Dynein light chain interacts with NRF-1 and EWG, structurally and functionally related transcription factors from humans and Drosophila

Nuclear respiratory factor-1 is a transcriptional activator that has been i mplicated in the nuclear control of respiratory chain expression. Yeast two -hybrid screens were performed to identify proteins that physically interac t with nuclear respiratory factor-1. Saturation screening of both mouse emb ryo and mouse testis libraries yielded 14 independent clones, all of which represented two different isoforms of dynein light chain. In addition to us ing the two-hybrid method, the specificity of the nuclear respiratory facto r-1/dynein light chain interaction was established by chemical crosslinking of the purified native proteins and by co-immunoprecipitation of nuclear r espiratory factor-1 and dynein light chain from mammalian cells, Both two-h ybrid and chemical crosslinking assays demonstrated that binding of dynein light chain required the first 26 amino acids of nuclear respiratory factor -1, Although dynein light chain is associated with dynein, a cytoplasmic mo tor molecule, immunolocalizations showed substantial nuclear staining using several different anti-dynein light chain antibodies, Moreover, fluorescen ce overlays of confocal images established that nuclear respiratory factor- 1 and dynein light chain displayed a very similar nuclear staining pattern, The significance of the nuclear respiratory factor-1/dynein light chain in teraction was investigated further by determining whether a similar interac tion was conserved between dynein light chain and the erect wing gene produ ct of Drosophila, a protein related to nuclear respiratory factor-1 through its DNA binding domain, Here, we establish that the erect wing gene produc t can bind and trans-activate transcription through authentic nuclear respi ratory factor-1 binding sites. Moreover, the erect wing gene product, like nuclear respiratory factor-1, interacted specifically with dynein light cha in both in vitro and in transfected cells. Thus, the interaction with dynei n light chain is conserved between transcription factors that are structura lly and functionally similar between humans and Drosophila.