2 proteins in trypanosoma brucei · page 3 of 33 25 abstract 26 trypanosoma brucei is the protozoan...
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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
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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
(which was not certified by peer review) is the author/funder.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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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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Page 16 of 33
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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Page 17 of 33
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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Page 18 of 33
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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Page 19 of 33
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
(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
Page 20 of 33
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
(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
Page 21 of 33
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
(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
Page 22 of 33
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
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35. Lam, S.S., J.D. Martell, K.J. Kamer, T.J. Deerinck, M.H. Ellisman, V.K. Mootha, and A.Y. Ting, 574
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41. Lacomble, S., N. Portman, and K. Gull, A protein-protein interaction map of the Trypanosoma 590
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42. Thomas, P.D., The Gene Ontology and the Meaning of Biological Function. Methods Mol Biol, 592
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44. Mick, D.U., R.B. Rodrigues, R.D. Leib, C.M. Adams, A.S. Chien, S.P. Gygi, and M.V. Nachury, 596
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compartment. Nat Cell Biol, 2014. 16(7): p. 663-72. 614
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trypanosomes. Mol Biol Cell, 2008. 19(3): p. 929-44. 617
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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
(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
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
(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
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.
(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
(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
(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
(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
(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
(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
(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
(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