Novel LC8 mutations have disparate effects on the assembly and stability of flagellar complexes.
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.
Yang
Pinfen
P
Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53233, USA. pinfen.yang@marquette.edu
Yang
Chun
C
Wirschell
Maureen
M
Davis
Stephanie
S
D020418:Algal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002696:Chlamydomonas / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005407:Flagella / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D055550:Protein Stability
D020418:Algal Proteins; D020409:Molecular Motor Proteins
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