RESUMO
Schizosaccharomyces pombe Sre1 is a membrane-bound transcription factor that controls adaptation to hypoxia. Like its mammalian homolog, sterol regulatory element-binding protein (SREBP), Sre1 activation requires release from the membrane. However, in fission yeast, this release occurs through a strikingly different mechanism that requires the Golgi Dsc E3 ubiquitin ligase complex and the proteasome. The mechanistic details of Sre1 cleavage, including the link between the Dsc E3 ligase complex and proteasome, are not well understood. Here, we present results of a genetic selection designed to identify additional components required for Sre1 cleavage. From the selection, we identified two new components of the fission yeast SREBP pathway: Dsc5 and Cdc48. The AAA (ATPase associated with diverse cellular activities) ATPase Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing protein, interact with known Dsc complex components and are required for SREBP cleavage. These findings provide a mechanistic link between the Dsc E3 ligase complex and the proteasome in SREBP cleavage and add to a growing list of similarities between the Dsc E3 ligase and membrane E3 ligases involved in endoplasmic reticulum-associated degradation.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Membrana/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Ubiquitina-Proteína Ligases/química , Proteínas de Transporte/química , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/química , Mutagênese , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Proteólise , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteína com ValosinaRESUMO
We present an automated, high throughput library construction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method.