protein components and dependences in a 543

eukaryotic flagellum. J Biol Chem, 2009. 284(9): p. 5610-9. 544

26. Sunter, J.D., C. Benz, J. Andre, S. Whipple, P.G. McKean, K. Gull, M.L. Ginger, and J. Lukes, 545

Modulation of flagellum attachment zone protein FLAM3 and regulation of the cell shape in 546

Trypanosoma brucei life cycle transitions. J Cell Sci, 2015. 128(16): p. 3117-30. 547

27. Hoog, J.L., S. Lacomble, C. Bouchet-Marquis, L. Briggs, K. Park, A. Hoenger, and K. Gull, 3D 548

Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction. 549

PLoS Negl Trop Dis, 2016. 10(1): p. e0004312. 550


https://doi.org/10.1101/2020.03.09.984815

Page 26 of 33

28. Rotureau, B. and J. Van Den Abbeele, Through the dark continent: African trypanosome 551

development in the tsetse fly. Front Cell Infect Microbiol, 2013. 3: p. 53. 552

29. Saada, E.A., Z.P. Kabututu, M. Lopez, M.M. Shimogawa, G. Langousis, M. Oberholzer, A. Riestra, 553

Z.O. Jonsson, J.A. Wohlschlegel, and K.L. Hill, Insect stage-specific receptor adenylate cyclases 554

are localized to distinct subdomains of the Trypanosoma brucei Flagellar membrane. Eukaryot 555

Cell, 2014. 13(8): p. 1064-76. 556

30. Broadhead, R., H.R. Dawe, H. Farr, S. Griffiths, S.R. Hart, N. Portman, M.K. Shaw, M.L. Ginger, S.J. 557

Gaskell, P.G. McKean, and K. Gull, Flagellar motility is required for the viability of the 558

bloodstream trypanosome. Nature, 2006. 440(7081): p. 224-7. 559

31. Varga, V., F. Moreira-Leite, N. Portman, and K. Gull, Protein diversity in discrete structures at the 560

distal tip of the trypanosome flagellum. Proc Natl Acad Sci U S A, 2017. 114(32): p. E6546-e6555. 561

32. Oberholzer, M., G. Langousis, H.T. Nguyen, E.A. Saada, M.M. Shimogawa, Z.O. Jonsson, S.M. 562

Nguyen, J.A. Wohlschlegel, and K.L. Hill, Independent analysis of the flagellum surface and 563

matrix proteomes provides insight into flagellum signaling in mammalian-infectious 564

Trypanosoma brucei. Mol Cell Proteomics, 2011. 10(10): p. M111.010538. 565

33. Shimogawa, M.M., E.A. Saada, A.A. Vashisht, W.D. Barshop, J.A. Wohlschlegel, and K.L. Hill, Cell 566

Surface Proteomics Provides Insight into Stage-Specific Remodeling of the Host-Parasite 567

Interface in Trypanosoma brucei. Mol Cell Proteomics, 2015. 14(7): p. 1977-88. 568

34. Subota, I., D. Julkowska, L. Vincensini, N. Reeg, J. Buisson, T. Blisnick, D. Huet, S. Perrot, J. Santi-569

Rocca, M. Duchateau, V. Hourdel, J.C. Rousselle, N. Cayet, A. Namane, J. Chamot-Rooke, et al., 570

Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel 571

flagellar proteins with unique sub-localization and dynamics. Mol Cell Proteomics, 2014. 13(7): 572

p. 1769-86. 573


https://doi.org/10.1101/2020.03.09.984815

Page 27 of 33

35. Lam, S.S., J.D. Martell, K.J. Kamer, T.J. Deerinck, M.H. Ellisman, V.K. Mootha, and A.Y. Ting, 574

Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods, 2015. 575

12(1): p. 51-4. 576

36. Hung, V., N.D. Udeshi, S.S. Lam, K.H. Loh, K.J. Cox, K. Pedram, S.A. Carr, and A.Y. Ting, Spatially 577

resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Nat Protoc, 578

2016. 11(3): p. 456-75. 579

37. Wirschell, M., H. Olbrich, C. Werner, D. Tritschler, R. Bower, W.S. Sale, N.T. Loges, P. 580

Pennekamp, S. Lindberg, U. Stenram, B. Carlen, E. Horak, G. Kohler, P. Nurnberg, G. Nurnberg, et 581

al., The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in 582

algae and humans. Nat Genet, 2013. 45(3): p. 262-8. 583

38. Oda, T., H. Yanagisawa, and M. Kikkawa, Detailed structural and biochemical characterization of 584

the nexin-dynein regulatory complex. Mol Biol Cell, 2015. 26(2): p. 294-304. 585

39. Oberholzer, M., M.A. Lopez, K.S. Ralston, and K.L. Hill, Approaches for functional analysis of 586

flagellar proteins in African trypanosomes. Methods Cell Biol, 2009. 93: p. 21-57. 587

40. Kabututu, Z.P., M. Thayer, J.H. Melehani, and K.L. Hill, CMF70 is a subunit of the dynein 588

regulatory complex. J Cell Sci, 2010. 123(Pt 20): p. 3587-95. 589

41. Lacomble, S., N. Portman, and K. Gull, A protein-protein interaction map of the Trypanosoma 590

brucei paraflagellar rod. PLoS One, 2009. 4(11): p. e7685. 591

42. Thomas, P.D., The Gene Ontology and the Meaning of Biological Function. Methods Mol Biol, 592

2017. 1446: p. 15-24. 593

43. Dean, S., J.D. Sunter, and R.J. Wheeler, TrypTag.org: A Trypanosome Genome-wide Protein 594

Localisation Resource. Trends Parasitol, 2017. 33(2): p. 80-82. 595

44. Mick, D.U., R.B. Rodrigues, R.D. Leib, C.M. Adams, A.S. Chien, S.P. Gygi, and M.V. Nachury, 596

Proteomics of Primary Cilia by Proximity Labeling. Dev Cell, 2015. 35(4): p. 497-512. 597


https://doi.org/10.1101/2020.03.09.984815

Page 28 of 33

45. Nager, A.R., J.S. Goldstein, V. Herranz-Perez, D. Portran, F. Ye, J.M. Garcia-Verdugo, and M.V. 598

Nachury, An Actin Network Dispatches Ciliary GPCRs into Extracellular Vesicles to Modulate 599

Signaling. Cell, 2017. 168(1-2): p. 252-263.e14. 600

46. He, M., S. Agbu, and K.V. Anderson, Microtubule Motors Drive Hedgehog Signaling in Primary 601

Cilia. Trends Cell Biol, 2017. 27(2): p. 110-125. 602

47. Blaineau, C., M. Tessier, P. Dubessay, L. Tasse, L. Crobu, M. Pages, and P. Bastien, A novel 603

microtubule-depolymerizing kinesin involved in length control of a eukaryotic flagellum. Curr 604

Biol, 2007. 17(9): p. 778-82. 605

48. Bertiaux, E., B. Morga, T. Blisnick, B. Rotureau, and P. Bastin, A Grow-and-Lock Model for the 606

Control of Flagellum Length in Trypanosomes. Curr Biol, 2018. 28(23): p. 3802-3814.e3. 607

49. Keeling, J., L. Tsiokas, and D. Maskey, Cellular Mechanisms of Ciliary Length Control. Cells, 2016. 608

5(1). 609

50. Fort, C. and P. Bastin, [Elongation of the axoneme and dynamics of intraflagellar transport]. Med 610

Sci (Paris), 2014. 30(11): p. 955-61. 611

51. He, M., R. Subramanian, F. Bangs, T. Omelchenko, K.F. Liem, Jr., T.M. Kapoor, and K.V. Anderson, 612

The kinesin-4 protein Kif7 regulates mammalian Hedgehog signalling by organizing the cilium tip 613

compartment. Nat Cell Biol, 2014. 16(7): p. 663-72. 614

52. Absalon, S., T. Blisnick, L. Kohl, G. Toutirais, G. Dore, D. Julkowska, A. Tavenet, and P. Bastin, 615

Intraflagellar transport and functional analysis of genes required for flagellum formation in 616

trypanosomes. Mol Biol Cell, 2008. 19(3): p. 929-44. 617

53. An, T. and Z. Li, An orphan kinesin controls trypanosome morphology transitions by targeting 618

FLAM3 to the flagellum. PLoS Pathog, 2018. 14(5): p. e1007101. 619

54. Liu, W., K. Apagyi, L. McLeavy, and K. Ersfeld, Expression and cellular localisation of calpain-like 620

proteins in Trypanosoma brucei. Mol Biochem Parasitol, 2010. 169(1): p. 20-6. 621


https://doi.org/10.1101/2020.03.09.984815

Page 29 of 33

55. Mavylutov, T., X. Chen, L. Guo, and J. Yang, APEX2- tagging of Sigma 1-receptor indicates 622

subcellular protein topology with cytosolic N-terminus and ER luminal C-terminus. Protein Cell, 623

2018. 9(8): p. 733-737. 624

56. Fisch, C. and P. Dupuis-Williams, Ultrastructure of cilia and flagella - back to the future! Biol Cell, 625

2011. 103(6): p. 249-70. 626

57. Edwards, B.F.L., R.J. Wheeler, A.R. Barker, F.F. Moreira-Leite, K. Gull, and J.D. Sunter, Direction of 627

flagellum beat propagation is controlled by proximal/distal outer dynein arm asymmetry. Proc 628

Natl Acad Sci U S A, 2018. 115(31): p. E7341-e7350. 629

58. Briggs, L.J., P.G. McKean, A. Baines, F. Moreira-Leite, J. Davidge, S. Vaughan, and K. Gull, The 630

flagella connector of Trypanosoma brucei: an unusual mobile transmembrane junction. J Cell Sci, 631

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

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

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

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

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

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

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

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

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

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

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

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

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


https://doi.org/10.1101/2020.03.09.984815

Page 30 of 33

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

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

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

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

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

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

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. 651

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

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

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

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

1816. 656

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

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

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

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

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

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

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

117-20. 664

665

Legends 666


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Page 31 of 33

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

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

(right). Major substructures are labeled. 669

670

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

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

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

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

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

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

677

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

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

cells. 680

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

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

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

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

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

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

687

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

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

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


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Page 32 of 33

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

indicated in black at left. 692

693

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

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

Streptavidin (red) and DAPI (blue). 696

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

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

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

of the field. 700

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

FS179-APEX2). 702

703

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

subdomains. 705

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

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

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

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

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

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

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

proteins identified in the AC1 proximity proteome. 713


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Page 33 of 33

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

proteins identified in the FS179 proximity proteome. 715

716

Figure 7. AC1s proximity proteome identifies tip proteins. 717

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

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

merged images. Bottom panel shows fluorescence image. 720

721

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

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

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

cells/mL in order to insure logarithmic growth. 725

726

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

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

729

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

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

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

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

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

735


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Table 1. Human homologues with disease association

T. brucei gene Human

homologue

Human gene product

description

Human disease phenotype e value

Tb927.3.4020 XP_016884319.1 PI4KA phosphatidylinositol 4-

kinase alpha isoform X4

Polymicrogyria, perisylvian, with cerebellar hypoplasia

and arthrogryposis

2.00411e-94

Tb927.5.3650 EAW71112.1 MLPH melanophilin, isoform

CRA_a

Griscelli syndrome type 3, identified in cancer

susceptibility loci

0.00600032

NP_055710.2 CEP162 centrosomal protein

of 162 kDa isoform a

Associated with diabetic retinopathy 2.69684e-17

Tb927.10.1510 AAH00779.2 CNOT1 protein, partial Associated with QT interval duration, PR interval, and

QRS duration

2.47459e-60

Tb927.10.5380 XP_005247666.1 IFT122 intraflagellar transport

protein 122 homolog isoform

X13

Craneoectodermal dysplasia 1 0.0

EAW91274.1 asp (abnormal spindle)-like,

microcephaly associated

Primary autosomal recessive microcephaly 5, lupus

nephritis susceptibility in women

0.000116493

Tb927.10.970 EAW91271.1 asp (abnormal spindle)-like,

microcephaly associated,

isoform CRA_b

Primary autosomal recessive microcephaly 5, lupus

nephritis susceptibility in women, locus associated with

ischemic stroke

4.26269e-12

Tb927.8.6870 BAG06714.1 MYO5B variant protein Congenital microvillous atrophy 9.0414e-05

Tb927.7.7260 XP_011512661.1 kinesin-like protein KIF6

isoform X4

ADHD, antibody response to smallpox vaccine, dental

caries

4.63801e-

135

Tb927.5.950 CAA09375.1 thioredoxin-like protein Susceptibility to HIV-1 1.87138e-32

Tb927.10.13000 EAW93986.1 PDE1C phosphodiesterase 1C,

calmodulin-dependent 70kDa,

isoform CRA_b

Endometriosis 8.12662e-66

AAD50326.1 truncated RAD50 protein Nijmegen breakage syndrome-like disorder 5.70368e-59

Tb927.9.9580 AAF23275.2 KIAA0998, partial (tubulin

tyrosine ligase like 5)

Cone-rod dystrophy 19, susceptibility to ischemic

stroke and coronary heart disease

7.80409e-56

EAW97654.1 UTP20, small subunit (SSU)

processome component,

isoform CRA_c

Influences neurodegeneration in Alzheimer’s disease, associated with subclinical atherosclerosis

2.98428e-20

Tb927.10.9380 BAC04112.1 UBXN11 UBX domain protein Pathophysiology of childhood obesity 1.17265e-23


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11

Tb927.8.5290 BAG64996.1 ITCH itchy E3 ubiquitin protein

ligase

Autoimmune disease 2.61032e-17

Tb927.11.3140 AAH31244.1 Dual-specificity tyrosine-(Y)-

phosphorylation regulated

kinase 4

Allergic rhinitis 5.35331e-

119

Tb927.5.1920 EAW64669.1 CCDC13 coiled-coil domain

containing 13, isoform CRA_a

Clozapine-induced agranulocytosis, chronic

periodontitis, pathophysiology of childhood obesity

6.40896e-13

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

associated factor 3

Schizophrenia, susceptibility to herpes simplex

encephalitis 3

0.0509298

Tb927.8.4510 AAH36816.1 Thioredoxin domain

containing 3

Primary ciliary dyskinesia 6, risk of fracture,

susceptibility for Alzheimer’s disease

2.4852e-06

BAG37189.1 CCDC65 coiled-coil domain

containing 65

Primary ciliary dyskinesia 27 1.04609e-36

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,

haploinsufficeincy

0.0592341

CAD38878.1 VWA8 von Willebrand factor A

domain containing 8

Emphysema, TB risk 1.15659e-

116

Tb927.5.4110 AAH50721.1 CEP104 centrosomal protein

104

Joubert syndrome 25 7.07883e-14

Tb927.3.1190 EAW64508.1 DLEC1 deleted in lung and

esophageal cancer 1, isoform

CRA_a

Lung cancer, malignant tumor of esophagus 0.00498254

Tb927.6.4750 BAG56787.1 BUB3, mitotic checkpoint

protein

Age of menarche and natural menopause 0.000868412

Tb927.11.6350 XP_006712094.1 ATAD2B ATPase family AAA

domain-containing protein 2B

isoform X10

Pathophysiology of childhood obesity 4.90261e-

102

Tb927.11.4210 AAC18038.1 antigen NY-CO-7 Autosomal recessive 16 spinocerebellar ataxia 0.00394461

Tb927.10.2900 BAG37316.1 KPNB1 karyopherin subunit

beta 1

Circulating levels of very long-chain saturated fatty

acids, multiple sclerosis susceptibility

5.10086e-78

Tb927.10.8390 AAH40929.1 HERC1 protein, partial Macrocephaly, dysmorphic facies, psychomotor 3.352e-64


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retardation, neuronal pattern development

Tb927.4.3810 BAF82512.1 POLR2B RNA polymerase II

subunit B

Age-related macular degeneration, adult height 0.0

Tb927.11.12430 AAT38107.1 CDC14A cell division cycle 14

homolog A

Autosomal recessive 32 deafness 4.10273e-46

Tb927.7.1560 AAI03916.1 TTC6 protein Allergy-specific susceptibility 1.23027e-09

Tb927.7.290 XP_005250264.1 FBXL13 F-box/LRR-repeat

protein 13 isoform X5

Blood pressure response to interventions 1.17865e-09

Tb927.11.14680 NP_001341508.1 serine/threonine-protein

kinase ATR isoform 2

Familial cutaneous telangiectasia and cancer

syndrome, Seckel syndrome 1

1.05991e-

113

Tb927.11.6430 XP_011524505.1 CCDC68 coiled-coil domain-

containing protein 68 isoform

X3

Schizophrenia, severe diabetic retinopathy, psychiatric

disorders

0.0250287


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Table 2. AC1s proximity proteome.

TriTryp GeneID TriTryp Annotation Localization* Source for location

data

AC1s/FS179s

avg ratio&

Tb927.2.5860 Hypothetical Tip TrypTag1 15.88

Tb927.2.5870 Hypothetical Tip TrypTag1 + 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 TrypTag1 + 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

Tb927.10.9380 SEP domain/UBX domain containing protein New Flagellum-distal points TrypTag1 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


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*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.


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2 proteins in Trypanosoma brucei · Page 3 of 33 25 ABSTRACT 26 Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne 27 disease. T