Three Members of the LC8/DYNLL Family Are Required for Outer Arm Dynein Motor Function

Abstract

The highly conserved LC8/DYNLL family proteins were originally identified in axonemal dyneins and subsequently found to function in multiple enzyme systems. Genomic analysis uncovered a third member (LC10) of this protein class in Chlamydomonas. The LC10 protein is extracted from flagellar axonemes with 0.6 M NaCl and cofractionates with the outer dynein arm in sucrose density gradients. Furthermore, LC10 is specifically missing only from axonemes of those strains that fail to assemble outer dynein arms. Previously, the oda12-1 insertional allele was shown to lack the Tctex2-related dynein light chain LC2. The LC10 gene is located ϳ2 kb from that of LC2 and is also completely missing from this mutant but not from oda12-2, which lacks only the 3 end of the LC2 gene. Although oda12-1 cells assemble outer arms that lack only LC2 and LC10, this strain exhibits a flagellar beat frequency that is consistently less than that observed for strains that fail to assemble the entire outer arm and docking complex (e.g., oda1). These results support a key regulatory role for the intermediate chain/light chain complex that is an integral and highly conserved feature of all oligomeric dynein motors.

Systematic Comparison of in Vitro Motile Properties between Chlamydomonas Wild-type and Mutant Outer Arm Dyneins Each Lacking One of the Three Heavy Chains

“…Strains and Media-C. reinhardtii mutants used in this study were oda12-1nit1 (a double mutant between oda12-1, which is a null mutant of the genes of two light chains LC2 and LC10 and lacks the entire outer arm, and the mutant nit1, which is deficient in nitrate reductase; this double mutant will be referred to as oda12) (10,11), oda11 (lacking the ␣ HC) (3), oda4-s7 (lacking the motor domain of the ␤ HC) (4), and oda2-t (lacking the motor domain of the ␥ HC) (5). The cells were grown in liquid Tris-acetate-phosphate medium with aeration on a 12 h of light/12 h of dark cycle.…”

“…This light chain is essential for proper OAD assembly, as evidenced by the fact that an LC2/LC10 null mutant, oda12, does not assemble functional OAD (10,26). Although oda12 lacks both LC2 and LC10, its motility defects can be rescued by transformation with a genomic fragment containing only the wild-type LC2 gene (10,11). We constructed a cDNA that encodes an LC2-BCCP fusion protein and used it to transform oda12.…”

Protein–protein interactions between intermediate chains and the docking complex of Chlamydomonas flagellar outer arm dynein

“…In Chlamydomonas OAD, several LCs have been shown to bind the ICs and form an IC-LC complex [30; 31]. Three of them (LC6, LC8, and LC10) belong to the LC8/DYNLL family [31]. Our data raise the possibility that IC1 and IC2 can heterodimerize without the dimerization enhancers.…”

“…Of these, LC8 is known to be a dimerization enhancer [28]. In Chlamydo-monas OAD, several LCs have been shown to bind the ICs and form an IC-LC complex [29,30]. Three of them (LC6, LC8, and LC10) belong to the LC8/DYNLL family [30].…”

Integrated control of axonemal dynein AAA+ motors

“…The oda6-r88 phenotype is likely due to the loss of either LC2 (a Tctex2-related protein) or LC9 (a Tctex1) as the LC6 protein, which appears to be Chlamydomonas -specific, is completely absent in oda13 which has wildtype beat frequency (DiBella et al , 2005). Although the oda12-1 strain which has a partial assembly defect and reduced beat frequency was originally described as lacking LC2 (Pazour et al , 1999) it was later found to also be a complete null for LC10 (Tanner et al , 2008) and thus to exhibit a synthetic phenotype due to the loss of both outer arm components.…”