Arp11 Affects Dynein–Dynactin Interaction and is Essential for Dynein Function in Aspergillus nidulans

Abstract

The dynactin complex contains proteins including p150 that interacts with cytoplasmic dynein and an actinrelated protein Arp1 that forms a minifilament. Proteins including Arp11 and p62 locate at the pointed end of the Arp1 filament, but their biochemical functions are unclear (Schroer TA. Dynactin. Annu Rev Cell Dev Biol 2004;20: 759-779). In Aspergillus nidulans, loss of Arp11 or p62 causes the same nuclear distribution (nud) defect displayed by dynein mutants, indicating that these pointed-end proteins are essential for dynein function. We constructed a strain with S-tagged p150 of dynactin that allows us to pull down components of the dynactin and dynein complexes. Surprisingly, while the ratio of pulled-down Arp1 to S-p150 in Arp11-depleted cells is clearly lower than that in wild-type cells, the ratio of pulled-down dynein to S-p150 is significantly higher. We further show that the enhanced dynein-dynactin interaction in Arp11-depleted cells is also present in the soluble fraction and therefore is not dependent upon the affinity of these proteins to the membrane. We suggest that loss of the pointed-end proteins alters the Arp1 filament in a way that affects the conformation of p150 required for its proper interaction with the dynein motor.

Identification of a Novel Site in the Tail of Dynein Heavy Chain Important for Dynein Function in Vivo

“…3C), which is expected because IC is the subunit that binds to dynactin (15)(16)(17) and because dynein forms normal plus-end comets (Fig. 1B), which is dynactin-dependent (49,(51)(52)(53). Moreover, the nudA F208V mutation does not affect the interaction between dynein and the LIS1 homolog NUDF (Fig.…”

“…Thymoglobulin (Sigma-Aldrich) was used as a 19 S marker. Antibodies against dynein HC, dynein IC, p150 dynactin, and NUDF/LIS1 were described previously (39,51,58). Anti-GFP antibodies from Covance (monoclonal) and Clontech (polyclonal) were also used for Western analyses.…”

Dynein Light Intermediate Chain in Aspergillus nidulans Is Essential for the Interaction between Heavy and Intermediate Chains

“…Construction of a Conditional Null Mutant of LIC-We made a conditional null mutant of LIC using a method similar to what has been previously described (38,39). The N-terminal LIC fragment of ϳ0.7 kb was obtained from A. nidulans genomic DNA by polymerase chain reaction using the following two primers: LIC5 (5Ј-AAGCGGCCGCTGTCTACGTCAAAAA-GATCC-3Ј) and LIC3 (5Ј-AACCCGGGTTCTTCCGCTCTC-TCCACTG-3Ј) (the NotI and SmaI sites are underlined).…”

“…nidulans strains used in this study are listed in Table 1. Growth media and growth conditions were as described previously (38).…”

“…Finally, the beads were boiled in the protein-loading buffer, and the proteins were subjected to Western analyses. Affinity-purified anti-HC, anti-IC, and antip150 antibodies were used as previously described (38,39).…”