SUMMARY Kes1, and other oxysterol binding protein (OSBP) superfamily members, are involved in membrane and lipid trafficking through trans-Golgi network (TGN) and endosomal systems. We demonstrate that Kes1 represents a sterol-regulated antagonist of TGN/endosomal phosphatidylinositol-4-phosphate signaling. This regulation modulates TOR activation by amino acids, and dampens gene expression driven by Gcn4; the primary transcriptional activator of the general amino acid control regulon. Kes1-mediated repression of Gcn4 transcription factor activity is characterized by nonproductive Gcn4 binding to its target sequences, involves TGN/endosome-derived sphingolipid signaling, and requires activity of the cyclin-dependent kinase 8 (CDK8) module of the enigmatic ‘large Mediator’ complex. These data describe a pathway by which Kes1 integrates lipid metabolism with TORC1 signaling and nitrogen sensing.

Previous work has shown that the N terminus of the Saccharomyces cerevisiae Sir3 protein is crucial for the function of Sir3 in transcriptional silencing. Here, we show that overexpression of N-terminal fragments of Sir3 in strains lacking the full-length protein can lead to some silencing of HML and HMR. Sir3 contains a BAH (bromo-adjacent homology) domain at its N terminus. Overexpression of this domain alone can lead to silencing as long as Sir1 is overexpressed and Sir2 and Sir4 are present. Overexpression of the closely related Orc1 BAH domain can also silence in the absence of any Sir3 protein. A previously characterized hypermorphic sir3 mutation, D205N, greatly improves silencing by the Sir3 BAH domain and allows it to bind to DNA and oligonucleosomes in vitro. A previously uncharacterized region in the Sir1 N terminus is required for silencing by both the Sir3 and Orc1 BAH domains. The structure of the Sir3 BAH domain has been determined. In the crystal, the molecule multimerizes in the form of a left-handed superhelix. This superhelix may be relevant to the function of the BAH domain of Sir3 in silencing.Epigenetic silencing is a term used to describe the heritable transmission of a transcriptionally inactive state. The silent mating type loci HML and HMR and telomeres of the budding yeast Saccharomyces cerevisiae are examples of loci that undergo this type of transcriptional silencing and have served as a paradigm for studying this process.HML and HMR harbor copies of the mating type information genes, ␣ and a, respectively. They are involved in mating type interconversion with the actively transcribed MAT locus. Transcriptional silencing at these loci relies on the existence of cis-acting DNA regulatory elements, termed silencers (E and I), which flank both loci. These elements recruit the DNA binding proteins Rap1, Abf1, and ORC, which then serve to recruit the silent information regulators (Sir) 1, 2, 3, and 4 (11, 13, 33). The widely accepted view of silencing at these loci (and at telomeres) is that histone tails are deacetylated through the action of Sir2, a NAD-dependent histone deacetylase, creating a binding surface on nucleosomes for the binding of Sir3 and Sir4. Multiple rounds of deacetylation lead to the formation of a Sir2/3/4 polymer that spreads on the nucleosomes of the silent region, altering the chromatin and making it unavailable for transcription. The detailed structure of silent chromatin is not known, and exactly how transcription is prevented is a matter of dispute (6, 34).Sir3 is essential for the establishment and maintenance of the silent state at the HM loci and telomeres. Genetic, twohybrid, and biochemical studies have identified interactions of Sir3 with histones H3 and H4, Sir4, Rap1, Abf1, and Sir3 itself (reviewed in references 11, 13, and 33). Interestingly, all these interactions are within the C-terminal two-thirds of the Sir3 protein. Nevertheless, expression of a Sir3 construct lacking the N-terminal region (hereafter referred to as the N terminus) is not suffici...

Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production, and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs, nor PITPs in general, have been exploited as targets for chemical inhibition for such purposes. Herein, we validate the first small molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and are effective inhibitors in vitro and in vivo. We further establish Sec14 is the sole essential NPPM target in yeast, that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects, and demonstrate NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof-of-concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies.

Sir3, a component of the transcriptional silencing complex in the yeast Saccharomyces cerevisiae, has an N-terminal BAH domain that is crucial for the protein's silencing function. Previous work has shown that the N-terminal alanine residue of Sir3 (Ala2) and its acetylation play an important role in silencing. Here we show that the silencing defects of Sir3 Ala2 mutants can be suppressed by mutations in histones H3 and H4, specifically, by H3 D77N and H4 H75Y mutations. Additionally, a mutational analysis demonstrates that three separate regions of the Sir3 BAH domain are important for its role in silencing. Many of these BAH mutations also can be suppressed by the H3 D77N and H4 H75Y mutations. In agreement with the results of others, in vitro experiments show that the Sir3 BAH domain can interact with partially purified nucleosomes. The silencing-defective BAH mutants are defective for this interaction. These results, together with the previously characterized interaction between the C-terminal region of Sir3 and the histone H3/H4 tails, suggest that Sir3 utilizes multiple domains to interact with nucleosomes.Transcriptional silencing in the yeast Saccharomyces cerevisiae occurs at the silent mating type loci, HML and HMR, at genes near telomeres, and at the ribosomal DNA. The silencer elements flanking the HM loci recruit the DNA binding proteins Rap1, Abf1, and Orc1, while telomeric sequences bind Rap1. These DNA-bound proteins in turn recruit the silent information regulator (SIR) proteins. Multiple protein-protein interactions lead to spreading of a Sir2, Sir3, and Sir4 complex to nearby nucleosomes (reviewed in references 11 and 35). Sir2 plays a crucial role in this spreading by deacetylating histone H4 K16, thus allowing Sir3 and Sir4 to bind to nucleosomes and allowing further spreading of the Sir complex (reviewed in references 11, 26, and 35). Sir3 protein levels seem to control the extent of silencing. Silencing normally spreads up to 4 kb from the telomeres but can go as far as 20 kb away from the telomeric ends when Sir3 is overexpressed (16,34). While the exact mechanism by which transcription is silenced at these loci is not yet clear, it seems to involve a specialized chromatin structure that prevents transcription at either initiation or elongation (6,36).The N-terminal tails of histones H4 and H3 are important for silencing. Mutations in these tail regions show loss of silencing because they cause reduced binding of the Sir2, -3, and -4 complex (17,19,41). In addition to the N-terminal tails, a group of residues in the core domain of H3 and H4 are important for silencing (31, 42). These residues cluster around H3 K79, a site of methylation by Dot1, forming a patch on the surface of the nucleosome that could be a potential site for interaction with the silencing complex (23,44,49). Silencing is restricted to silent loci by many redundant mechanisms that prevent the spread of the silencing complex from the silent chromatin to active chromatin. Acetylation at H4 K16, methylation at H3 K4 and K7...

Proteins of the Sec14 superfamily regulate phosphoinositide signaling, and dysfunction of individual members of this superfamily results in a variety of human diseases. This study uses a directed evolution approach as a novel prism through which the functional engineering of a Sec14-like phosphatidylinositol transfer protein can be observed.