title: Novel LC8 mutations have disparate effects on the assembly and stability of flagellar complexes.
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.
authors.affiliation: Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53233, USA. pinfen.yang@marquette.edu
authors.forename: Pinfen
authors.initials: P
authors.lastname: Yang
authors.forename: Chun
authors.initials: C
authors.lastname: Yang
authors.forename: Maureen
authors.initials: M
authors.lastname: Wirschell
authors.forename: Stephanie
authors.initials: S
authors.lastname: Davis
mesh_terms: 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
chemical_list: D020418:Algal Proteins; D020409:Molecular Motor Proteins
references.citation: Biochemistry. 2008 Jan 15;47(2):503-8
references.pmid: 18092820
references.citation: J Cell Biol. 2007 Feb 12;176(4):473-82
references.pmid: 17296796
references.citation: J Cell Biol. 1981 Jan;88(1):80-8
references.pmid: 7204490
references.citation: Mol Biol Cell. 2004 Oct;15(10):4382-94
references.pmid: 15269286
references.citation: J Cell Sci. 2007 Oct 15;120(Pt 20):3653-65
references.pmid: 17895364
references.citation: Int Rev Cytol. 2002;219:115-55
references.pmid: 12211628
references.citation: J Cell Biol. 1998 Mar 9;140(5):1137-47
references.pmid: 9490726
references.citation: Mol Biol Cell. 2004 Dec;15(12):5431-42
references.pmid: 15469982
references.citation: Mol Cell. 1999 Mar;3(3):287-96
references.pmid: 10198631
references.citation: J Biol Chem. 1979 Apr 25;254(8):3084-90
references.pmid: 155062
references.citation: Mol Biol Cell. 2009 Jul;20(13):3044-54
references.pmid: 19420136
references.citation: Dev Cell. 2007 Oct;13(4):523-38
references.pmid: 17925228
references.citation: Cancer Cell. 2004 Jun;5(6):575-85
references.pmid: 15193260
references.citation: Proc Natl Acad Sci U S A. 2007 Jun 12;104(24):10028-33
references.pmid: 17551010
references.citation: J Biol Chem. 1997 Aug 15;272(33):20929-35
references.pmid: 9252421
references.citation: Mol Biol Cell. 1995 Jun;6(6):697-711
references.pmid: 7579689
references.citation: J Cell Biol. 2001 Jun 11;153(6):1315-26
references.pmid: 11402073
references.citation: J Biol Chem. 2008 Feb 15;283(7):4004-13
references.pmid: 18084006
references.citation: Mol Biol Cell. 2003 May;14(5):2041-56
references.pmid: 12802074
references.citation: Mol Cell Biol. 1996 May;16(5):1966-77
references.pmid: 8628263
references.citation: Science. 1996 Nov 1;274(5288):774-7
references.pmid: 8864115
references.citation: Nat Cell Biol. 2007 Jul;9(7):788-96
references.pmid: 17546040
references.citation: J Cell Biol. 2004 Jan 19;164(2):255-66
references.pmid: 14718520
references.citation: J Cell Sci. 2006 Mar 15;119(Pt 6):1165-74
references.pmid: 16507594
references.citation: J Biol Chem. 2005 Mar 4;280(9):8172-9
references.pmid: 15611139
references.citation: J Biol Chem. 2006 Apr 7;281(14):9552-9
references.pmid: 16452477
references.citation: Mol Biol Cell. 2005 Jul;16(7):3107-16
references.pmid: 15829565
references.citation: Mol Biol Cell. 2009 Jul;20(13):3055-63
references.pmid: 19420135
references.citation: J Cell Biol. 2008 Jan 28;180(2):403-15
references.pmid: 18227282
references.citation: J Mol Biol. 2001 Feb 9;306(1):97-108
references.pmid: 11178896
references.citation: Mol Biol Cell. 1998 Dec;9(12):3335-49
references.pmid: 9843573
references.citation: FEBS Lett. 2001 Aug 17;503(2-3):135-41
references.pmid: 11513870
references.citation: J Cell Biol. 1998 May 18;141(4):979-92
references.pmid: 9585416
references.citation: J Biol Chem. 1995 May 12;270(19):11445-52
references.pmid: 7744782
references.citation: PLoS Genet. 2006 Jan;2(1):e1
references.pmid: 16440056
references.citation: Proc Natl Acad Sci U S A. 2007 Apr 24;104(17):7229-34
references.pmid: 17438267
references.citation: J Biol Chem. 2003 Oct 17;278(42):41491-9
references.pmid: 12904292
references.citation: Mol Biol Cell. 2005 Dec;16(12):5661-74
references.pmid: 16195342
references.citation: Nat Struct Biol. 1999 Aug;6(8):735-40
references.pmid: 10426949
references.citation: J Biol Chem. 2007 Jun 8;282(23):17272-9
references.pmid: 17428790