Three-dimensional structures of the TAFII-containing complexes TFIID and TFTC

TBP (TATA-binding protein)-associated factors (TAF(II)s) are components of large multiprotein complexes such as TFIID, TFTC, STAGA, PCAF/GCN5, and SAGA, which play a key role in the regulation of gene expression by RNA polymerase II. The structures of TFIID and TFTC have been determined at 3.5-nanometer resolution by electron microscopy and digital image analysis of single particles. Human TFIID resembles a macromolecular clamp that contains four globular domains organized around a solvent-accessible groove of a size suitable to bind DNA. TFTC is larger and contains five domains, four of which are similar to TFIID.     

Reversible interconversion of two forms of a valyl-tRNA synthetase-containing protein complex

When an enzyme-containing complex from yeast was incubated in a buffered solution at room temperature, the valyl-transfer RNA synthetase activity and total protein oscillated synchronously between two physical states. This observation suggests a regulatory process that controls a number of enzymes as a group, an integrated function of a kind not heretofore recognized. The two forms of the complex were separated by ammonium sulfate precipitation of one of them in samples withdrawn from the incubated solution every 30 seconds. Glutathione and dithiothreitol in high concentrations (50 mM) enhance formation of the 50% saturated ammonium sulfate-soluble form. Oxidized glutathione, diphosphopyridine nucleotide, triphosphopyridine nucleotide, and a mercurial thiol binding agent in moderate concentrations (0.1 to 1.0 mM) shift the distribution toward the precipitable form. It is suggested that the two forms represent functional and nonfunctional complex-bound enzymes which are interconverted in response to oxidoreductive signals.     

CLAVATA3, a multimeric ligand for the CLAVATA1 receptor-kinase

The CLAVATA1 (CLV1) and CLAVATA3 (CLV3) proteins form a potential receptor and ligand pair that regulates the balance between cell proliferation and differentiation at the shoot meristem of Arabidopsis. CLV1 encodes a receptor-kinase, and CLV3 encodes a predicted small, secreted polypeptide. We demonstrate that the CLV3 and CLV1 proteins coimmunoprecipitate in vivo, that yeast cells expressing CLV1 and CLV2 bind to CLV3 from plant extracts, and that binding requires CLV1 kinase activity. CLV3 only associates with the presumed active CLV1 protein complex in vivo. More than 75% of CLV3 in cauliflower extracts is bound with CLV1, consistent with hypotheses of ligand sequestration. Soluble CLV3 was found in an approximately 25-kilodalton multimeric complex.     

Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity

Rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1), extends the life spans of yeast, flies, and mice. Calorie restriction, which increases life span and insulin sensitivity, is proposed to function by inhibition of mTORC1, yet paradoxically, chronic administration of rapamycin substantially impairs glucose tolerance and insulin action. We demonstrate that rapamycin disrupted a second mTOR complex, mTORC2, in vivo and that mTORC2 was required for the insulin-mediated suppression of hepatic gluconeogenesis. Further, decreased mTORC1 signaling was sufficient to extend life span independently from changes in glucose homeostasis, as female mice heterozygous for both mTOR and mLST8 exhibited decreased mTORC1 activity and extended life span but had normal glucose tolerance and insulin sensitivity. Thus, mTORC2 disruption is an important mediator of the effects of rapamycin in vivo.