Summary The conserved transcriptional regulator Heat Shock Factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol, yet its regulation is poorly understood. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC and Hsf1 senses an RQC-mediated translation stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.

We have purified distinct complexes of nine to 12 proteins [referred to as BRG1‐associated factors (BAFs)] from several mammalian cell lines using an antibody to the SWI2‐SNF2 homolog BRG1. Microsequencing revealed that the 47 kDa BAF is identical to INI1. Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene. A group of BAF47‐associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co‐purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI‐SNF complex. Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4‐VP16 to a nucleosomal template similar to the yeast SWI‐SNF complex. Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition. The two SWI‐SNF2 homologs, BRG1 and hbrm, were found in separate complexes. Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI‐SNF complex may be tailored to the needs of a differentiated cell type.

The SWI/SNF complex in yeast facilitates the function of transcriptional activators by opposing chromatin-dependent repression of transcription. We demonstrate that in mammals SWI/SNF complexes are present in multiple forms made up of 9-12 proteins that we refer to as BRGl-associated factors (BAFs) ranging from 47 to 250 kD. We have isolated cDNAs for human BAF155, BAF170, and BAF60. BAF155 and BAF170 are encoded by separate genes that are both homologs of yeast SWI3. Both contain a region of similarity to the DNA binding domain of myb, but lack the basic residues known to be necessary for interaction with DNA. The two SWI3 homologs copurify on antibody columns specific for either BAF155 or BAF170, indicating that they are in the same complex. BAF60 is encoded by a novel gene family. An open reading frame from yeast, which is highly homologous, encodes the previously uncharacterized 73=kD subunit of the yeast SWI/SNF complex required for transcriptional activation by the glucocorticoid receptor (Cairns et al., this issue). BAF60a is expressed in all tissues examined, whereas BAF60b and BAF60c are expressed preferentially in muscle and pancreas, respectively. BAF60a is present within the 2000-kD BRG1 complex, whereas BAF60b is in a distinct complex that shares some but not all subunits with the BRG1 complex. The observed similarity between mammalian BAF190, BAF170, BAF155, BAF60, and BAF47 and yeast SNF2/SWI2, SWI3, SWI3, SWP73, and SNF5, respectively, underscores the similarity of the mammalian and yeast complexes. However, the complexes in mammals are more diverse than the SWI/SNF complex in yeast and are likely dedicated to developmentally distinct functions.

y g by addition of nonrecombinant pCDNA3, as necessary. Results represent three t o six different transfected cultures, with the experiments performed on at least two different days. For dark conditions, plates of NIH 313 cells were wrapped in foil immediately after transfection until the time of harvesting (48 hours), except for a single brief medium change performed under dim red light (Kodak Safelight, Wratten Series 1A) 24 hours before harvesting. 18. E. A. Griffin Jr., D. Staknis, C. J. Weitz, unpublished data. (17) and light conditions were processed in parallel and incubated together. For light conditions, cells were illuminated (Fiber-Lite High Intensity Illuminator, Series 180; set at 10 relative illumination units) immediately after transfection until the time of harvesting. Plates were positioned 15 t o 30 cm from the tips of the twin fiberoptic guides. Transfected cells under dark