Point Mutations in the Stem Region and the Fourth AAA Domain of Cytoplasmic Dynein Heavy Chain Partially Suppress the Phenotype of NUDF/LIS1 Loss in Aspergillus nidulans

Abstract

Cytoplasmic dynein performs multiple cellular tasks but its regulation remains unclear. The dynein heavy chain has a N-terminal stem that binds to other subunits and a C-terminal motor unit that contains six AAA (ATPase associated with cellular activities) domains and a microtubule-binding site located between AAA4 and AAA5. In Aspergillus nidulans, NUDF (a LIS1 homolog) functions in the dynein pathway, and two nudF6 partial suppressors were mapped to the nudA dynein heavy chain locus. Here we identified these two mutations. The nudAL1098F mutation resides in the stem region, and nudAR3086C is in the end of AAA4. These mutations partially suppress the phenotype of nudF deletion but do not suppress the phenotype exhibited by mutants of dynein intermediate chain and Arp1. Surprisingly, the stronger DnudF suppressor, nudAR3086C, causes an obvious decrease in the basal level of dynein's ATPase activity and an increase in dynein's distribution along microtubules. Thus, suppression of the DnudF phenotype may result from mechanisms other than simply the enhancement of dynein's ATPase activity. The fact that a mutation in the end of AAA4 negatively regulates dynein's ATPase activity but partially compensates for NUDF loss indicates the importance of the AAA4 domain in dynein regulation in vivo.

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

“…F208V Mutation and Creation of the nudA F208I Mutation-The nudA sequence of the 4-63 mutant corresponding to the wild type nudA fragment that complemented the 4-63 mutant was sequenced using primers as described previously (57), which led to the identification of the nudA F208V mutation. To construct the mutant strain carrying the nudA F208I mutation, we first made the DNA fragment containing this mutation using fusion PCR.…”

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

“…The 0.7-kb fragment was digested by NotI and SmaI, and ligated into the corresponding sites of the LB01 vector (41). The resultant plasmid was transformed into the LZ26 strain containing GFP-nudA (dynein HC) and S-tagged IC (42). In the expected alcA-LIC strain, the full-length fusion gene is under the control of the regulatable promoter alcA, which can be shut off by glucose but can be induced by glycerol to express a downstream gene at a moderate level.…”

“…nidulans protein extract was obtained from an overnight culture of 1 liter using the liquid nitrogen grinding method for breaking the hyphae, which was similar to what has been described previously (43), except that the protein isolation buffer contains 25 mM Tris, pH 8.0, 0.4% Triton X-100, 1 mM dithiothreitol, and a protease inhibitor mixture (Sigma). The construction of an Stagged dynein intermediate chain strain (S-IC) and the method for dynein purification were described previously (42). For purification of A. nidulans dynein from S-IC strains, about 30 ml of a protein extract (about 10 mg/ml) was incubated for a half-hour at room temperature with 0.5 ml of S-protein beads (Novagen, Inc. Madison, WI).…”

“…Thus, we determined whether HC-IC association is affected by the deletion of these LCs. We have previously made a strain containing a functional S-tagged dynein IC (S-IC), and the S-IC is able to pull down the HC proteins (42). In this work, we introduced the S-IC into the ⌬nudG and ⌬robA backgrounds by genetic crosses and examined the HC-IC association at 42°C.…”

“…5). This alcA-LIC strain was made in the background of LZ26 that contains S-IC and GFP-HC under the control of its endogenous promoter (42). Thus, the effect of LIC depletion on FIGURE 2.…”