Interactions of cytoplasmic dynein light chains Tctex-1 and LC8 with the intermediate chain IC74.
The interactions of three subunits of cytoplasmic dynein from Drosophila melanogaster, LC8, Tctex-1, and the N-terminal domain of IC74 (N-IC74, residues 1-289), were characterized in vitro by affinity methods, limited proteolysis, and circular dichroism spectroscopy. These subunits were chosen for study because they are presumed to promote the assembly of the complex and to be engaged in the controlled binding and release of cargo. Limited proteolysis and mass spectrometry of N-IC74 in the presence of LC8 and Tctex-1 localized binding of Tctex-1 to the vicinity of K104 and K105, and localized binding of LC8 to the region downstream of K130. Circular dichroism, fluorescence, sedimentation velocity, and proteolysis studies indicate that N-IC74 has limited secondary and tertiary structure at near physiological solution conditions. Upon addition of LC8, N-IC74 undergoes a significant conformational change from largely unfolded to a more ordered structure. This conformational change is reflected in increased global protection of N-IC74 from proteolytic digestion following the interaction, and in a significant change in the CD signal. A smaller but reproducible change in the CD spectra was observed upon Tctex-1 binding as well. The increased structure introduced into N-IC74 upon light chain binding suggests a mechanism by which LC8 and Tctex-1 may regulate the assembly of the dynein complex.
Makokha
Moses
M
Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701 and Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455.
Hare
Michael
M
Li
Mingang
M
Hays
Thomas
T
Barbar
Elisar
E
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000737:chemistry; D004398:Dyneins / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000494:pharmacology; D019791:Guanidine / Q000737:chemistry; D013058:Mass Spectrometry; D008868:Microtubule Proteins / Q000737:chemistry* / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D029867:Xenopus Proteins; D056427:t-Complex Genome Region
D002352:Carrier Proteins; D029721:Drosophila Proteins; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D029867:Xenopus Proteins; C542608:ctp protein, Drosophila; D019286:Endopeptidase K; D004398:Dyneins; D019791:Guanidine