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The effects of retinoids on gene regulation are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Here, we provide the first biochemical evidence that, in vitro, ligand governs the transcriptional activity of RXRa/RARa by inducing conformational changes in the ligand-binding domains. Using limited proteolytic digestion we show that binding of the cognate ligand causes a conformational change in the carboxy-terminal part of the receptor. We also show that recombinant RXRa/RARa is partially active in the absence of exogenously added ligand. Trans-activation depends critically on the ligand-dependent transcriptional activation function AF-2 of RARer. Full activation by recombinant RXRoL/RAR~, however, requires the addition of either all-trans RA, 9-cis RA, or other RAR-specific agonists, whereas an RARa-specific antagonist abolishes trans-activation. Intriguingly, the ligand-dependent AF-2 of RXR does not contribute to the level of transcription from the RARI32 promoter in vitro even when the cognate ligand (9-cis RA1 is bound. Thus, the major role of RXR in trans-activation of the RARI32 promoter is to serve as an auxiliary factor required for the binding of RAR which, in turn, is directly responsible for transcriptional activity.

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We have identified and isolated mutations in the first Drosophila gene encoding a subunit of the Sec61 protein translocation channel, DSec61beta. While neither the Saccharomyces cerevisiae Sec61beta nor its functional Escherichia coli homologue are essential for viability or for protein translocation, we show that DSec61beta is essential for embryonic development. Homozygous mutant embryos die at the end of embryogenesis and are impaired in the secretion of cuticle proteins from the epidermis. DSec61beta germ line clones, result in defects in dorso-ventral patterning of the egg and are consistent with affected secretion of the protein Gurken from the oocyte to the follicle cells. Clonal analyses in the imaginal discs reveal defects in adult structures, including rhabdomere morphogenesis and a reduction of the size of tarsal segments in the leg. This is the first in vivo study of a component of the protein translocation machinery in higher eukaryotes, and illustrates how a protein that has an inessential, kinetic function in single-cell organisms can become critical for the complex development of a multicellular organism.