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Published on: May 5, 2020
Novel LC8 mutations have disparate effects on the assembly and stability of flagellar complexes
Pinfen Yang1, Chun Yang, Maureen Wirschell
1Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53233, USA. pinfen.yang@marquette.edu
Abstract:
LC8 functions as a dimer crucial for a variety of molecular motors and non-motor complexes. Emerging models, founded on structural studies, suggest that the LC8 dimer promotes the stability and refolding of dimeric target proteins in molecular complexes, and its interactions with selective target proteins, including dynein subunits, is regulated by LC8 phosphorylation, which is proposed to prevent LC8 dimerization. To test these hypotheses in vivo, we determine the impacts of two new LC8 mutations on the assembly and stability of defined LC8-containing complexes in Chlamydomonas flagella. The three types of dyneins and the radial spoke are disparately affected by dimeric LC8 with a C-terminal extension. The defects include the absence of specific subunits, complex instability, and reduced incorporation into the axonemal super complex. Surprisingly, a phosphomimetic LC8 mutation, which is largely monomeric in vitro, is still dimeric in vivo and does not significantly change flagellar generation and motility. The differential defects in these flagellar complexes support the structural model and indicate that modulation of target proteins by LC8 leads to the proper assembly of complexes and ultimately higher level complexes. Furthermore, the ability of flagellar complexes to incorporate the phosphomimetic LC8 protein and the modest defects observed in the phosphomimetic LC8 mutant suggest that LC8 phosphorylation is not an effective mechanism for regulating molecular complexes.
Insights
Light chain 8 (LC8) dimer stability is crucial for molecular complex assembly in Chlamydomonas flagella. Mutations reveal LC8
Area of Science:
- Cell Biology
- Structural Biology
- Biochemistry
Background:
- The LC8 (light chain 8) dimer is essential for various molecular motors and complexes.
- LC8 is proposed to stabilize dimeric target proteins and its interactions are regulated by phosphorylation.
- Phosphorylation is hypothesized to prevent LC8 dimerization, impacting complex formation.
Purpose of the Study:
- To investigate the in vivo effects of LC8 mutations on the assembly and stability of LC8-containing complexes.
- To test the hypothesis that LC8 phosphorylation regulates its dimerization and function.
- To examine the impact of LC8 mutations on Chlamydomonas flagellar structure and motility.
Main Methods:
- Introduction of two novel LC8 mutations into Chlamydomonas.
- Analysis of LC8-containing complexes, including dyneins and radial spokes, in flagella.
- Assessment of subunit composition, complex stability, and incorporation into the axonemal superstructure.
Main Results:
- Dimeric LC8 with a C-terminal extension differentially affected dyneins and radial spokes, causing subunit absence and instability.
- A phosphomimetic LC8 mutation, monomeric in vitro, remained dimeric in vivo with minimal impact on flagellar function.
- Flagellar complexes incorporated the phosphomimetic LC8, indicating functional dimeric state in vivo.
Conclusions:
- Differential defects observed in flagellar complexes support structural models of LC8 function.
- LC8's modulation of target proteins is critical for proper assembly of complexes.
- LC8 phosphorylation appears to be an ineffective mechanism for regulating molecular complexes in vivo.
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