The anaphase-promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators. Cdh1p is an APC coactivator that directly binds APC substrates. A genetic screen in budding yeast identified residues within Cdh1p critical for its function. Cdh1p proteins containing mutations within the "C box" or the "IR" motif could bind substrate, but not the APC, whereas mutants that only bound the APC were not identified, suggesting an ordered assembly of the ternary APC-Cdh1p-substrate complex. Supporting this hypothesis, we found that substrate binding to wild-type Cdh1p enhanced its association with the APC in yeast cells. We used peptide competition assays to demonstrate that Cdh1p interacts directly with the D box and the KEN box, two motifs within APC substrates known to be required for APC-mediated degradation. Moreover, an intact D box domain within a substrate was required to stimulate the association between the Cdh1p-substrate complex and the APC.
BackgroundMicrobial lipids are produced by many oleaginous organisms including the well-characterized yeast Yarrowia lipolytica, which can be engineered for increased lipid yield by up-regulation of the lipid biosynthetic pathway and down-regulation or deletion of competing pathways.ResultsWe describe a strain engineering strategy centered on diacylglycerol acyltransferase (DGA) gene overexpression that applied combinatorial screening of overexpression and deletion genetic targets to construct a high lipid producing yeast biocatalyst. The resulting strain, NS432, combines overexpression of a heterologous DGA1 enzyme from Rhodosporidium toruloides, a heterlogous DGA2 enzyme from Claviceps purpurea, and deletion of the native TGL3 lipase regulator. These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation. In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h.ConclusionsThe yields, productivities, and titers reported in this study may further support the applied goal of cost-effective, large -scale microbial lipid production for use as biofuels and biochemicals.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0492-3) contains supplementary material, which is available to authorized users.
Gene targeting is a challenge in organisms where non-homologous end-joining is the predominant form of recombination. We show that cell division cycle synchronization can be applied to significantly increase the rate of homologous recombination during transformation. Using hydroxyurea-mediated cell cycle arrest, we obtained improved gene targeting rates in Yarrowia lipolytica, Arxula adeninivorans, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris demonstrating the broad applicability of the method. Hydroxyurea treatment enriches for S-phase cells that are active in homologous recombination and enables previously unattainable genomic modifications.
The pre-replicative complex (pre-RC) is formed at all potential origins of replication through the action of the origin recognition complex (ORC), Cdc6, Cdt1, and the Mcm2-7 complex. The end result of pre-RC formation is the loading of the Mcm2-7 replicative helicase onto origin DNA. We examined pre-RC formation in vitro and found that it proceeds through separable binding events. Origin-bound ORC recruits Cdc6, and this ternary complex then promotes helicase loading in the presence of a pre-formed Mcm2-7-Cdt1 complex. Using a stepwise pre-RC assembly assay, we investigated the fate of pre-RC components during later stages of the reaction. We determined that helicase loading is accompanied by dissociation of ORC, Cdc6, and Cdt1 from origin DNA. This dissociation requires ATP hydrolysis at a late stage of pre-RC assembly. Our results indicate that pre-RC formation is a dynamic process.Eukaryotic DNA replication initiates at origins of replication throughout the genome. The first event in this tightly regulated process is origin selection. In the budding yeast Saccharomyces cerevisiae, specific nucleotide sequences mark sites where replication can initiate. These sites are bound by the origin recognition complex (ORC) 3 (1, 2). As cells enter G 1 , ORC recruits Cdc6, Cdt1, and the Mcm2-7 replicative helicase to the origin DNA resulting in pre-replicative complex (pre-RC) formation (3). Activation of S phase kinases leads to the recruitment of numerous additional factors to the Mcm2-7 complex resulting in the formation of a pair of bi-directional replisomes (4). Multiple mechanisms regulate pre-RC formation to ensure that each origin can only initiate once per cell cycle (5). Thus, the pre-RC marks all potential sites of replication initiation in G 1 , forms the foundation for the assembly of the replisome, and is tightly regulated to ensure complete genome replication.Studies of S. cerevisiae DNA replication initiation have been greatly facilitated by the short (80 -120 bp), well defined origins of replication of this organism (6). These sequences include a conserved 11-bp autonomous replicating sequence consensus sequence and multiple 10 -15-bp B-elements. Much of our understanding of the pre-RC has come from in vitro origin binding assays. It was shown that the ORC-origin interaction is ATP-dependent (7) and that Cdc6 cooperatively binds the ORC-DNA complex (8). This ternary complex is also regulated by Cdc6 ATPase activity (9). In vitro pre-RC assembly in budding yeast extracts indicates sequential association of ORC, Cdc6, Cdt1, and Mcm2-7 with the origin (10, 11), and ATP hydrolysis by Cdc6 is required for .In this study, we examine pre-RC formation in vitro and find that the process can be separated into three distinct sequential binding events. The final step of Mcm2-7 complex loading correlates with reduced origin association of ORC, Cdc6, and Cdt1. The dissociation of these proteins from the origin requires ATP hydrolysis at a late step in pre-RC assembly. Our findings indicate that the pre-RC proteins as...
The perinuclear localization of myosin-V was investigated in a variety of cultured mammalian cells and in primary cultures of rat hippocampus. In all cells investigated, myosin-V immunoreactivity was associated with the centrosome. In interphase cells, myosin-V was found in pericentriolar material, and in both mother and daughter centrioles. These results were obtained by using two different fixation protocols with three different affinity-purified antibodies that recognized a single band in Western blots. During cell division, myosin-V staining was intense throughout the cytoplasm and was concentrated in a trail between migrating centrioles and in the mitotic spindle poles and spindle fibers. The centrosome targeting site was determined to reside within the globular tail domain, because centrosome association also was observed in living cells transfected with DNA encoding the tail domain fused with a green f luorescent protein tag, but not in cells transfected with the vector encoding green f luorescent protein by itself.The ability to move and position organelles is essential to eukaryotic cellular physiology. Motility is accomplished by mechanochemical ATPases, dyneins, and kinesins that translocate on microtubules, and myosins that translocate on actin filaments. At least 15 classes of myosins have been identified (1, 2).The range of functions of most of the novel myosins have yet to be fully characterized. Myosin-V is a dimeric, nonfilamentous myosin with calmodulin light chains and actin-dependent ATPase activity showing an absolute requirement for calcium (3, 4). Class V myosins, for which full-length sequences have been determined, include the protein products of the mouse Myo5a gene (dilute locus) (5) and the yeast MYO2 (6) and MYO4 (7) genes, chicken myosin-Va (8, 9), human myosin-Va (MYH12, GenBank accession no. Y07759), and rat myosin-Vb (myr6) (10).Phenotypic analyses of yeast myo2 and myo4 and mouse dilute mutants have shown that myosin-V is required for the polarized transport of organelles and other cytoplasmic components. Myo2-66 yeast cells, which express a temperaturesensitive mutation of a myosin-V gene (6), cannot progress through the cell cycle at restrictive temperatures, but remain as large, unbudded cells with numerous small vesicles in the cytoplasm. The defective gene, Myo2p, is required for vacuole inheritance and for the polarized localization of chitin synthase Chs3p and SEC4 in the budding yeast. Myo4p, another isoform of myosin-V in yeast, is required for the restricted localization of ASH1 mRNA to the daughter cell. As a consequence, Ash1p, a repressor of mating type switching, is selectively expressed in the daughter cell (for review see ref. 1).Mutation of the mouse Myo5a gene results in defective pigment granule transfer (11) and, in such homozygous mutants as d l20J (dilute-lethal), may lead to neurological impairment that culminates in early postnatal death (5). Lack of smooth endoplasmic reticulum in the dendritic spines of Purkinje cells has been demonstrated in th...
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