Investigation of the evolution of intermediate filament-forming proteins in Eukaryotes
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Abstract
The remarkable increased complexity of eukaryotes' cytoskeleton represents one of the major
transitions in life. Inherently intertwined with eukaryogenesis itself, molecular innovations that
emerged during cytoskeleton evolution are pivotal for understanding Eukaryotes` evolutionary history.
Despite distant homologs for two major cytoskeletal components, actin, and tubulin, identified in
prokaryotes, no clear homolog for intermediate filaments has been found. Intermediate filaments
represent crucial components in the eukaryotic cytoskeleton, being regarded as one of the major
molecular innovations that evolved in this kingdom. Long evolutionary distances, poor sequence
conservation, and many other hurdles render tracing the evolution of such proteins a daunting task.
One possible route to circumvent such limitations is to investigate single-cell eukaryotes, namely
protists, whose cell biology is expected to entail many overlooked fiber-forming, intermediate
filament-like proteins.
The filament-forming mitochondrial citrate synthase described in the series of works that prompted
this study was an ideal candidate for studying the emergence of intermediate filament proteins over a
known evolutionary interval. The study of such multifunctional proteins is important for several
reasons. As part of a well-conserved protein family, the mitochondrial citrate synthase (mCS) makes it
tractable to study transitions over known and relatively short evolutionary intervals in a phylogenetic
tree. Furthermore, mCS evolution of fiber formation in protists could be the product of convergent
evolution, giving rise to novel IF structures and potentially elucidating mechanisms for filament
formation in non-metazoan lineages. Another possible role of fiber formation is linked to metabolism
regulation. Therefore, studying the evolutionary interval along which such behavior emerged would
allow us to pinpoint the evolutionary forces acting on enzyme fiber formation and their possible
recruitment for structural functions.
Here, we characterized the properties of in vitro fiber formation of the recombinant mitochondrial
citrate synthase of Tetrahyemna thermophila. We provide orthogonal evidence for this behavior to be
a buffer-driven artifact caused by a specific chemical moiety that is most likely absent in the native
biological context. We also show that buffer-induced aggregation is a feature common to eukaryotic
mitochondrial citrate synthase but absent in bacterial homologs.
Using an in-vivo approach, we investigated the protein's localization using Strep and GFP-fibre
deficient tagged mutants. We show that the protein colocalizes with mitochondria and does not form
clear foci during starvation, which indicates the absence of metabolic regulation via filamentation.
Furthermore, we monitored the protein's localization during conjugation, reporting the absence of
any extramitochondrial structure.
Finally, we show how MES-induced ordered fibers emerged in the last common ancestor of
Hymenostomatida while other species and ancestral proteins form amorphous aggregates. We
thereby conclude that ordered fibers likely are a product of neutral processes.
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Dates
Created: 2024Issued: 2024-11-18Updated: 2024-11-18
Faculty
Fachbereich Chemie
Publisher
Philipps-Universität Marburg
Language
eng
Data types
DoctoralThesis
Keywords
BiochemieEvolutionsbiologieBiochemistryEvolutionary Biology
DDC-Numbers
540
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Lometto, Stefano: Investigation of the evolution of intermediate filament-forming proteins in Eukaryotes. : Philipps-Universität Marburg 2024-11-18. DOI: https://doi.org/10.17192/z2024.0486.