The 8-kDa dynein light chain binds to its targets via a conserved (K/R)XTQT motif.
Cytoplasmic dynein is a large, multisubunit molecular motor that translocates cargoes toward the minus ends of microtubules. Proper functioning of the dynein motor requires precise assembly of its various subunits. Using purified recombinant proteins, we show that the highly conserved 8-kDa light chain (DLC8) binds to the intermediate chain of the dynein complex. The DLC8-binding region was mapped to a highly conserved 10-residue fragment (amino acid sequence SYSKETQTPL) C-terminal to the second alternative splicing site of dynein intermediate chain. Yeast two-hybrid screening using DLC8 as bait identified numerous additional DLC8-binding proteins. Biochemical and mutational analysis of selected DLC8-binding proteins revealed that DLC8 binds to a consensus sequence containing a (K/R)XTQT motif. The (K/R)XTQT motif interacts with the common target-accepting grooves of DLC8 dimer. The role of each conserved amino acid residue in this pentapeptide motif in supporting complex formation with DLC8 was systematically studied using site-directed mutagenesis.
Lo
K W
KW
Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China.
Naisbitt
S
S
Fan
J S
JS
Sheng
M
M
Zhang
M
M
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001667:Binding, Competitive; D017124:Conserved Sequence; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D056445:Cytoplasmic Dyneins; D004252:DNA Mutational Analysis; D019281:Dimerization; D004398:Dyneins / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D010455:Peptides; D011994:Recombinant Proteins; D005982:Glutathione Transferase; C427758:DYNLL1 protein, human; C486069:Dynll1 protein, rat; D056445:Cytoplasmic Dyneins; D004398:Dyneins