The activity of the stress-responsive factor, E , is induced by the extracytoplasmic accumulation of misfolded or unfolded protein. The inner membrane protein RseA is the central regulatory molecule in this signal transduction cascade and acts as a E -specific anti-factor. Here we show that E activity is primarily determined by the ratio of RseA to E . RseA is rapidly degraded in response to extracytoplasmic stress, leading to an increase in the free pool of E and initiation of the stress response. We present evidence that the putative inner membrane serine protease, DegS, is responsible for this regulated degradation of RseA.

Talin is a high-molecular-weight cytoskeletal protein concentrated at regions of cell-substratum contact and, in lymphocytes, at cell-cell contacts. Integrin receptors are involved in the attachment of adherent cells to extracellular matrices and of lymphocytes to other cells. In these situations, talin codistributes with concentrations of integrins in the cell surface membrane. Furthermore, in vitro binding studies suggest that integrins bind to talin, although with low affinity. Talin also binds with high affinity to vinculin, another cytoskeletal protein concentrated at points of cell adhesion. Finally, talin is a substrate for the Ca2(+)-activated protease, calpain II, which is also concentrated at points of cell-substratum contact. To learn more about the structure of talin and its involvement in transmembrane connections between extracellular adhesions and the cytoskeleton, we have cloned and sequenced murine talin. We describe a model for the structure of talin based on this sequence and other data. Homologies between talin and other proteins define a novel family of submembranous cytoskeleton-associated proteins all apparently involved in connections to the plasma membrane.

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To assess the importance of residue 50 in determining the binding specificity of the homeodomain from the engrailed transcription factor of Drosophila, the DNA-binding properties of isolated homeodomains containing glutamine (wild type), alanine, and lysine at this position have been studied. In binding site selection experiments using the wild-type engrailed homeodomain, TAATTA was identified as a high-affinity, consensus binding site. When the glutamine at position 50 was replaced by a lysine (QK50), the binding site preference changed to TAATCC. The half-life and affinity of the complex between the QK50 protein and a DNA site containing TAATCC were increased significantly compared to the half-life and affinity of the complex between the wild-type protein and a TAATTA site. This suggests that Lys50 forms a more favorable interaction with the TAATCC DNA than Gln50 does with the TAATTA site. In fact, the wild-type Gln50 side chain (which forms a hydrophobic interaction with the last A:T base pair of the TAATTA site in the cocrystal structure [Kissinger, C. R., Liu, B., Martin-Blanco, E., Kornberg, T. B., & Pabo, C. O. (1990) Cell 63, 579-590]) appears to play only a small role in determining binding affinity and specificity for the TAATTA site, as the QA50 mutant has only a 2-fold reduced affinity for the TAATTA site and discriminates between the TAATTA and TAATCC sites as well as the wild-type protein. As a result, determinants in addition to Gln50 must be involved in establishing the differential binding specificity of the engrailed homeodomain.