IRC3 Regulates Mitochondrial Translation in Response to Metabolic Cues in Saccharomyces cerevisiae.

Mitochondrial oxidative phosphorylation (OXPHOS) enzymes have a dual genetic origin. Mechanisms regulating the expression of nucleus-encoded OXPHOS subunits in response to metabolic cues (glucose versus glycerol) are well understood, while the regulation of mitochondrially encoded OXPHOS subunits is poorly defined. Here, we show that IRC3, a DEAD/H box helicase gene, previously implicated in mitochondrial DNA maintenance, is central to integrating metabolic cues with mitochondrial translation. Irc3 associates with mitochondrial small ribosomal subunits in cells consistent with its role in regulating translation elongation based on the Arg8m reporter system. IRC3-deleted cells retained mitochondrial DNA despite a growth defect on glycerol plates. Glucose-grown Δirc3ρ+ and irc3 temperature-sensitive cells at 37°C have reduced translation rates from the majority of mRNAs. In contrast, when galactose was the carbon source, a reduction in mitochondrial translation was observed predominantly from Cox1 mRNA in Δirc3ρ+ cells but no defect was observed in irc3 temperature-sensitive cells, at 37°C. In support of a model whereby IRC3 responds to metabolic cues to regulate mitochondrial translation, Δirc3 suppressor strains isolated for restoration of growth on glycerol medium restore mitochondrial protein synthesis differentially in the presence of glucose versus glycerol.

MRX8, the conserved mitochondrial YihA GTPase family member, is required for de novo Cox1 synthesis at suboptimal temperatures in Saccharomyces cerevisiae.

The synthesis of Cox1, the conserved catalytic-core subunit of Complex IV, a multisubunit machinery of the mitochondrial oxidative phosphorylation (OXPHOS) system under environmental stress, has not been sufficiently addressed. In this study, we show that the putative YihA superfamily GTPase, Mrx8, is a bona fide mitochondrial protein required for Cox1 translation initiation and elongation during suboptimal growth condition at 16°C. Mrx8 was found in a complex with mitochondrial ribosomes, consistent with a role in protein synthesis. Cells expressing mutant Mrx8 predicted to be defective in guanine nucleotide binding and hydrolysis were compromised for robust cellular respiration. We show that the requirement of Pet309 and Mss51 for cellular respiration is not bypassed by overexpression of Mrx8 and vice versa. Consistently the ribosomal association of Mss51 is independent of Mrx8. Significantly, we find that GTPBP8, the human orthologue, complements the loss of cellular respiration in Δmrx8 cells and GTPBP8 localizes to the mitochondria in mammalian cells. This strongly suggests a universal role of the MRX8 family of proteins in regulating mitochondrial function.

NET37, a nuclear envelope transmembrane protein with glycosidase homology, is involved in myoblast differentiation.

The nuclear lamina and its associated proteins are important for nuclear structure and chromatin organization and also have been implicated in the regulation of cell signaling and gene expression. In this study we demonstrate that the lamina-associated nuclear envelope transmembrane protein NET37 is required for myogenic differentiation of C2C12 cells. NET37, a member of glycosidase family 31, is highly expressed in mouse skeletal muscle and is strongly up-regulated during C2C12 differentiation. By protease mapping we show that its glycosidase homology domain is located in the lumen of the nuclear envelope/endoplasmic reticulum. When NET37 is depleted from proliferating myoblasts by RNAi, myogenic differentiation is significantly impaired, and there is a concomitant delay in up-regulation of the late myogenic transcription factor myogenin. We expressed silencing-resistant NET37 mutated at a conserved residue in the glycosidase domain and found that this predicted catalytically inactive protein is unable to support myogenesis in cells depleted of wild type NET37. Therefore, the enzymatic function of NET37 appears to be important for myogenic differentiation. C2C12 cells depleted of NET37 have reduced activation of Akt after shifting to differentiation medium and are defective in insulin like growth factor-II (IGF-II) secretion, an autocrine/paracrine factor involved in Akt activation. We also observed that pro-IGF-II co-immunoprecipitates with NET37. Based on our results, we propose that NET37 has a role in IGF-II maturation in the secretory pathway during myoblast differentiation. The localization of NET37 at the nuclear envelope raises the possibility that it may coordinate myogenic events between the nuclear interior and the endoplasmic reticulum lumen via transmembrane communication.

The yeast GTPase Mtg2p is required for mitochondrial translation and partially suppresses an rRNA methyltransferase mutant, mrm2.

The assembly of ribosomes involves the coordinated processing and modification of rRNAs with the temporal association of ribosomal proteins. This process is regulated by assembly factors such as helicases, modifying enzymes, and GTPases. In contrast to the assembly of cytoplasmic ribosomes, there is a paucity of information concerning the role of assembly proteins in the biogenesis of mitochondrial ribosomes. In this study, we demonstrate that the Saccharomyces cerevisiae GTPase Mtg2p (Yhr168wp) is essential for mitochondrial ribosome function. Cells lacking MTG2 lose their mitochondrial DNA, giving rise to petite cells. In addition, cells expressing a temperature-sensitive mgt2-1 allele are defective in mitochondrial protein synthesis and contain lowered levels of mitochondrial ribosomal subunits. Significantly, elevated levels of Mtg2p partially suppress the thermosensitive loss of mitochondrial DNA in a 21S rRNA methyltransferase mutant, mrm2. We propose that Mtg2p is involved in mitochondrial ribosome biogenesis. Consistent with this role, we show that Mtg2p is peripherally localized to the mitochondrial inner membrane and associates with the 54S large ribosomal subunit in a salt-dependent manner.

Atomic-level structural correlations across the morphotropic phase boundary of a ferroelectric solid solution: xBiMg1/2Ti1/2O3-(1 - x)PbTiO3.

Revelation of unequivocal structural information at the atomic level for complex systems is uniquely important for deeper and generic understanding of the structure property connections and a key challenge in materials science. Here we report an experimental study of the local structure by applying total elastic scattering and Raman scattering analyses to an important non-relaxor ferroelectric solid solution exhibiting the so-called composition-induced morphotropic phase boundary (MPB), where concomitant enhancement of physical properties have been detected. The powerful combination of static and dynamic structural probes enabled us to derive direct correspondence between the atomic-level structural correlations and reported properties. The atomic pair distribution functions obtained from the neutron total scattering experiments were analysed through big-box atom-modelling implementing reverse Monte Carlo method, from which distributions of magnitudes and directions of off-centred cationic displacements were extracted. We found that an enhanced randomness of the displacement-directions for all ferroelectrically active cations combined with a strong dynamical coupling between the A- and B-site cations of the perovskite structure, can explain the abrupt amplification of piezoelectric response of the system near MPB. Altogether this provides a more fundamental basis in inferring structure-property connections in similar systems including important implications in designing novel and bespoke materials.

Erlins restrict SREBP activation in the ER and regulate cellular cholesterol homeostasis.

Cellular cholesterol levels are controlled by endoplasmic reticulum (ER) sterol sensing proteins, which include Scap and Insig-1. With cholesterol sufficiency, Insig inhibits the activation of sterol regulatory element binding proteins (SREBPs), key transcription factors for cholesterol and fatty acid biosynthetic genes, by associating with Scap-SREBP complexes to promote their ER retention. Here we show that the multimeric ER proteins erlins-1 and -2 are additional SREBP regulators. Depletion of erlins from cells grown with sterol sufficiency led to canonical activation of SREBPs and their target genes. Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins. Erlins bound cholesterol with specificity and strong cooperativity and responded to ER cholesterol changes with altered diffusional mobility, suggesting that erlins themselves may be regulated by cholesterol. Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery. We speculate that erlins promote stability of the SREBP-Scap-Insig complex and may contribute to the highly cooperative control of this system.

Regulation of myoblast differentiation by the nuclear envelope protein NET39.

Recently, several transmembrane proteins of the nuclear envelope have been implicated in regulation of signaling and gene expression. Here we demonstrate that the nuclear lamina-associated nuclear envelope transmembrane protein NET39 (Ppapdc3) functions as a negative regulator of myoblast differentiation, in part through effects on mTOR signaling. We found that NET39 is highly expressed in cardiac and skeletal muscle tissues and becomes strongly upregulated during cultured myoblast differentiation. Knockdown of NET39 by RNA interference in myoblasts strongly promoted differentiation, whereas overexpression of NET39 repressed myogenesis. Proteomic analysis of NET39 complexes immunoprecipitated from myotubes, in combination with other methods, identified mTOR as an interaction partner of NET39. We found that ectopic expression of NET39 in myoblasts negatively regulated myogenesis by diminishing mTOR activity, which in turn decreased insulin-like growth factor II production and autocrine signaling. Our results indicate that NET39 is part of the regulatory machinery for myogenesis and raise the possibility that it may be important for muscle homeostasis.

In vivo functional characterization of the Saccharomyces cerevisiae 60S biogenesis GTPase Nog1.

The Saccharomyces cerevisiae Nog1 GTPase is critical for assembly of the large ribosomal subunit. Mutations in conserved residues in the GTP-binding pocket cause defects in cell growth and 60S ribosome assembly but mutant proteins retain their ability to associate with the pre-60S. Association of Nog1 with the pre-60S is independent of guanine nucleotide added to cell extracts. Thus, it appears that nucleotide occupancy does not substantially affect Nog1 association with pre-60S particles. Somewhat surprisingly, neither of the conserved threonines in the G2 motif of the GTPase domain is essential for Nog1 function. Neither the steady-state rRNA levels nor the protein composition (as determined by isobaric labeling and identification by mass spectrometry of peptides) of the pre-60S particles in the nog1P176V mutant are grossly perturbed, although levels of four proteins (Nog1, Nop2, Nop15, and Tif6) are modestly reduced in pre-60S particles isolated from the mutant. Deletion analysis revealed that the C-terminal 168 amino acids are not required for function; however, the N-terminal 126 amino acids are required. Optimal association with pre-60S particles requires sequences between amino acids 347-456. Several conserved charge-to-alanine substitutions outside the GTPase domain display modest growth phenotypes indicating that these residues are not critical for function.

The Escherichia coli GTPase CgtAE is involved in late steps of large ribosome assembly.

The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtA(E), in late steps of large ribosomal subunit biogenesis. CgtA(E) belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtA(E) cause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accumulate in a cgtA(E) mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtA(E) is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtA(E). We also show that purified CgtA(E) associates with purified ribosomal particles in the GTP-bound form. Finally, CgtA(E) cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants.

The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels.

The Obg subfamily of bacterial GTP-binding proteins are biochemically distinct from Ras-like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, we generated mutations in the Caulobacter crescentus obg gene (cgtAC) which, in Ras-like proteins, would result in either activating or dominant negative phenotypes. In C. crescentus, a P168V mutant is not activating in vivo, although in vitro, the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Furthermore, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP-binding pocket were, however, important for function. To examine the in vivo consequences of depleting CgtAC, we generated a temperature-sensitive mutant, G80E. At the permissive temperature, G80E cells grow slowly and have reduced levels of 50S ribosomal subunits, indicating that CgtAC is important for 50S assembly and/or stability. Surprisingly, at the non-permissive temperature, G80E cells rapidly lose viability and yet do not display an additional ribosome defect. Thus, the essential nature of the cgtAC gene does not appear to result from its ribosome function. G80E cells arrest as predivisional cells and stalkless cells. Flow cytometry on synchronized cells reveals a G1-S arrest. Therefore, CgtAC is necessary for DNA replication and progression through the cell cycle.