The primary structure of the aspartate transcarbamylase region of the URA2 gene product in Saccharomyces cerevisiae. Features involved in activity and nuclear localization

The yeast URA2 locus encodes a multifunctional protein which possesses the carbamylphosphate synthetase and aspartate transcarbamylase activities and which catalyzes the first two reactions of the pyrimidine pathway. We report here the nucleotide sequence of the central and the 3' region of this locus. The latter encodes that part of the multifunctional protein which has the aspartate transcarbamylase activity. The deduced amino acid sequence shows a high degree of homology with the known aspartate transcarbamylases of various organisms from Escherichia coli to mammals. The amino acid residues that have been shown to be involved in the catalytic site of the E. coli enzyme are all conserved suggesting that, in the more complex structure of the yeast protein, the catalytic sites are also located at subunit interfaces. There is also an important conservation of the amino acid pairs that, in E. coli, are implicated in intra- and interchain interactions. As well as the oligomeric structure suggested by these two features, the three-dimensional structure of the yeast enzyme must also be organized to account for the channeling of carbamylphosphate, from the carbamylphosphate synthetase catalytic site to that of aspartate transcarbamylase, and for the concomitant feedback inhibition of the two activities by the end product UTP. The URA2 gene product was shown to be localized in the nucleus. With the aim of identifying the regions that may be involved in this transport, we have determined by electron microscopy the subcellular distribution of aspartate transcarbamylase in three strains expressing different fragments of the URA2 locus. In the first strain the protein lacks 190 residues at the N terminus, but accumulates normally in the nucleus. In the second strain the protein lacks 382 residues in the central part and seems impaired in the nuclear transport process. In the third strain the 476-residue protein encoded by the 3' region of URA2 locus and catalyzing the aspartate transcarbamylase reaction is able by itself to migrate to and accumulate in the nucleus. This suggests that two regions are involved in the nuclear accumulation. On the basis of their conservation in analogous proteins of other eukaryotes and their similarity to sequences already identified as nuclear location signals, a sequence in the central region of the protein and two short sequences in the C-terminal region are good candidates for the nuclear location signal involved in the targeting of the URA2 product.     

Clathrin-dependent APP endocytosis and Aβ secretion are highly sensitive to the level of plasma membrane cholesterol

Several lines of evidence support a strong relationship between cholesterol and Alzheimer's disease pathogenesis. Membrane cholesterol is known to modulate amyloid precursor protein (APP) endocytosis and amyloid-β (Aβ) secretion. Here we show in a human cell line model of endocytosis (HEK293 cells) that cholesterol exerts these effects in a dose-dependent and linear manner, over a wide range of concentrations (-40% to + 40% variations of plasma membrane cholesterol induced by methyl-beta-cyclodextrin (MBCD) and MBCD-cholesterol complex respectively). We found that the gradual effect of cholesterol is inhibited by small interference RNA-mediated downregulation of clathrin. Modulation of clathrin-mediated APP endocytosis by cholesterol was further demonstrated using mutants of proteins involved in the formation of early endosomes (dynamin2, Eps15 and Rab5). Importantly we show that membrane proteins other than APP are not affected by cholesterol to the same extent. Indeed clathrin-dependent endocytosis of transferrin and cannabinoid1 receptors as well as internalization of surface proteins labelled with a biotin derivative (sulfo-NHS-SS-biotin) were not sensitive to variations of plasma membrane cholesterol from -40% to 40%. In conclusion clathrin-dependent APP endocytosis appears to be very sensitive to the levels of membrane cholesterol. These results suggest that cholesterol increase in AD could be responsible for the enhanced internalization of clathrin-, dynamin2-, Eps15- and Rab5-dependent endocytosis of APP and the ensuing overproduction of Aβ.     

Genetic analysis of yeast strains lacking negative feedback control: a one-step method for positive selection and cloning of carbamoylphosphate synthetase-aspartate transcarbamoylase mutants unable to respond to UTP

We have undertaken an in vivo genetic approach to the analysis of negative feedback control by uridine triphosphate (UTP) of the yeast carbamoylphosphate synthetase-aspartate transcarbamoylase multifunctional protein (CPSase-ATCase). Using an analog of uracil, 5-fluorouracil, we have constructed a screening system leading, in one step, to selection and cloning of a functional aspartate transcarbamoylase that is defective in negative feedback control by UTP. Due to the nature of the screen, spontaneous or UV-induced mutants could be recovered. Well-characterized cloned mutants have been sequenced and reveal one or two modifications in single codons leading to single amino acid replacements. These amino acid changes occurred either in the CPSase or ATCase domains, abolishing their sensitivity to regulation but not their catalytic activities. Hence the regulatory and catalytic sites are distinct. With the same screening system, it may also be possible to enlarge the scope of the molecular study of the feedback processes to include equivalent proteins in fungi as well as higher eukaryotes.     

Involvement of very short DNA tandem repeats and the influence of the RAD52 gene on the occurrence of deletions in Saccharomyces cerevisiae

Chromosomal rearrangements, such as deletions, duplications, or Ty transposition, are rare events. We devised a method to select for such events as Ura(+) revertants of a particular ura2 mutant. Among 133 Ura(+) revertants, 14 were identified as the result of a deletion in URA2. Of seven classes of deletions, six had very short regions of identity at their junctions (from 7 to 13 bp long). This strongly suggests a nonhomologous recombination mechanism for the formation of these deletions. The total Ura(+) reversion rate was increased 4.2-fold in a rad52Delta strain compared to the wild type, and the deletion rate was significantly increased. All the deletions selected in the rad52Delta context had microhomologies at their junctions. We propose two mechanisms to explain the occurrence of these deletions and discuss the role of microhomology stretches in the formation of fusion proteins.     

Genetic selection for reciprocal translocation at chosen chromosomal sites in Saccharomyces cerevisiae.

We have constructed viable Saccharomyces cerevisiae strains containing a reciprocal translocation between the URA2 site of chromosome X and the HIS3 site of chromosome XV. Our methodology is an extension of the method originally developed to introduce an altered cloned sequence at the chromosomal location from which the parent sequence was derived (S. Scherer and R.W. Davis, Proc. Natl. Acad. Sci. U.S.A. 76:4951-4955, 1979). It comprises three essential steps. First, a nonreverting ura2- strain was constructed by deleting a 3.7-kilobase fragment from the coding sequence of the wild-type URA2 gene. Second, part of the coding sequence of the wild-type URA2 gene (without promotor) was inserted at the HIS3 locus of the ura2- strain. Third, after several generations of growth on uracil-supplemented medium, ura2+ colonies were selected which resulted from mitotic recombination between the nonoverlapping deletions of URA2 located on chromosomes X and XV.     

A critical role for the glial-derived neuromodulator D-serine in the age-related deficits of cellular mechanisms of learning and memory

Age-associated deficits in learning and memory are closely correlated with impairments of synaptic plasticity. Analysis of N-methyl-D-aspartate receptor (NMDAr)-dependent long-term potentiation (LTP) in CA1 hippocampal slices indicates that the glial-derived neuromodulator D-serine is required for the induction of synaptic plasticity. During aging, the content of D-serine and the expression of its synthesizing enzyme serine racemase are significantly decreased in the hippocampus. Impaired LTP and NMDAr-mediated synaptic potentials in old rats are rescued by exogenous D-serine. These results highlight the critical role of glial cells and presumably astrocytes, through the availability of D-serine, in the deficits of synaptic mechanisms of learning and memory that occur in the course of aging.     

Modifications of the molecular weight of membrane-bound nonspecific β-glucosidase in type 1 gaucher disease determined in situ by the radiation inactivation method

The radiation inactivation method has been used to compare the molecular weight of the nonspecific membrane-bound β-glucosidase in situ in normal human spleen and in that of two patients with Gaucher diease type 1. We report, in type 1 Gaucher spleen, the presence of a high molecular weight component (557 000) in addition to the normal low molecular weight component (97 800). The various possible hypotheses explaining this high molecular weight component are discussed.     

Identifying gastropod spawn from DNA barcodes: possible but not yet practicable

Identifying life stages of species with complex life histories is problematic as species are often only known and/or described from a single stage. DNA barcoding has been touted as an important tool for linking life-history stages of the same species. To test the current efficacy of DNA barcodes for identifying unknown mollusk life stages, 24 marine gastropod egg capsules were collected off the Philippines in deep water and sequenced for partial fragments of the COI, 16S and 12S mitochondrial genes. Two egg capsules of known shallow-water Mediterranean species were used to calibrate the method. These sequences were compared to those available in GenBank and the Barcode of Life Database (BOLD). Using COI sequences alone, only a single Mediterranean egg capsule was identified to species, and a single Philippine egg capsule was identified tentatively to genus; all other COI sequences recovered matches between 76% and 90% with sequences from BOLD and GenBank. Similarity-based identification using all three markers confirmed the Mediterranean specimens' identifications. A phylogenetic approach was also implemented to confirm similarity-based identifications and provide a higher-taxonomic identification when species-level identifications were not possible. Comparison of available GenBank sequences to the diversity curve of a well-sampled coral reef habitat in New Caledonia highlights the poor taxonomic coverage achieved at present in existing genetic databases, emphasizing the need to develop DNA barcoding projects for megadiverse and often taxonomically challenging groups such as mollusks, to fully realize its potential as an identification and discovery tool.