Dynein light chain LC8 promotes assembly of the coiled-coil domain of swallow protein.
LC8 is a highly conserved light-chain subunit of cytoplasmic dynein that is thought to play a fundamental role in both the assembly of the motor complex and the recruitment of cargo. An interaction between LC8 and the Drosophila protein swallow has been previously characterized and supports a role for dynein in the localization of maternal morphogens during oogenesis. Swallow is required for the proper localization of bicoid mRNA, the anterior determinant that plays a critical role in establishment of the Drosophila embryonic axis. In this work, we prepared constructs of swallow, each containing a predicted coiled-coil domain and variable surrounding segments that lie within the domain proposed to interact with LC8. The interaction between LC8 and swallow domains was characterized by glutathione S-transferase (GST) pull-down assays, limited proteolysis followed by mass spectrometry, and circular dichroic spectroscopy. Hydrodynamic measurements, covalent cross-linking, and circular dichroic spectroscopy show that this domain of swallow is an unstable dimeric coiled-coil. Upon LC8 binding, however, the coiled-coil becomes significantly more stable. A possible general role for LC8 in macromolecular assembly is discussed.
Wang
Lei
L
Department of Biological Sciences, Ohio University, Athens, Ohio 45701, USA.
Hare
Michael
M
Hays
Thomas S
TS
Barbar
Elisar
E
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004398:Dyneins; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000378:metabolism; D013816:Thermodynamics
D002352:Carrier Proteins; D029721:Drosophila Proteins; D010446:Peptide Fragments; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C542608:ctp protein, Drosophila; C073970:swa protein, Drosophila; D005982:Glutathione Transferase; D004398:Dyneins