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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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Official URL: http://dx.doi.org/10.1128/mSphere.01090-20

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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
Subjects: Q Science > QH Natural history
Q Science > QL Zoology
Q Science > QP Physiology
Q Science > QR Microbiology
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:
DateEvent
February 2021Published
10 February 2021Available
26 December 2020Accepted
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:
Project/Grant IDRIOXX Funder NameFunder ID
AI052348National Institutes of Healthhttp://dx.doi.org/10.13039/100000002
AI142544National Institutes of Healthhttp://dx.doi.org/10.13039/100000002
GM089778National Institutes of Healthhttp://dx.doi.org/10.13039/100000002
104627/Z/14/ZWellcome Trusthttp://dx.doi.org/10.13039/100010269
UNSPECIFIEDUniversity of California, Los Angeleshttp://dx.doi.org/10.13039/100007185
Fulbright-García Robles scholarship Comisión México-Estados Unidos para el Intercambio Educativo y CulturalUNSPECIFIED
CVU 325790Consejo Nacional de Ciencia y Tecnologíahttp://dx.doi.org/10.13039/501100003141
UNSPECIFIEDUniversity of California Institute for Mexico and the United Stateshttp://dx.doi.org/10.13039/100005909
Is Part Of: 1

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