DNA-bound Bas1 recruits Pho2 to activate ADE genes in Saccharomyces cerevisiae.

Expression of the genes in the ADE regulon of Saccharomyces cerevisiae is repressed by the presence of purine bases in the extracellular medium and derepressed when cells are grown in the absence of purines. Derepression requires the transcriptional activators Bas1 and Pho2, as well as the biosynthetic intermediates 5'-phosphoribosyl-4-succinocarboxamide-5-aminoimidazole (SAICAR) and 5'-phosphoribosyl-4-carboxamide- 5-aminoimidazole (AICAR). In this study, we investigated if nuclear localization and binding to promoter DNA by the activators are regulated by purines. Using indirect immunofluorescence, we found that Bas1 is localized to the nucleus under both repressing and derepressing conditions. Importantly, we detected Bas1 bound to promoter DNA under both conditions using chromatin immunoprecipitation assays at several ADE promoters (ADE1, ADE2, ADE4, and ADE5,7) and HIS4. We analyzed the binding of Bas1 to wild-type and mutant sequences of the ADE5,7 promoters in vivo, and found that Bas1 binds independently to each of its two binding sites. Pho2 was not required for the association of Bas1 with chromosomal DNA, but it was required for an increase in Bas1-immunoprecipitated DNA. The presence of Pho2 at promoters was dependent on Bas1 and occurred only under derepressing conditions when the ADE genes are transcribed at elevated levels. We propose a model for regulation of the ADE genes in which DNA-bound Bas1 is inactive due to masking of its activation domain and Pho2 binds poorly to promoters when cells have sufficient purine nucleotides. Upon limitation for purines, the SAICAR/AICAR regulatory signal is transmitted to the nucleus to increase Bas1 and Pho2 interaction, recruiting Pho2 to promoters and freeing the activation domains for transactivation.

Functional mapping of Bas2. Identification of activation and Bas1-interaction domains.

The transcriptional activator protein Bas2 is required to express more than 20 genes in pathways for purine nucleotide and histidine biosynthesis, phosphate utilization, and the HO endonuclease by acting with co-regulator proteins Bas1, Pho4, and Swi5. The role that Bas2 plays in transcriptional activation may be to unmask latent activation domains in the co-regulator and to promote ternary complex formation between Bas2, the co-regulator, and DNA. We show that Bas2 also contributes to transcriptional activation by providing an activation domain. We localize this domain in Bas2 to the C-terminal 156 amino acids using deletion analysis and fusion to a heterologous DNA binding domain. Additionally, we show that Bas2 makes direct contacts with Bas1. This interaction is detected by co-immunoprecipitation and by two-hybrid analysis. We localize the interaction region to the central portion of Bas2, from amino acids 112 to 404.

The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting.

Membrane proteins that are degraded in the vacuole of Saccharomyces cerevisiae are sorted into discrete intralumenal vesicles, analogous to the internal membranes of multi-vesiculated bodies (MVBs). Recently, it has shown that the attachment of ubiquitin (Ub) mediates sorting into lumenal membranes. We describe a complex of Vps27p and Hse1p that localizes to endosomal compartments and is required for the recycling of Golgi proteins, formation of lumenal membranes and sorting of ubiquitinated proteins into those membranes. The Vps27p-Hse1p complex binds to Ub and requires multiple Ub Interaction Motifs (UIMs). Mutation of these motifs results in specific defects in the sorting of ubiquitinated proteins into the vacuolar lumen. However, the recycling of Golgi proteins and the generation of lumenal membranes proceeds normally in Delta UIM mutants. These data support a model in which the Vps27p-Hse1p complex has multiple functions at the endosome, one of which is as a sorting receptor for ubiquitinated membrane proteins destined for degradation.