RNA polymerase I stability couples cellular growth to metal availability.

Zinc is an essential cofactor of all major eukaryotic RNA polymerases. How the activity of these enzymes is coordinated or regulated according to cellular zinc levels is largely unknown. Here we show that the stability of RNA polymerase I (RNAPI) is tightly coupled to zinc availability in vivo. In zinc deficiency, RNAPI is specifically degraded by proteolysis in the vacuole in a pathway dependent on the export in Xpo1p and deubiquitination of the RNAPI large subunit Rpa190p by Ubp2p and Ubp4p. RNAPII is unaffected, which allows for the expression of genes required in zinc deficiency. RNAPI export to the vacuole is required for survival during zinc starvation, suggesting that degradation of zinc-binding subunits might provide a last resort zinc reservoir. These results reveal a hierarchy of cellular transcriptional activities during zinc starvation and show that degradation of the most active cellular transcriptional machinery couples cellular growth and proliferation to zinc availability.

Sphingolipid signaling mediates iron toxicity.

Iron constitutes a major source of toxicity due to its ability to generate reactive oxygen species that can damage cellular macromolecules. However, the precise mechanism by which exposure to high iron concentrations results in cellular toxicity remains unknown. Here we identify sphingolipid synthesis and signaling as a major mediator of iron toxicity in S. cerevisiae. Inhibition of sphingolipid synthesis by myriocin treatment or after overexpression of the negative regulator Orm2p confers resistance to high iron. High iron conditions upregulate sphingolipid synthesis, and increasing sphingolipid levels by inactivating Orm2p exacerbates sensitivity to iron. Toxicity is mediated by sphingolipid signaling, as inactivation of the sphingolipid-activated protein kinases Pkh1p and Ypk1p and of the transcription factor Smp1p also enhances resistance to high iron conditions. These results demonstrate an unexpected connection between sphingolipid flux and iron toxicity and show that activation of a signal transduction cascade contributes to iron-mediated cellular toxicity.

Polymer-driven crystallization.

Obtaining well-diffracting crystals of macromolecules remains a significant barrier to structure determination. Here we propose and test a new approach to crystallization, in which the crystallization target is fused to a polymerizing protein module, so that polymer formation drives crystallization of the target. We test the approach using a polymerization module called 2TEL, which consists of two tandem sterile alpha motif (SAM) domains from the protein translocation Ets leukemia (TEL). The 2TEL module is engineered to polymerize as the pH is lowered, which allows the subtle modulation of polymerization needed for crystal formation. We show that the 2TEL module can drive the crystallization of 11 soluble proteins, including three that resisted prior crystallization attempts. In addition, the 2TEL module crystallizes in the presence of various detergents, suggesting that it might facilitate membrane protein crystallization. The crystal structures of two fusion proteins show that the TELSAM polymer is responsible for the majority of contacts in the crystal lattice. The results suggest that biological polymers could be designed as crystallization modules.