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Endoplasmic reticulum (ER)-localized enzymes synthesize the vast majority of cellular lipids. The ER therefore has a major influence on cellular lipid biomass and balances the production of different lipid categories, classes, and species. Signals from outside and inside the cell are directed to ER-localized enzymes, and lipid enzyme activities are defined by the integration of internal, homeostatic, and external information. This allows ER-localized lipid synthesis to provide the cell with membrane lipids for growth, proliferation, and differentiation-based changes in morphology and structure, and to maintain membrane homeostasis across the cell. ER enzymes also respond to physiological signals to drive carbohydrates and nutritionally derived lipids into energy-storing triglycerides. In this review, we highlight some key regulatory mechanisms that control ER-localized enzyme activities in animal cells. We also discuss how they act in concert to maintain cellular lipid homeostasis, as well as how their dysregulation contributes to human disease. ispartof: EMBO Reports vol:18 issue:11 pages:1905-1921 ispartof: location:England status: published

The biogenesis of autophagosomes depends on the conjugation of Atg8-like proteins with phosphatidylethanolamine. Atg8 processing by the cysteine protease Atg4 is required for its covalent linkage to phosphatidylethanolamine, but it is also necessary for Atg8 deconjugation from this lipid to release it from membranes. How these two cleavage steps are coordinated is unknown. Here we show that phosphorylation by Atg1 inhibits Atg4 function, an event that appears to exclusively occur at the site of autophagosome biogenesis. These results are consistent with a model where the Atg8-phosphatidylethanolamine pool essential for autophagosome formation is protected at least in part by Atg4 phosphorylation by Atg1 while newly synthesized cytoplasmic Atg8 remains susceptible to constitutive Atg4 processing., The protease Atg4 mediates Atg8 lipidation, required for autophagosome biogenesis, but also triggers Atg8 release from the membranes, however is unclear how these steps are coordinated. Here the authors show that phosphorylation by Atg1 inhibits Atg4 at autophagosome formation sites.

Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3-interacting region (LIR) at the C-terminus of the protein and a novel motif at the N-terminus. Although both sites are important for Atg4-Atg8 interaction in vivo, only the new N-terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE-bound Atg8.

26 p.-1 fig.-1 tab. Galluzzi, Lorenzo et al., Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.

Plants contain both cytosolic and chloroplastic GAPDHs (glyceraldehyde-3-phosphate dehydrogenases). In Arabidopsis thaliana, cytosolic GAPDH is involved in the glycolytic pathway and is represented by two differentially expressed isoforms (GapC1 and GapC2) that are 98% identical in amino acid sequence. In the present study we show that GapC1 is a phosphorylating NAD-specific GAPDH with enzymatic activity strictly dependent on Cys(149). Catalytic Cys(149) is the only solvent-exposed cysteine of the protein and its thiol is relatively acidic (pK(a)=5.7). This property makes GapC1 sensitive to oxidation by H(2)O(2), which appears to inhibit enzyme activity by converting the thiolate of Cys(149) (-S-) into irreversible oxidized forms (-SO(2)(-) and -SO(3)(-)) via a labile sulfenate intermediate (-SO(-)). GSH (reduced glutathione) prevents this irreversible process by reacting with Cys(149) sulfenates to give rise to a mixed disulfide (Cys(149)-SSG), as demonstrated by both MS and biotinylated GSH. Glutathionylated GapC1 can be fully reactivated either by cytosolic glutaredoxin, via a GSH-dependent monothiol mechanism, or, less efficiently, by cytosolic thioredoxins physiologically reduced by NADPH:thioredoxin reductase. The potential relevance of these findings is discussed in the light of the multiple functions of GAPDH in eukaryotic cells (e.g. glycolysis, control of gene expression and apoptosis) that appear to be influenced by the redox state of the catalytic Cys(149).

Rice blast fungus ( Magnaporthe grisea) forms a highly specialized infection structure for plant penetration, the appressorium, the formation and growth of which are regulated by the Mst11- Mst7- Pmk1 mitogen- activated protein kinase cascade. We characterized the MST50 gene that directly interacts with both MST11 and MST7. Similar to the mst11 mutant, the mst50 mutant was defective in appressorium formation, sensitive to osmotic stresses, and nonpathogenic. Expressing a dominant active MST7 allele in mst50 complemented its defects in appressorium but not lesion formation. The sterile alpha-motif ( SAM) domain of Mst50 was essential for its interaction with Mst11 and for appressorium formation. Although the SAM and Ras-association domain ( RAD) of Mst50 were dispensable for its interaction with Mst7, deletion of RAD reduced appressorium formation and virulence on rice ( Oryza sativa) seedlings. The interaction between Mst50 and Mst7 or Mst11 was detected by coimmunoprecipitation assays in developing appressoria. Mst50 also interacts with Ras1, Ras2, Cdc42, and Mgb1 in yeast two- hybrid assays. Expressing a dominant active RAS2 allele in the wild- type strain but not in mst50 stimulated abnormal appressorium formation. These results indicate that MST50 functions as an adaptor protein interacting with multiple upstream components and plays critical roles in activating the Pmk1 cascade for appressorium formation and plant infection in M. grisea.

We investigated the role in cell morphogenesis and pathogenicity of the Candida albicans GPR1 gene, encoding the G protein-coupled receptor Gpr1. Deletion of C. albicans GPR1 has only minor effects in liquid hypha-inducing media but results in strong defects in the yeast-to-hypha transition on solid hypha-inducing media. Addition of cAMP, expression of a constitutively active allele of the G alpha protein Gpa2 or of the catalytic protein kinase A subunit TPKI restores the wild-type phenotype of the CaGPR1-deleted strain. Overexpression of HST7, encoding a component of the mitogen-activated protein kinase pathway, does not suppress the defect in filamentation. These results indicate that CaGpr1 functions upstream in the cAMP-protein kinase A (PKA) pathway. We also show that, in the presence of glucose, CaGpr1 is important for amino acid-induced transition from yeast to hyphal cells. Finally, as opposed to previous reports, we show that CaGpa2 acts downstream of CaGpr1 as activator of the cAMP-PKA pathway but that deletion of neither CaGpr1 nor CaGpa2 affects glucose-induced cAMP signaling. In contrast, the latter is abolished in strains lacking CaCdc25 or CaRas1, suggesting that the CaCdc25-CaRas1 rather than the CaGpr1-CaGpa2 module mediates glucose-induced cAMP signaling in C. albicans. ispartof: Molecular biology of the cell vol:16 issue:4 pages:1971-1986 ispartof: location:United States status: published

A sugar-transporter-encoding gene, mstA, which is a member of the major facilitator superfamily, has been cloned from a genomic DNA library of the filamentous fungus Aspergillus niger. To enable the functional characterization of MSTA, a full-length cDNA was expressed in a Saccharomyces cerevisiae strain deficient in hexose uptake. Uptake experiments using 14C-labelled monosaccharides demonstrated that although able to transport D-fructose ( K(m), 4.5+/-1.0 mM), D-xylose ( K(m), 0.3+/-0.1 mM) and D-mannose ( K(m), 60+/-20 microM), MSTA has a preference for D-glucose (K(m), 25+/-10 microM). pH changes associated with sugar transport indicate that MSTA catalyses monosaccharide/H+ symport. Expression of mstA in response to carbon starvation and upon transfer to poor carbon sources is consistent with a role for MSTA as a high-affinity transporter for D-glucose, D-mannose and D-xylose. Northern analysis has shown that mstA is subject to CreA-mediated carbon catabolite repression and pH regulation mediated by PacC. A. niger strains in which the mstA gene had been disrupted are phenotypically identical with isogenic reference strains when grown on 0.1-60 mM D-glucose, D-mannose, D-fructose or D-xylose. This indicates that A. niger possesses other transporters capable of compensating for the absence of MSTA.

The human gene RPMS12 encodes a protein similar to bacterial ribosomal protein S12 and is proposed to represent the human mitochondrial orthologue, RPMS12 reporter gene expression in cultured human cells supports the idea that the gene product is mitochondrial and is localized to the inner membrane. Human cells contain at least four structurally distinct RPMS12 mRNAs that differ in their 5'-untranslated region (5'-UTR) as a result of alternate splicing and of 5' end heterogeneity. All of them encode the same polypeptide, The full 5'-UTR contains two types of sequence element implicated elsewhere in translational regulation as follows: a short upstream open reading frame and an oligopyrimidine tract similar to that found at the 5' end of mRNAs encoding other growth-regulated proteins, including those of cytosolic ribosomes, The fully spliced (short) mRNA is the predominant form in all cell types studied and is translationally down-regulated in cultured cells in response to serum starvation, even though it lacks both of the putative translational regulatory elements. By contrast, other splice variants containing one or both of these elements are not translationally regulated by growth status but are translated poorly in both growing and non-growing cells. Reporter analysis identified a 26-nucleotide tract of the 5'-UTR of the short mRNA that is essential for translational down-regulation in growth-inhibited cells. Such experiments also confirmed that the 5'-UTR of the longer mRNA variants contains negative regulatory elements for translation. Tissue representation of RPMS12 mRNA is highly variable, following a typical mitochondrial pattern, but the relative levels of the different splice variants are similar in different tissues. These findings indicate a complex, multilevel regulation of RPMS12 gene expression in response to signals mediating growth, tissue specialization, and probably metabolic needs. ispartof: Journal of biological chemistry vol:274 issue:45 pages:31853-31862 ispartof: location:United States status: published

GAR1 is a 25-kDa nucleolar protein that is essential for yeast cell growth. The protein is associated with a subset of small nucleolar RNAs and is required for pre-rRNA processing. By expressing in yeast various deletions of GAR1 fused to a reporter protein, we have searched for which particular domain of GAR1 can account for its nucleolar localization. We report here that the glycine/arginine-rich domains of GAR1, which are shared by several other nucleolar proteins, are neither sufficient nor required for the steady-state accumulation of the fusion protein in the nucleolus. We further demonstrate that the central domain of GAR1 is both sufficient to target the beta-galactosidase to the yeast nucleolus and to restore the growth of a strain deficient in GAR1. As opposed to the other characterized nucleolar proteins, the nucleolar targeting domain of GAR1 does not exhibit any homology with the SV40 T-antigen-type nuclear localization sequence. Moreover, none of the modified GAR1 proteins that we examined has allowed us to distinguish the nuclear and nucleolar targeting domains. The presence in GAR1 of a single domain that is responsible for both nuclear entry and nucleolar accumulation suggests that GAR1 either could be carried piggyback by another nucleolar component, possibly as part of a small nucleolar ribonucleoprotein particle, or could be transported to the nucleolus by using a pathway different from the other nucleolar proteins. ispartof: Journal of biological chemistry vol:269 issue:28 pages:18499-18506 ispartof: location:United States status: published

Many cyclic hydrocarbons, e.g. aromatics, cycloalkanes, and terpenes, are toxic to microorganisms. The primary site of the toxic action is probably the cytoplasmic membrane, but the mechanism of the toxicity is still poorly understood. The effects of cyclic hydrocarbons were studied in liposomes prepared from Escherichia coli phospholipids. The membrane-buffer partition coefficients of the cyclic hydrocarbons revealed that these lipophilic compounds preferentially reside in the membrane. The partition coefficients closely correlated with the partition coefficients of these compounds in a standard octanol-water system. The accumulation of hydro carbon molecules resulted in swelling of the membrane bilayer, as assessed by the release of fluorescence self-quenching of fluorescent fatty acid and phospholipid analogs. Parallel to the expansion of the membrane, an increase in membrane fluidity was observed. These effects on the integrity of the membrane caused an increased passive flux of protons and carboxyfluorescein. In cytochrome c oxidase containing proteoliposomes, both components of the proton motive force, the pH gradient and the electrical potential, were dissipated with increasing concentrations of cyclic hydrocarbons. The dissipating effect was primarily the result of an increased permeability of the membrane for protons (ions). At higher concentrations, cytochrome c oxidase was also inactivated. The effective concentrations of the different cyclic hydrocarbons correlated with their partition coefficients between the membrane and aqueous phase. The impairment of microbial activity by the cyclic hydrocarbons most likely results from hydrophobic interaction with the membrane, which affects the functioning of the membrane and membrane-embedded proteins.

The organization of multicellular organisms depends on cell-cell communication. The signal molecules are often soluble components in the extracellular fluid, but also include odors and light. A large array of surface receptors is involved in the detection of these signals. Signals are then transduced across the plasma membrane so that enzymes at the inner face of the membrane are activated, producing second messengers, which by a complex network of interactions activate target proteins or genes. Vertebrate cells have been used to study hormone and neurotransmitter action, vision, the regulation of cell growth and differentiation. Sensory transduction in lower eukaryotes is predominantly used for other functions, notably cell attraction for mating and food seeking. By comparing sensory transduction in lower and higher eukaryotes general principles may be recognized that are found in all organisms and deviations that are present in specialised systems. This may also help to understand the differences between cell types within one organism and the importance of a particular pathway that may or may not be general. In a practical sense, microorganisms have the advantage of their easy genetic manipulation, which is especially advantageous for the identification of the function of large families of signal transducing components.