Production and analysis of a mammalian septin hetero-octamer complex.
GTP-binding proteins
biochemical reconstitution
cytoskeleton
polymerization
protein complex
septins
Journal
Cytoskeleton (Hoboken, N.J.)
ISSN: 1949-3592
Titre abrégé: Cytoskeleton (Hoboken)
Pays: United States
ID NLM: 101523844
Informations de publication
Date de publication:
11 2020
11 2020
Historique:
received:
29
06
2020
revised:
26
10
2020
accepted:
08
11
2020
pubmed:
14
11
2020
medline:
4
8
2021
entrez:
13
11
2020
Statut:
ppublish
Résumé
The septins are filament-forming proteins found in diverse eukaryotes from fungi to vertebrates, with roles in cytokinesis, shaping of membranes and modifying cytoskeletal organization. These GTPases assemble into rod-shaped soluble hetero-hexamers and hetero-octamers in mammals, which polymerize into filaments and higher order structures. While the cell biology and pathobiology of septins are advancing rapidly, mechanistic study of the mammalian septins is limited by a lack of recombinant hetero-octamer materials. We describe here the production and characterization of a recombinant mammalian septin hetero-octamer of defined stoichiometry, the SEPT2/SEPT6/SEPT7/SEPT3 complex. Using a fluorescent protein fusion to the complex, we observed filaments assembled from this complex. In addition, we used this novel tool to resolve recent questions regarding the organization of the soluble septin complex. Biochemical characterization of a SEPT3 truncation that disrupts SEPT3-SEPT3 interactions is consistent with SEPT3 occupying a central position in the complex while the SEPT2 subunits are at the ends of the rod-shaped octameric complexes. Consistent with SEPT2 being on the complex ends, we find that our purified SEPT2/SEPT6/SEPT7/SEPT3 hetero-octamer copolymerizes into mixed filaments with separately purified SEPT2/SEPT6/SEPT7 hetero-hexamer. We expect this new recombinant production approach to lay essential groundwork for future studies into mammalian septin mechanism and function.
Identifiants
pubmed: 33185030
doi: 10.1002/cm.21643
pmc: PMC7845785
mid: NIHMS1660272
doi:
Substances chimiques
Septins
EC 3.6.1.-
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
485-499Subventions
Organisme : NIGMS NIH HHS
ID : R35 GM136337
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM097664
Pays : United States
Informations de copyright
© 2020 Wiley Periodicals LLC.
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