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APEX2 proximity proteomics resolves flagellum subdomains and identifies flagellum tip-specific proteins in Trypanosoma brucei
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Vélez-Ramírez, Daniel E., Shimogawa, Michelle M., Ray, Sunayan S., Lopez, Andrew, Rayatpisheh, Shima, Langousis, Gerasimos, Gallagher-Jones, Marcus, Dean, Samuel, Wohlschlegel, James A., Hill, Kent L. and Phillips, Margaret (2021) APEX2 proximity proteomics resolves flagellum subdomains and identifies flagellum tip-specific proteins in Trypanosoma brucei. mSphere, 6 (1). e01090-20. doi:10.1128/mSphere.01090-20 ISSN 2379-5042.
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WRAP-APEX2-proximity-proteomics-resolves-flagellum-subdomains-identifies-flagellum-tip-specific-proteins-Trypanosoma-brucei-Dean-2021.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution 4.0. Download (1838Kb) | Preview |
Official URL: http://dx.doi.org/10.1128/mSphere.01090-20
Abstract
Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum (cilium) that plays critical roles in transmission and pathogenesis. An emerging concept is that the flagellum is organized into subdomains, each having specialized composition and function. The overall flagellum proteome has been well studied, but a critical knowledge gap is the protein composition of individual subdomains. We have tested whether APEX-based proximity proteomics could be used to examine the protein composition of T. brucei flagellum subdomains. As APEX-based labeling has not previously been described in T. brucei, we first fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, a well-characterized axonemal complex. We found that DRC1-APEX2 directs flagellum-specific biotinylation, and purification of biotinylated proteins yields a DRC1 “proximity proteome” having good overlap with published proteomes obtained from purified axonemes. Having validated the use of APEX2 in T. brucei, we next attempted to distinguish flagellar subdomains by fusing APEX2 to a flagellar membrane protein that is restricted to the flagellum tip, AC1, and another one that is excluded from the tip, FS179. Fluorescence microscopy demonstrated subdomain-specific biotinylation, and principal-component analysis showed distinct profiles between AC1-APEX2 and FS179-APEX2. Comparing these two profiles allowed us to identify an AC1 proximity proteome that is enriched for tip proteins, including proteins involved in signaling. Our results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve the proteome composition of flagellum subdomains that cannot themselves be readily purified.
| Item Type: | Journal Article | |||||||||||||||||||||||||||
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| Subjects: | Q Science > QH Natural history Q Science > QL Zoology Q Science > QP Physiology Q Science > QR Microbiology |
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| Divisions: | Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School | |||||||||||||||||||||||||||
| Library of Congress Subject Headings (LCSH): | Trypanosoma, Trypanosoma brucei , Cellular signal transduction , Flagella (Microbiology), Proteomics | |||||||||||||||||||||||||||
| Journal or Publication Title: | mSphere | |||||||||||||||||||||||||||
| Publisher: | American Society for Microbiology | |||||||||||||||||||||||||||
| ISSN: | 2379-5042 | |||||||||||||||||||||||||||
| Official Date: | February 2021 | |||||||||||||||||||||||||||
| Dates: |
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| Volume: | 6 | |||||||||||||||||||||||||||
| Number: | 1 | |||||||||||||||||||||||||||
| Article Number: | e01090-20 | |||||||||||||||||||||||||||
| DOI: | 10.1128/mSphere.01090-20 | |||||||||||||||||||||||||||
| Status: | Peer Reviewed | |||||||||||||||||||||||||||
| Publication Status: | Published | |||||||||||||||||||||||||||
| Access rights to Published version: | Open Access (Creative Commons) | |||||||||||||||||||||||||||
| Date of first compliant deposit: | 3 November 2022 | |||||||||||||||||||||||||||
| Date of first compliant Open Access: | 4 November 2022 | |||||||||||||||||||||||||||
| RIOXX Funder/Project Grant: |
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| Is Part Of: | 1 |
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