The role of preassembled cytoplasmic complexes in assembly of flagellar dynein subunits

Abstract

Previous work has revealed a cytoplasmic pool of flagellar precursor proteins capable of contributing to the assembly of new flagella, but how and where these components assemble is unknown. We tested Chlamydomonas outer-dynein arm subunit stability and assembly in the cytoplasm of wild-type cells and 11 outer dynein arm assembly mutant strains (oda1-oda11) by Western blotting of cytoplasmic extracts, or immunoprecipitates from these extracts, with five outer-row dynein subunit-specific antibodies. Western blots reveal that at least three oda mutants (oda6, oda7, and oda9) alter the level of a subunit that is not the mutant gene product. Immunoprecipitation shows that large preassembled flagellar complexes containing all five tested subunits (three heavy chains and two intermediate chains) exist within wild-type cytoplasm. When the preassembly of these subunits was examined in oda strains, we observed three patterns: complete coassembly (oda 1, 3, 5, 8, and 10), partial coassembly (oda7 and oda11), and no coassembly (oda2, 6, and 9) of the four tested subunits with HCbeta. Our data, together with previous studies, suggest that flagellar outer-dynein arms preassemble into a complete Mr approximately 2 x 10(6) dynein arm that resides in a cytoplasmic precursor pool before transport into the flagellar compartment.

Figure 1

Diagram illustrating the relationship between outer-row dynein arms in situ and after dissociation by 0.6 M NaCl into 7S, 12S, and 18S particles. The loci (ODA1, ODA2, etc.) that are known to encode subunits of each particle are also indicated.

Figure 2

Specificity of antibody B3B. WT and oda11 flagella, run on 5% gels and blotted to PVDF membrane, were probed with C11.6 (left panel), which detected equal levels of HCβ in both samples. A parallel blot probed with antibody B3B (right panel) detected a single band in WT flagella that is missing in flagella of HCα assembly mutant oda11. Antibodies were detected with a peroxidase-linked secondary antibody and chemiluminescent detection.

Figure 3

Western blots of HCα in WT and oda cytoplasmic extracts. Samples of cytoplasmic extracts from wild-type and oda1-oda11 cells were separated on 7% gels and blotted to PVDF membrane. Each lane was loaded with 20 μg of total protein. (A) Gel stained with Coomassie blue. (B) Western blot probed with B3B (anti-HCα). No HCα was detectable in either oda7 or oda11; levels in oda5 are 50% of WT.

Figure 4

Western blots of WT and oda cytoplasmic extracts test subunit stability. Cytoplasmic extracts of WT and oda1-oda11 prepared as in Figure 2 were probed with mAbs C11.6 (anti-HCβ), 25–8 (anti-HCγ), 1878A (anti-IC78), and 1869A (anti-IC70). (A) Outer dynein arm proteins (indicated along the right margin) present in cytoplasmic extracts of WT and oda1–11. Most oda mutant samples display antigen levels similar to WT, but there are several exceptions as discussed in text. Multiple bands below HCβ in WT, oda1, and oda3 are due to the breakdown of HCβ. No detectable HCβ was found in oda4, no HCγ was found in oda2, no IC78 was found in oda9, and no IC70 was found in oda6 (at this exposure; see text and Figure 3C). 1878A (anti-IC70) cross-reacted with cytoplasm proteins migrating faster than 78 kDa (asterisk) that are apparently unrelated to flagellar dynein. (B) Western blots of a second set of independently prepared cytoplasmic extracts from oda5-oda10 probed with 1878A (anti-IC78) and 1869A (anti-IC70) that illustrate the variability in the reductions of IC70 in oda9 and IC78 in oda6 (compare with bottom two panels in panel A). (C) Upper and lower panels show the results of a 10-fold increase in exposure time for lanes oda5–oda7 from the bottom panels of Figure 3A and Figure 3B, respectively, and reveal an IC70 band in oda6 cytoplasmic extracts.

Figure 5

Fragmentation of 70-kDa antigens by acid hydrolysis. Proteins of ∼70 kDa were purified by SDS-PAGE from WT axonemes, oda6 cytoplasmic extract, and pf28 cytoplasmic extract, digested by partial hydrolysis with formic acid, separated on a 12% gel, blotted to PVDF membrane, and probed with 1869A (anti-IC70). All three samples generate similar fragmentation patterns.

Figure 6

Immunoprecipitation of preassembled complexes. Immunoprecipitates with C11.6 (anti-HCβ) or no antibody (control) from WT and oda1–oda11 cytoplasmic extracts were separated on 7% gels, blotted to PVDF membrane, and probed with B3B (anti-HCα), C11.6 (anti-HCβ), 25–8 (anti-HCγ), 1878A (anti-IC78), or 1869A (anti-IC70). Immunoprecipitates of oda1 extracts showed the presence of all five major outer-dynein arm proteins at equivalent levels to WT, as did immunoprecipitates of oda3, 5, 8, and 10. The immunoprecipitate of oda11 lacked only HCα; the immunoprecipitate of oda7 lacked HCα and had greatly reduced levels of IC70 and IC78, whereas no proteins coprecipitated with HCβ in oda2, oda6, or oda9. The oda4 lanes serve as a no-antigen control.

Figure 7

Model of the dynein assembly process. Solid arrows indicate steps thought to occur in wild-type cells, where all of the subunits are hypothesized to preassemble in the cytoplasm (left side) before moving into the flagellum (right side). An intermediate compartment (IFT) may be required for transport between the cytoplasm and flagellum. Assembly mutants oda1–oda11 block this process at the steps indicated by each number in the diagram and affect the preassembly of one of three complexes, an IC–HC complex (top), a LC complex (middle), or a docking complex (bottom). Dashed arrows show assembly pathways occurring during cytoplasmic complementation of assembly mutants.