The Palmitoylation Two-step♦ : Mutational Analysis of Saccharomyces cerevisiae Erf2 Reveals a Two-step Reaction Mechanism for Protein Palmitoylation by DHHC Enzymes
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PROVIDER: S-EPMC2992312 | biostudies-literature | 2010 Dec
REPOSITORIES: biostudies-literature
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Project description:DHHC protein acyltransferases (PATs) catalyze the palmitoylation of eukaryotic proteins through an enzymatic mechanism that remains largely unexplored. In this study we have combined genetic and biochemical approaches to examine the molecular mechanism of palmitate transfer of the yeast Ras PAT, which is composed of Erf2 and Erf4. The palmitoylation reaction consists of two steps; they are autopalmitoylation of the enzyme to create a palmitoyl-Erf2 intermediate followed by the transfer of the palmitoyl moiety to the Ras substrate. Palmitoyl-CoA serves as the palmitate donor. To elucidate the kinetic properties of the Erf2·Erf4 PAT, we have developed a coupled enzyme assay that monitors the turnover of the palmitoyl-enzyme species indirectly by measuring the rate of CoASH release. Mutational analysis indicates that the DHHC motif constitutes the catalytic core of the enzyme required for autopalmitoylation and palmitoyl transfer to the Ras2 substrate. In the absence of Ras2, the palmitoyl-Erf2·Erf4 complex undergoes a cycle of hydrolysis and re-palmitoylation, implying that in the presence of palmitoyl-CoA, the complex is autopalmitoylated and competent to transfer palmitate to a protein substrate.
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Project description:Plasma membrane localization of Ras requires posttranslational addition of farnesyl and palmitoyl lipid moieties to a C-terminal CaaX motif (C is cysteine, a is any aliphatic residue, X is the carboxy terminal residue). To better understand the relationship between posttranslational processing and the subcellular localization of Ras, a yeast genetic screen was undertaken based on the loss of function of a palmitoylation-dependent RAS2 allele. Mutations were identified in an uncharacterized open reading frame (YLR246w) that we have designated ERF2 and a previously described suppressor of hyperactive Ras, SHR5. ERF2 encodes a 41-kDa protein with four predicted transmembrane (TM) segments and a motif consisting of the amino acids Asp-His-His-Cys (DHHC) within a cysteine-rich domain (CRD), called DHHC-CRD. Mutations within the DHHC-CRD abolish Erf2 function. Subcellular fractionation and immunolocalization experiments reveal that Erf2 tagged with a triply iterated hemagglutinin epitope is an integral membrane protein that colocalizes with the yeast endoplasmic reticulum marker Kar2. Strains lacking ERF2 are viable, but they have a synthetic growth defect in the absence of RAS2 and partially suppress the heat shock sensitivity resulting from expression of the hyperactive RAS2(V19) allele. Ras2 proteins expressed in an erf2Delta strain have a reduced level of palmitoylation and are partially mislocalized to the vacuole. Based on these observations, we propose that Erf2 is a component of a previously uncharacterized Ras subcellular localization pathway. Putative members of an Erf2 family of proteins have been uncovered in yeast, plant, worm, insect, and mammalian genome databases, suggesting that Erf2 plays a role in Ras localization in all eucaryotes.
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Project description:The dopamine transporter (DAT) is a plasma membrane protein that mediates the reuptake of extracellular dopamine (DA) and controls the spatiotemporal dynamics of dopaminergic neurotransmission. The transporter is subject to fine control that tailors clearance of transmitter to physiological demands, and dysregulation of reuptake induced by psychostimulant drugs, transporter polymorphisms, and signaling defects may impact transmitter tone in disease states. We previously demonstrated that DAT undergoes complex regulation by palmitoylation, with acute inhibition of the modification leading to rapid reduction of transport activity and sustained inhibition of the modification leading to transporter degradation and reduced expression. Here, to examine mechanisms and outcomes related to increased modification, we coexpressed DAT with palmitoyl acyltransferases (PATs), also known as DHHC enzymes, which catalyze palmitate addition to proteins. Of 12 PATs tested, DAT palmitoylation was stimulated by DHHC2, DHHC3, DHHC8, DHHC15, and DHHC17, with others having no effect. Increased modification was localized to previously identified palmitoylation site Cys580 and resulted in upregulation of transport kinetics and elevated transporter expression mediated by reduced degradation. These findings confirm palmitoylation as a regulator of multiple DAT properties crucial for appropriate DA homeostasis and identify several potential PAT pathways linked to these effects. Defects in palmitoylation processes thus represent possible mechanisms of transport imbalances in DA disorders.
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Project description:Gene tagging with fluorescent proteins is commonly applied to investigate the localization and dynamics of proteins in their cellular environment. Ideally, a fluorescent tag is genetically inserted at the endogenous locus at the N- or C- terminus of the gene of interest without disrupting regulatory sequences including the 5' and 3' untranslated region (UTR) and without introducing any extraneous unwanted "scar" sequences, which may create unpredictable transcriptional or translational effects. We present a reliable, low-cost, and highly efficient method for the construction of such scarless C-terminal and N-terminal fusions with fluorescent proteins in yeast. The method relies on sequential positive and negative selection and uses an integration cassette with long flanking regions, which is assembled by two-step PCR, to increase the homologous recombination frequency. The method also enables scarless tagging of essential genes with no need for a complementing plasmid. To further ease high-throughput strain construction, we have computationally automated design of the primers, applied the primer design code to all open reading frames (ORFs) of the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the fission yeast Schizosaccharomyces pombe (S. pombe), and provide here the computed sequences. To illustrate the scarless N- and C-terminal gene tagging methods in S. cerevisiae, we tagged various genes including the E3 ubiquitin ligase RSP5, the proteasome subunit PRE1, and the eleven Rab GTPases with yeast codon-optimized mNeonGreen or mCherry; several of these represent essential genes. We also implemented the scarless C-terminal gene tagging method in the distantly related organism S. pombe using kanMX6 and HSV1tk as positive and negative selection markers, respectively, as well as ura4. The scarless gene tagging methods presented here are widely applicable to visualize and investigate the functional roles of proteins in living cells.
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Project description:Compartmentation via filamentation has recently emerged as a novel mechanism for metabolic regulation. In order to identify filament-forming metabolic enzymes systematically, we performed a genome-wide screening of all strains available from an open reading frame-GFP collection in Saccharomyces cerevisiae. We discovered nine novel filament-forming proteins and also confirmed those identified previously. From the 4159 strains, we found 23 proteins, mostly metabolic enzymes, which are capable of forming filaments in vivo. In silico protein-protein interaction analysis suggests that these filament-forming proteins can be clustered into several groups, including translational initiation machinery and glucose and nitrogen metabolic pathways. Using glutamine-utilising enzymes as examples, we found that the culture conditions affect the occurrence and length of the metabolic filaments. Furthermore, we found that two CTP synthases (Ura7p and Ura8p) and two asparagine synthetases (Asn1p and Asn2p) form filaments both in the cytoplasm and in the nucleus. Live imaging analyses suggest that metabolic filaments undergo sub-diffusion. Taken together, our genome-wide screening identifies additional filament-forming proteins in S. cerevisiae and suggests that filamentation of metabolic enzymes is more general than currently appreciated.
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Project description:Fks1, with orthologs in nearly all fungi as well as plants and many protists, plays a central role in fungal cell wall formation as the putative catalytic component of β-1,3-glucan synthase. It is also the target for an important new antifungal group, the echinocandins, as evidenced by the localization of resistance-conferring mutations to Fks1 hot spots 1, 2, and 3 (residues 635 to 649, 1354 to 1361, and 690 to 700, respectively). Since Fks1 is an integral membrane protein and echinocandins are cyclic peptides with lipid tails, Fks1 topology is key to understanding its function and interaction with echinocandins. We used hemagglutinin (HA)-Suc2-His4C fusions to C-terminally truncated Saccharomyces cerevisiae Fks1 to experimentally define its topology and site-directed mutagenesis to test function of selected residues. Of the 15 to 18 transmembrane helices predicted in silico for Fks1 from evolutionarily diverse fungi, 13 were experimentally confirmed. The N terminus (residues 1 to 445) is cytosolic and the C terminus (residues 1823 to 1876) external; both are essential to Fks1 function. The cytosolic central domain (residues 715 to 1294) includes newly recognized homology to glycosyltransferases, and residues potentially involved in substrate UDP-glucose binding and catalysis are essential. All three hot spots are external, with hot spot 1 adjacent to and hot spot 3 largely embedded within the outer leaflet of the membrane. This topology suggests a model in which echinocandins interact through their lipid tails with hot spot 3 and through their cyclic peptides with hot spots 1 and 2.
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Project description:In addition to environmental factors and intrinsic variations in base substitution rates, specific genome-destabilizing mutations can shape the mutational trajectory of genomes. How specific alleles influence the nature and position of accumulated mutations in a genomic context is largely unknown. Understanding the impact of genome-destabilizing alleles is particularly relevant to cancer genomes where biased mutational signatures are identifiable. We first created a more complete picture of cellular pathways that impact mutation rate using a primary screen to identify essential Saccharomyces cerevisiae gene mutations that cause mutator phenotypes. Drawing primarily on new alleles identified in this resource, we measure the impact of diverse mutator alleles on mutation patterns directly by whole-genome sequencing of 68 mutation-accumulation strains derived from wild-type and 11 parental mutator genotypes. The accumulated mutations differ across mutator strains, displaying base-substitution biases, allele-specific mutation hotspots, and break-associated mutation clustering. For example, in mutants of POLα and the Cdc13-Stn1-Ten1 complex, we find a distinct subtelomeric bias for mutations that we show is independent of the target sequence. Together our data suggest that specific genome-instability mutations are sufficient to drive discrete mutational signatures, some of which share properties with mutation patterns seen in tumors. Thus, in a population of cells, genome-instability mutations could influence clonal evolution by establishing discrete mutational trajectories for genomes.
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Project description:Vitamin C (VC) is comprehensively applied in foods, cosmetics, pharmaceuticals, and especially clinical medicine. Nowadays, the industrial production of VC mainly relies on the classic two-step fermentation route, and researchers have explored the way for one-step fermentation of VC in recent years. In this study, a VC biosynthesis pathway that directly produced VC from glucose was reconstructed in Saccharomyces cerevisiae, and the protein engineering and metabolic engineering strategies were adopted to improve it. First, five exogenous modules from Arabidopsis were introduced into the chassis cells by synthetic biology approaches to obtain the strain YLAA harboring VC biosynthesis. In addition, L-galactose dehydrogenase (L-GalDH) and L-galactono-1,4-lactone dehydrogenase (L-GLDH) were fused and expressed in S. cerevisiae cells for the first time, which increased the intracellular VC accumulation by 2.78-fold, reaching 9.97 ± 0.09 mg/L. Through copy number engineering, it was further confirmed that the last step catalyzed by L-GLDH is the rate-limiting step. GDP-L-galactose phosphorylase (GPP) encoded by vtc2 is another rate-limiting enzyme confirmed by GAL1p overexpression results. Finally, by balancing gene expression and cell growth, the highest production strain with overexpressing vtc2 by multicopy plasmids was constructed. The VC accumulation reached 24.94 ± 1.16 mg/L, which was currently the highest production from glucose in S. cerevisiae. The production of the recombinant strain reached nearly 44 mg/L with the exogenous addition of L-galactose or glutathione. The results further emphasized the importance of the step catalyzed by GPP. The investigation provided experience for the efficient biosynthesis of VC and the determination of rate-limiting steps.
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Project description:Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the PIKK family, which contain a C-terminal phosphatidylinositol 3-kinase-like (PI3K) domain followed by a 35-residue FATC domain. Single residue changes of L3733A and F3744A, within the FATC domain, resulted in transcriptional changes and phenotypes that were similar but not identical to those caused by mutations in the PI3K domain or deletions of other SAGA or NuA4 components. The distinct nature of the FATC mutations was also apparent from the additive effect of tra1-L3733A with SAGA, NuA4, and tra1 PI3K domain mutations. Tra1-L3733A associates with SAGA and NuA4 components and with the Gal4 activation domain, to the same extent as wild-type Tra1; however, steady-state levels of Tra1-L3733A were reduced. We suggest that decreased stability of Tra1-L3733A accounts for the phenotypes since intragenic suppressors of tra1-L3733A restored Tra1 levels, and reducing wild-type Tra1 led to comparable growth defects. Also supporting a key role for the FATC domain in the structure/function of Tra1, addition of a C-terminal glycine residue resulted in decreased association with Spt7 and Esa1, and loss of cellular viability. These findings demonstrate the regulatory potential of mechanisms targeting the FATC domains of PIKK proteins.
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Project description:The characterization of mutational spectra is usually carried out in one of three ways-by direct observation through mutation accumulation (MA) experiments, through parent-offspring sequencing, or by indirect inference from sequence data. Direct observations of spontaneous mutations with MA experiments are limited, given (i) the rarity of spontaneous mutations, (ii) applicability only to laboratory model species with short generation times, and (iii) the possibility that mutational spectra under lab conditions might be different from those observed in nature. Trio sequencing is an elegant solution, but it is not applicable in all organisms. Indirect inference, usually from divergence data, faces no such technical limitations, but rely upon critical assumptions regarding the strength of natural selection that are likely to be violated. Ideally, new mutational events would be directly observed before the biased filter of selection, and without the technical limitations common to lab experiments. One approach is to identify very young mutations from population sequencing data. Here we do so by leveraging two characteristics common to all new mutations-new mutations are necessarily rare in the population, and absent in the genomes of immediate relatives. From 132 clinical yeast strains, we were able to identify 1,425 putatively new mutations and show that they exhibit extremely low signatures of selection, as well as display a mutational spectrum that is similar to that identified by a large scale MA experiment. We verify that population sequencing data are a potential wealth of information for inferring mutational spectra, and should be considered for analysis where MA experiments are infeasible or especially tedious.
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