Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex.

Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

Yeast sterol regulatory element-binding protein (SREBP) cleavage requires Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing subunit of the Golgi Dsc E3 ligase.

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.

Degradation of sterol regulatory element-binding protein precursor requires the endoplasmic reticulum-associated degradation components Ubc7 and Hrd1 in fission yeast.

Sre1, the fission yeast sterol regulatory element-binding protein (SREBP), is an endoplasmic reticulum (ER) membrane-bound transcription factor that is a principal regulator of hypoxic gene expression. Under low oxygen, Sre1 is cleaved from its inactive ER precursor form to generate an active nuclear transcription factor that up-regulates genes required for low oxygen growth. To maintain a constant supply of Sre1, Sre1 precursor synthesis must be regulated to replenish Sre1 precursor lost to proteolytic cleavage under low oxygen. In this study, we investigated the mechanisms controlling Sre1 precursor levels. We found that positive feedback regulation at the sre1(+) promoter increases the synthesis of the Sre1 precursor under low oxygen and that this regulation is required for maximal Sre1 activation and target gene expression. We also demonstrate that the Sre1 precursor is rapidly degraded by the proteasome in the absence of its binding partner Scp1, which is required for oxygen-regulated Sre1 cleavage. Degradation of Sre1 in the absence of Scp1 requires the ER-associated degradation (ERAD) components Ubc7, an E2 ubiquitin conjugating enzyme, and Hrd1, an E3 ubiquitin ligase. We conclude that positive feedback regulation to up-regulate Sre1 precursor synthesis under low oxygen is essential for Sre1 function and propose that excess Sre1 precursor is removed by ERAD to ensure complex formation between Sre1 and its binding partner Scp1. Thus, Sre1 is a new example of an endogenous ERAD substrate, establishing fission yeast as an organism for the study of this important degradative pathway.

A Screen for Candidate Targets of Lysine Polyphosphorylation Uncovers a Conserved Network Implicated in Ribosome Biogenesis.

Polyphosphates (polyP) are chains of inorganic phosphates found in all cells. Previous work has implicated these chains in diverse functions, but the mechanism of action is unclear. A recent study reports that polyP can be non-enzymatically and covalently attached to lysine residues on yeast proteins Nsr1 and Top1. One question emerging from this work is whether so-called "polyphosphorylation" is unique to these proteins or instead functions as a global regulator akin to other lysine post-translational modifications. Here, we present the results of a screen for polyphosphorylated proteins in yeast. We uncovered 15 targets including a conserved network of proteins functioning in ribosome biogenesis. Multiple genes contribute to polyphosphorylation of targets by regulating polyP synthesis, and disruption of this synthesis results in translation defects as measured by polysome profiling. Finally, we identify 6 human proteins that can be modified by polyP, highlighting the therapeutic potential of manipulating polyphosphorylation in vivo.

Nicotinamide Suppresses the DNA Damage Sensitivity of Saccharomyces cerevisiae Independently of Sirtuin Deacetylases.

Nicotinamide is both a reaction product and an inhibitor of the conserved sirtuin family of deacetylases, which have been implicated in a broad range of cellular functions in eukaryotes from yeast to humans. Phenotypes observed following treatment with nicotinamide are most often assumed to stem from inhibition of one or more of these enzymes. Here, we used this small molecule to inhibit multiple sirtuins at once during treatment with DNA damaging agents in the Saccharomyces cerevisiae model system. Since sirtuins have been previously implicated in the DNA damage response, we were surprised to observe that nicotinamide actually increased the survival of yeast cells exposed to the DNA damage agent MMS. Remarkably, we found that enhanced resistance to MMS in the presence of nicotinamide was independent of all five yeast sirtuins. Enhanced resistance was also independent of the nicotinamide salvage pathway, which uses nicotinamide as a substrate to generate NAD+, and of a DNA damage-induced increase in the salvage enzyme Pnc1 Our data suggest a novel and unexpected function for nicotinamide that has broad implications for its use in the study of sirtuin biology across model systems.

Analyzing mRNA localization to the endoplasmic reticulum via cell fractionation.

The partitioning of secretory and membrane protein-encoding mRNAs to the endoplasmic reticulum (ER), and their translation on ER-associated ribosomes, governs access to the secretory/exocytic pathways of the cell. As mRNAs encoding secretory and membrane proteins comprise approximately 30% of the transcriptome, the localization of mRNAs to the ER represents an extraordinarily prominent, ubiquitous, and yet poorly understood RNA localization phenomenon.The partitioning of mRNAs to the ER is generally thought to be achieved by the signal recognition particle (SRP) pathway. In this pathway, mRNA localization to the ER is determined by the translation product - translation yields an N-terminal signal sequence or a topogenic signal that is recognized by the SRP and the resulting mRNA-ribosome-SRP complex is then recruited to the ER membrane. Recent studies have demonstrated that mRNAs can be localized to the ER via a signal sequence and/or translation-independent pathway(s) and that discrete sets of cytosolic protein-encoding mRNAs are enriched on the ER membrane, though they lack an encoded signal sequence. These key findings reopen investigations into the mechanism(s) that govern mRNA localization to the ER. In this contribution, we describe two independent methods that can be utilized to study this important and poorly understood aspect of eukaryotic cell biology. These methods comprise two independent means of fractionating tissue culture cells to yield free/cytosolic polyribosomes and ER membrane-bound polyribosomes. Detailed methods for the fractionation and characterization of the two polyribosome pools are provided.