Iron (Fe) is an essential cofactor for a wide range of cellular processes. We have previously demonstrated in yeast that Cth2 is expressed during Fe deficiency and promotes degradation of a battery of mRNAs leading to reprogramming of Fe-dependent metabolism and Fe storage. We report here that the Cth2-homologous protein Cth1 is transiently expressed during Fe deprivation and participates in the response to Fe deficiency through the degradation of mRNAs primarily involved in mitochondrially localized activities including respiration and amino acid biosynthesis. In parallel, wild-type cells, but not cth1Deltacth2Delta cells, accumulate mRNAs encoding proteins that function in glucose import and storage and store high levels of glycogen. In addition, Fe deficiency leads to phosphorylation of Snf1, an AMP-activated protein kinase family member required for the cellular response to glucose starvation. These studies demonstrate a metabolic reprogramming as a consequence of Fe starvation that is dependent on the coordinated activities of two mRNA-binding proteins.

Iron is an essential nutrient that participates as a redox cofactor in a broad range of cellular processes. In response to iron deficiency, the budding yeast Saccharomyces cerevisiae induces the expression of the Cth1 and Cth2 mRNA-binding proteins to promote a genome-wide remodeling of cellular metabolism that contributes to the optimal utilization of iron. Cth1 and Cth2 proteins bind to specific AU-rich elements within the 3-untranslated region of many mRNAs encoding proteins involved in iron-dependent pathways, thereby promoting their degradation. Here, we show that the DEAD box Dhh1 helicase plays a crucial role in the mechanism of Cth2-mediated mRNA turnover. Yeast two-hybrid experiments indicate that Cth2 protein interacts in vivo with the carboxyl-terminal domain of Dhh1. We demonstrate that the degradation of succinate dehydrogenase SDH4 mRNA, a known target of Cth2 on iron-deficient conditions, depends on Dhh1. In addition, we localize the Cth2 protein to cytoplasmic processing bodies in strains defective in the 5 to 3 mRNA decay pathway. Finally, the degradation of trapped SDH4 mRNA intermediates by Cth2 supports the 5 to 3 directionality of mRNA turnover. Taken together, these results suggest that Cth2 protein recruits the Dhh1 helicase to ARE-containing mRNAs to promote mRNA decay.

Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au146(p-MBA)57 (p-MBA: para-mercaptobenzoic acid), solved by electron diffraction (MicroED) to subatomic resolution (0.85 Å) and by X-ray diffraction at atomic resolution (1.3 Å). The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure whereas the surface gold atoms follow a C2 rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au146(p-MBA)57 is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault.

Vacuolar H؉ -ATPases (V-ATPases) are highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although it is generally believed that V-ATPases transport protons by a rotary catalytic mechanism analogous to that used by F 1 F 0 -ATPases, the structure and subunit composition of the central or peripheral stalk of the multisubunit complex are not well understood. We searched for proteins that bind to the E subunit of V-ATPase using the yeast two-hybrid assay and identified the H subunit as an interacting partner. Physical association between the E and H subunits of V-ATPase was confirmed in vitro by precipitation assays. Deletion mapping analysis revealed that a 78-amino acid fragment at the amino terminus of the E subunit was sufficient for binding to the H subunit. Expression of the amino-terminal fragments of the E subunits from human and yeast as dominantnegative mutants resulted in dramatic decreases in bafilomycin A 1 -sensitive ATP hydrolysis and proton transport activities of V-ATPase. Our data demonstrate the physiological significance of the interaction between the E and H subunits of V-ATPase and extend previous studies on the arrangement of subunits on the peripheral stalk of V-ATPase. Vacuolar Hϩ -ATPases (V-ATPases) 1 energize and acidify intracellular compartments of the vacuolar system of eukaryotic cells. They are essential for the normal function of secretory vesicles, the trans-Golgi network, endosomes, lysosomes, the yeast vacuole, and other intracellular membrane compartments (1, 2). In some specialized cells such as the intercalated cells of the kidney and the osteoclasts, V-ATPases reside at high levels on the plasma membrane, where they are responsible for transepithelial or cellular proton transport required for normal acid-base homeostasis and bone remodeling (2). Despite their wide range of physiological functions, V-ATPases share a highly conserved structure and common enzymatic properties that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol (3). They contain two macrodomains or sectors: V 1 , a catalytic domain composed of peripheral membrane proteins, and V 0 , a transmembrane domain composed of intrinsic membrane proteins that transmits protons through the lipid bilayer (4). The V 1 domain attaches to the V 0 domain at the cytoplasmic face of the membrane. In Saccharomyces cerevisiae, the V 1 domain is composed of eight distinct polypeptide chains, and the V 0 domain contains five (4).V-ATPases are evolutionarily related and structurally similar to F 1 F 0 -ATPases (F-ATPases) of bacteria, chloroplasts, and mitochondria (3). F-ATPases also have two sectors: F 1 , a peripherally attached complex composed of a catalytic head and a stalk, and F 0 , composed of intrinsic membrane subunits and a stator arm. F-ATPases have a rotary catalytic mechanism (5-7). The proton electrochemical gradient across the membrane drives translocation of protons through a pathway composed of the a and c subunits in the F 0 sector,...

iron (Fe) deficiency. Mammalian members of this family of proteins are known to undergo nucleocytoplasmic shuttling, but little is known about the role of shuttling in the mechanism of ARE-dependent mRNA decay. Here we demonstrate that, like its mammalian homologues, Cth2 is a nucleocytoplasmic shuttling protein whose nuclear export depends on mRNA transport to the cytosol. The nuclear import information of Cth2 is contained within its tandem zinc finger domain, but it is independent of mRNA-binding function. Moreover, we also demonstrate that nucleocytoplasmic shuttling of Cth2 requires active transcription and that disruption of shuttling leads to defects in Cth2 function in mRNA decay under Fe deficiency. Taken together, our data suggest that under conditions of Fe deficiency Cth2 travels into the nucleus to recruit target mRNAs, perhaps cotranscriptionally, that are destined for cytosolic degradation as part of the mechanism of adaptation to growth under Fe limitation. These data also suggest an important role for nucleocytoplasmic shuttling in this conserved family of proteins in the mechanism of ARE-mediated mRNA decay.Iron (Fe) participates in numerous biochemical processes, of which many are essential. Thus, under conditions of Fe scarcity, cells must ensure the appropriate allocation of Fe in order to meet essential metabolic needs while lowering its incorporation into proteins that participate in nonessential processes or proteins involved in metabolic pathways for which there are alternative salvage mechanisms (9,16,20,23,24,26,32). The Saccharomyces cerevisiae Cth1 and Cth2 proteins play a pivotal role in the cellular adaptation to Fe limitation by posttranscriptionally regulating Fe metabolism (23, 24).Cth1 and Cth2 belong to the TTP (Tristetraprolin) family of mRNA-destabilizing proteins. Members of this family are characterized by an RNA-binding motif consisting of two tandem zinc fingers (TZFs) of the CX 8 CX 5 CX 3 H type, which directly interact with AU-rich elements (AREs) within the 3Ј untranslated region of select groups of mRNAs (2,3,28,29). This interaction leads to the rapid destabilization of the bound transcript in a process termed ARE-mediated mRNA decay (AMD). In response to Fe starvation, yeast Cth1 and Cth2 coordinately promote AMD of select groups of mRNAs, many of which encode proteins with functions in highly Fe-demanding processes such the tricarboxylic acid cycle, the mitochondrial electron transport chain, heme biogenesis, and Fe-S-containing proteins, presumably to allow prioritization of Fe utilization (23,24,32).Studies have demonstrated that yeast AREs modulate poly(A) tail removal of a reporter mRNA, followed by decapping (31), and recent studies have demonstrated that Cth2-dependent AMD is catalyzed from the 5Ј end to the 3Ј end by the cytoplasmic exonuclease Xrn1 (20,22). Similarly, studies have demonstrated that mammalian TTP promotes AMD by inducing poly(A) tail removal, followed by decapping and mRNA decay from both 5Ј33Ј and 3Ј35Ј directions (2,4,7,12,13,15), sugges...

b Iron (Fe) is an essential element for all eukaryotic organisms because it functions as a cofactor in a wide range of biochemical processes. Cells have developed sophisticated mechanisms to tightly control Fe utilization in response to alterations in cellular demands and bioavailability. In response to Fe deficiency, the yeast Saccharomyces cerevisiae activates transcription of the CTH1 and CTH2 genes, which encode proteins that bind to AU-rich elements (AREs) within the 3= untranslated regions (3=UTRs) of many mRNAs, leading to metabolic reprogramming of Fe-dependent pathways and decreased Fe storage. The precise mechanisms underlying Cth1 and Cth2 function and regulation are incompletely understood. We report here that the Cth1 and Cth2 proteins specifically bind in vivo to AREs located at the 3=UTRs of their own transcripts in an auto-and cross-regulated mechanism that limits their expression. By mutagenesis of the AREs within the CTH2 transcript, we demonstrate that a Cth2 negativefeedback loop is required for the efficient decline in Cth2 protein levels observed upon a rapid rise in Fe availability. Importantly, Cth2 autoregulation is critical for the appropriate recovery of Fe-dependent processes and resumption of growth in response to a change from Fe deficiency to Fe supplementation.

Adeno-associated virus was used to transduce primary mouse osteoclasts with the B1 isoform of vacuolar H + -ATPase. B1, which is not normally expressed in osteoclasts, was correctly targeted to ruffled membranes of resorbing osteoclasts. Mutant subunit B1 that lacked a functional actin-binding site did not accumulate in ruffled membranes.

Metallic nanoparticles display unique optical, electronic, and chemical properties compared to their bulk counterparts. These properties are influenced by the internal structure of nanoparticles. Therefore, atomic structural characterization of nanoparticles is of paramount importance in nanotechnology. In this work, we present the synthesis, mass spectrometry, and structural characterization of highly monodisperse thiolate-protected gold nanoparticles (∼3.8 nm) using aberration-corrected scanning transmission electron microscopy (STEM). Mass spectrometry reveals the composition to be Au ∼2000 (SC 6 H 13 ) ∼290 . The images registered in the high-angle annular dark field detector (HAADF−STEM) showed the presence of decahedral and single-crystal facecentered cubic (fcc) nanoparticles as well as fcc structures with multiple planar defects. We also observed nanoparticles with an inner grain boundary corresponding to a highangle grain boundary classified as Σ9 under the coincidence site lattice notation. Experimental structural analysis and characterization of grain boundaries were correlated with simulated HAADF−STEM images of structural models for Σ9. The present report demonstrates the coexistence of two crystallites within thiolate-protected nanoparticles separated by high-angle grain boundaries.