<named-content content-type="genus-species">Trypanosoma brucei</named-content> RAP1 Has Essential Functional Domains That Are Required for Different Protein Interactions

ABSTRACT RAP1 is a telomere protein that is well conserved from protozoa to mammals. It plays important roles in chromosome end protection, telomere length control, and gene expression/silencing at both telomeric and nontelomeric loci. Interaction with different partners is an important mechanism by...

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Autores principales: Marjia Afrin, Hanadi Kishmiri, Ranjodh Sandhu, M. A. G. Rabbani, Bibo Li
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Publicado: American Society for Microbiology 2020
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spelling oai:doaj.org-article:80c4e73956134255abe3a3f04b1cd2ba2021-11-15T15:27:53Z<named-content content-type="genus-species">Trypanosoma brucei</named-content> RAP1 Has Essential Functional Domains That Are Required for Different Protein Interactions10.1128/mSphere.00027-202379-5042https://doaj.org/article/80c4e73956134255abe3a3f04b1cd2ba2020-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00027-20https://doaj.org/toc/2379-5042ABSTRACT RAP1 is a telomere protein that is well conserved from protozoa to mammals. It plays important roles in chromosome end protection, telomere length control, and gene expression/silencing at both telomeric and nontelomeric loci. Interaction with different partners is an important mechanism by which RAP1 executes its different functions in yeast. The RAP1 ortholog in Trypanosoma brucei is essential for variant surface glycoprotein (VSG) monoallelic expression, an important aspect of antigenic variation, where T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. Like other RAP1 orthologs, T. brucei RAP1 (TbRAP1) has conserved functional domains, including BRCA1 C terminus (BRCT), Myb, MybLike, and RAP1 C terminus (RCT). To study functions of various TbRAP1 domains, we established a strain in which one endogenous allele of TbRAP1 is flanked by loxP repeats, enabling its conditional deletion by Cre-mediated recombination. We replaced the other TbRAP1 allele with various mutant alleles lacking individual functional domains and examined their nuclear localization and protein interaction abilities. The N terminus, BRCT, and RCT of TbRAP1 are required for normal protein levels, while the Myb and MybLike domains are essential for normal cell growth. Additionally, the Myb domain of TbRAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor (TbTRF), while the BRCT domain is required for its self-interaction. Furthermore, the TbRAP1 MybLike domain contains a bipartite nuclear localization signal that is required for its interaction with importin α and its nuclear localization. Interestingly, RAP1’s self-interaction and the interaction between RAP1 and TRF are conserved from kinetoplastids to mammals. However, details of the interaction interfaces have changed throughout evolution. IMPORTANCE Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. TbRAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although TbRAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for TbRAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of TbRAP1 point mutation phenotypes. We have also determined functional domains of TbRAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of TbRAP1-mediated VSG silencing.Marjia AfrinHanadi KishmiriRanjodh SandhuM. A. G. RabbaniBibo LiAmerican Society for MicrobiologyarticleRAP1telomereTrypanosoma bruceiprotein-protein interactionRAP1MicrobiologyQR1-502ENmSphere, Vol 5, Iss 1 (2020)
institution DOAJ
collection DOAJ
language EN
topic RAP1
telomere
Trypanosoma brucei
protein-protein interaction
RAP1
Microbiology
QR1-502
spellingShingle RAP1
telomere
Trypanosoma brucei
protein-protein interaction
RAP1
Microbiology
QR1-502
Marjia Afrin
Hanadi Kishmiri
Ranjodh Sandhu
M. A. G. Rabbani
Bibo Li
<named-content content-type="genus-species">Trypanosoma brucei</named-content> RAP1 Has Essential Functional Domains That Are Required for Different Protein Interactions
description ABSTRACT RAP1 is a telomere protein that is well conserved from protozoa to mammals. It plays important roles in chromosome end protection, telomere length control, and gene expression/silencing at both telomeric and nontelomeric loci. Interaction with different partners is an important mechanism by which RAP1 executes its different functions in yeast. The RAP1 ortholog in Trypanosoma brucei is essential for variant surface glycoprotein (VSG) monoallelic expression, an important aspect of antigenic variation, where T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. Like other RAP1 orthologs, T. brucei RAP1 (TbRAP1) has conserved functional domains, including BRCA1 C terminus (BRCT), Myb, MybLike, and RAP1 C terminus (RCT). To study functions of various TbRAP1 domains, we established a strain in which one endogenous allele of TbRAP1 is flanked by loxP repeats, enabling its conditional deletion by Cre-mediated recombination. We replaced the other TbRAP1 allele with various mutant alleles lacking individual functional domains and examined their nuclear localization and protein interaction abilities. The N terminus, BRCT, and RCT of TbRAP1 are required for normal protein levels, while the Myb and MybLike domains are essential for normal cell growth. Additionally, the Myb domain of TbRAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor (TbTRF), while the BRCT domain is required for its self-interaction. Furthermore, the TbRAP1 MybLike domain contains a bipartite nuclear localization signal that is required for its interaction with importin α and its nuclear localization. Interestingly, RAP1’s self-interaction and the interaction between RAP1 and TRF are conserved from kinetoplastids to mammals. However, details of the interaction interfaces have changed throughout evolution. IMPORTANCE Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. TbRAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although TbRAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for TbRAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of TbRAP1 point mutation phenotypes. We have also determined functional domains of TbRAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of TbRAP1-mediated VSG silencing.
format article
author Marjia Afrin
Hanadi Kishmiri
Ranjodh Sandhu
M. A. G. Rabbani
Bibo Li
author_facet Marjia Afrin
Hanadi Kishmiri
Ranjodh Sandhu
M. A. G. Rabbani
Bibo Li
author_sort Marjia Afrin
title <named-content content-type="genus-species">Trypanosoma brucei</named-content> RAP1 Has Essential Functional Domains That Are Required for Different Protein Interactions
title_short <named-content content-type="genus-species">Trypanosoma brucei</named-content> RAP1 Has Essential Functional Domains That Are Required for Different Protein Interactions
title_full <named-content content-type="genus-species">Trypanosoma brucei</named-content> RAP1 Has Essential Functional Domains That Are Required for Different Protein Interactions
title_fullStr <named-content content-type="genus-species">Trypanosoma brucei</named-content> RAP1 Has Essential Functional Domains That Are Required for Different Protein Interactions
title_full_unstemmed <named-content content-type="genus-species">Trypanosoma brucei</named-content> RAP1 Has Essential Functional Domains That Are Required for Different Protein Interactions
title_sort <named-content content-type="genus-species">trypanosoma brucei</named-content> rap1 has essential functional domains that are required for different protein interactions
publisher American Society for Microbiology
publishDate 2020
url https://doaj.org/article/80c4e73956134255abe3a3f04b1cd2ba
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