Protein prenylation: genes, enzymes, targets, and functions.
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Amino Acid Sequence↗
Biomedical subjects
J Rine
Publications and source records attributed to J Rine.
Gene overexpression in studies of Saccharomyces cerevisiae.
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Cloning, Molecular↗
SUM1-1: a suppressor of silencing defects in Saccharomyces cerevisiae.
The repression of transcription of the silent mating-type locus HMRa in the yeast Saccharomyces cerevisiae requires the four SIR proteins, histone H4 and a flanking site designated HMR-E. The SUM1-1 mutation alleviated the need for many of these components in transcriptional repression. In the absence of each of the SIR proteins, SUM1-1 restored repression in MAT alpha strains; thus, SUM1-1 appeared to bypass the need for the SIR genes in repression of HMRa. Repression was not specific to the genes normally present at HMR, since the TRP1 gene placed at HMR was repressed by SUM1-1 in a sir3 strain. Therefore, like the mechanisms of silencing normally used at HMR, silencing by SUM1-1 was gene-nonspecific. SUM1-1 suppressed point mutations in histone H4, but failed to suppress strongly a deletion mutation in histone H4. Similarly, SUM1-1 suppressed mutations in the three known elements of HMR-E, but was unable to suppress a deletion of HMR-E. These epistasis analyses implied that the functions required for repression at HMR can be ordered, with the SIR genes and silencer elements acting upstream of SUM1-1. SUM1-1 itself may function at the level of chromatin in the assembly of inactive DNA at the silent mating-type loci.
Alleles↗
A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae.
Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.
Alleles↗
A role for CDC7 in repression of transcription at the silent mating-type locus HMR in Saccharomyces cerevisiae.
The mating-type genes at MAT in Saccharomyces cerevisiae are expressed, whereas the same genes located at HML and HMR are transcriptionally repressed. The DNA element responsible for repression at HMR has been termed a silencer and contains an autonomous replication sequence, a binding site for GRFI/RAPI, and a binding site for ABFI. A double-mutant HMR-E silencer that contains single nucleotide substitutions in both the GRFI/RAPI- and ABFI-binding sites no longer binds either factor in vitro, nor represses transcription at HMR in vivo. In MAT alpha cells, this derepression of a information results in a nonmating phenotype. Second-site suppressor mutations were isolated that restored the alpha mating phenotype to MAT alpha cells containing the double-mutant silencer. One of these suppressors, designated sas1-1, conferred a temperature-sensitive lethal phenotype to the cell. SAS1 was found to be identical to CDC7, a gene which encodes a protein kinase required for the initiation of DNA replication. This new allele of CDC7 was designated cdc7-90. cdc7-90 restored the alpha mating phenotype by restoring silencing. The original allele of CDC7, isolated on the basis of the cell cycle phenotype it confers, also restored silencing, and overexpression of CDC7 interfered with silencing. cdc7-90 did not restore detectable binding of GRFI/RAPI or ABFI to the double-mutant silencer in vitro. These results indicate that a reduced level of CDC7 function restores silencing to a locus defective in binding two factors normally required for silencing.
Crosses, Genetic↗
Enzymatic coupling of cholesterol intermediates to a mating pheromone precursor and to the ras protein.
The post-translational processing of the yeast a-mating pheromone precursor, Ras proteins, nuclear lamins, and some subunits of trimeric G proteins requires a set of complex modifications at their carboxyl termini. This processing includes three steps: prenylation of a cysteine residue, proteolytic processing, and carboxymethylation. In the yeast Saccharomyces cerevisiae, the product of the DPR1-RAM1 gene participates in this type of processing. Through the use of an in vitro assay with peptide substrates modeled after a presumptive a-mating pheromone precursor, it was discovered that mutations in DPR1-RAM1 cause a defect in the prenylation reaction. It was further shown that DPR1-RAM1 encodes an essential and limiting component of a protein prenyltransferase. These studies also implied a fixed order of the three processing steps shared by prenylated proteins: prenylation, proteolysis, then carboxymethylation. Because the yeast protein prenyltransferase could also prenylate human H-ras p21 precursor, the human DPR1-RAM1 analogue may be a useful target for anticancer chemotherapy.
Amino Acid Sequence↗
Common modifications of trimeric G proteins and ras protein: involvement of polyisoprenylation.
The heterotrimeric guanine nucleotide-binding regulatory proteins act at the inner surface of the plasma membrane to relay information from cell surface receptors to effectors inside the cell. These G proteins are not integral membrane proteins, yet are membrane associated. The processing and function of the gamma subunit of the yeast G protein involved in mating-pheromone signal transduction was found to be affected by the same mutations that block ras processing. The nature of these mutations implied that the gamma subunit was polyisoprenylated and that this modification was necessary for membrane association and biological activity. A microbial screen was developed for pharmacological agents that inhibit polyisoprenylation and that have potential application in cancer therapy.
Amino Acid Sequence↗
Prenylation of mammalian Ras protein in Xenopus oocytes.
Ras protein requires an intermediate of the cholesterol biosynthetic pathway for posttranslational modification and membrane anchorage. This step is necessary for biological activity. Maturation of Xenopus laevis oocytes induced by an oncogenic human Ras protein can be inhibited by lovastatin or compactin, inhibitors of the synthesis of mevalonate, an intermediate of cholesterol biosynthesis. This inhibition can be overcome by mevalonic acid or farnesyl diphosphate, a cholesterol biosynthetic intermediate downstream of mevalonate, but not by squalene, an intermediate after farnesyl pyrophosphate in the pathway. This study supports the idea that in Xenopus oocytes, the Ras protein is modified by a farnesyl moiety or its derivative. Furthermore, an octapeptide with the sequence similar to the C-terminus of the c-H-ras protein inhibits the biological activity of Ras proteins in vivo, suggesting that it competes for the enzyme or enzymes responsible for transferring the isoprenoid moiety (prenylation) in the oocytes. This inhibition of Ras prenylation by the peptide was also observed in vitro, using both Saccharomyces cerevisiae and Xenopus oocyte extracts. These observations show that Xenopus oocytes provide a convenient in vivo system for studies of inhibitors of the posttranslational modification of the Ras protein, especially for inhibitors such as peptides that do not penetrate cell membranes.
Amino Acid Sequence↗
Genetic and biochemical evaluation of eucaryotic membrane protein topology: multiple transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl coenzyme A reductase.
Both 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase isozymes of the yeast Saccharomyces cerevisiae are predicted to contain seven membrane-spanning domains. Previous work had established the utility of the histidinol dehydrogenase protein domain, encoded by HIS4C, as a topologically sensitive monitor that can be used to distinguish between the lumen of the endoplasmic reticulum and the cytoplasm. This study directly tested the structural predictions for HMG-CoA reductase by fusing the HIS4C domain to specific sites in the HMG-CoA reductase isozymes. Yeast cells containing the HMG-CoA reductase-histidinol dehydrogenase fusion proteins grew on histidinol-containing medium if the HIS4C domain was present on the cytoplasmic side of the endoplasmic reticulum membrane but not if the HIS4C domain was targeted to the endoplasmic reticulum lumen. Systematic exchanges of transmembrane domains between the isozymes confirmed that both isozymes had equivalent membrane topologies. In general, deletion of an even number of putative transmembrane domains did not interfere with the topology of the protein, but deletion or duplication of an odd number of transmembrane domains inverted the orientation of the protein. The data confirmed the earlier proposed topology for yeast HMG-CoA reductase, demonstrated that the yeast enzymes are core glycosylated, and provided in vivo evidence that the properties of transmembrane domains were, in part, dependent upon their context within the protein.
Amino Acids↗
Cell-type control of membrane biogenesis induced by HMG-CoA reductase.
Quantitative increases in HMG-CoA reductase, the rate-limiting enzyme in sterol biosynthesis, induce membrane biogenesis in both yeast and mammalian cells. The subcellular organization of the resulting membrane differs in the two cell types: mammalian cells generate crystalloid endoplasmic reticulum whereas yeast cells assemble karmellae. We examined the consequences of heterologous expression of HMG-CoA reductase to distinguish features of this response that were cell-type specific from those that were isozyme-specific. This analysis demonstrated that membrane proliferation was induced in both mammalian and yeast cells by HMG-CoA reductase from either organism. However, the morphology of the induced membranes was determined by the cell type rather than the particular isozyme. Thus, both yeast and mammalian HMG-CoA reductase contained functional signals for membrane proliferation that were operational in either cell type, but the qualitative response to those signals was cell-type specific.
Animals↗
A role for isoprenoid lipids in the localization and function of an oncoprotein.
Intermediates of the cholesterol biosynthetic pathway are covalently attached to a number of eukaryotic proteins, including the Ras oncoprotein. Ras protein is post-translationally processed at its carboxyl terminus in three steps, resulting in a COOH-terminal cysteine residue to which a polyisoprenoid moiety, probably farnesyl, is attached in a thioether linkage. Polyisoprenylation of Ras protein is required for its membrane association and for the oncogenicity of mutant forms of the protein. Inhibition of polyisoprenylation may offer a route by which Ras-mediated tumors can be pharmacologically suppressed. Other proteins that are polyisoprenylated include nuclear lamin B, fungal mating factors, and subunits of trimeric guanine nucleotide-binding proteins. A consensus sequence for polyisoprenylation (Cys-aliphatic-aliphatic-X) has been identified at the COOH-terminus of modified proteins. Recent evidence indicates that proteins can be modified by several different polyisoprenoids.
Amino Acid Sequence↗
Epigenetic inheritance of transcriptional states in S. cerevisiae.
SIR1, one of several genes required for repression of yeast silent mating type loci, has a unique role in repression of the HML alpha locus. Single-cell assays revealed that cells with mutant alleles of SIR1, including presumptive null alleles, existed as populations of genetically identical cells whose members were in one of two different regulatory states. A minority of cells had a repressed HML alpha locus whereas the majority had a derepressed HML alpha locus. The two states were mitotically stable, although rare changes in state were observed during mitotic growth, possibly reflecting heritable changes to the HML alpha locus at or before replication. Analysis of changes in state suggests that SIR1 protein has a role in the establishment but not the maintenance of repression of silent mating type genes, whereas SIR2, SIR3, and SIR4 are required for maintenance.
Alleles↗
Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans.
The activity of an oncoprotein and the secretion of a pheromone can be affected by an unusual protein modification. Specifically, posttranslational modification of yeast a-factor and Ras protein requires an intermediate of the cholesterol biosynthetic pathway. This modification is apparently essential for biological activity. Studies of yeast mutants blocked in sterol biosynthesis demonstrated that the membrane association and biological activation of the yeast Ras2 protein require mevalonate, a precursor of sterols and other isoprenes such as farnesyl pyrophosphate. Furthermore, drugs that inhibit mevalonate biosynthesis blocked the in vivo action of oncogenic derivatives of human Ras protein in the Xenopus oocyte assay. The same drugs and mutations also prevented the posttranslational processing and secretion of yeast a-factor, a peptide that is farnesylated. Thus, the mevalonate requirement for Ras activation may indicate that attachment of a mevalonate-derived (isoprenoid) moiety to Ras proteins is necessary for membrane association and biological function. These observations establish a connection between the cholesterol biosynthetic pathway and transformation by the ras oncogene and offer a novel pharmacological approach to investigating, and possibly controlling, ras-mediated malignant transformations.
Amino Acid Sequence↗
Transmission electron microscopy and immunocytochemical studies of yeast: analysis of HMG-CoA reductase overproduction by electron microscopy.
The results and anecdotes presented here are intended only as a general guide to other would-be immunocytochemists, because other proteins will undoubtedly respond at least somewhat differently than does HMG-CoA reductase. Nevertheless, based on these experiences, we offer the following suggestions: 1. Antiserum of high specificity should be raised and affinity-purified. Using this antiserum, immunofluorescence microscopy should be attempted before resorting to electron microscopic localization. In the absence of immunolocalization at the light-microscope level, it may be a waste of time to pursue the problem to higher levels of resolution. 2. Cells should be prefixed in 1% formaldehyde-1% glutaraldehyde. Direct fixation of the growing culture and use of phosphate buffer are recommended. The prefixed sample can then be divided into two or three aliquots. One aliquot should receive no postfixation (for optimal immunoreactivity), while the others can be postfixed in osmium-potassium ferricyanide (for possible immunolocalization) or permanganate (for ultrastructural analysis). Because of its ease of use, Spurr's resin should be tried initially. If immunocytochemistry is successful, no further preparations are necessary. If unsuccessful, LR White resin is recommended, but the sample must be treated to remove the cell wall. Electron microscopy and immunocytochemistry offer views into the molecular arrangement of individual cells, a view not easily obtained by other means. It is satisfying and often enlightening to be able to see the extremes as well as the average. In studies of the organization of karmellae, for example, ultrastructural analysis easily revealed the asymmetric segregation pattern, while immunoblots and cell fractionation could not even demonstrate the existence of this membrane organization. The richness of the information available to those who can avert reductionist tendencies, even for a short time, is remarkable.
Histocytochemistry↗
Genetic and molecular characterization of suppressors of SIR4 mutations in Saccharomyces cerevisiae.
In order to learn more about other proteins that may be involved in repression of HML and HMR in Saccharomyces cerevisiae, extragenic suppressor mutations were identified that could restore repression in cells defective in SIR4, a gene required for function of the silencer elements flanking HML and HMR. These suppressor mutations, which define at least three new genes, SAN1, SAN2 and SAN3, arose at the frequency expected for loss-of-function mutations following mutagenesis. All san mutations were recessive. Suppression by san1 was allele-nonspecific, since san1 could suppress two very different alleles of SIR4, and was locus-specific since san1 was unable to suppress a SIR3 mutation or a variety of mutations conferring auxotrophies. The SAN1 gene was cloned, sequenced, and used to construct a null allele. The null allele had the same phenotype as the EMS-induced mutations and exhibited no pleiotropies of its own. Thus, the SAN1 gene was not essential. SAN1-mediated suppression was neither due to compensatory mutations in interacting proteins, nor to translational missense suppression. SAN1 may act posttranslationally to control the stability or activity of the SIR4 protein.
Alleles↗
Positive and negative transcriptional control by heme of genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.
Responses of the yeast genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, HMG1 and HMG2, to in vivo changes in heme concentrations were investigated. Expression of the genes was determined by direct measurement of the mRNA transcribed from each gene, by direct assay of the enzyme activity encoded by each gene, and by measurement of the expression of lacZ fusions to the control regions of each gene. These studies indicated that expression of HMG1 was stimulated by heme, whereas expression of HMG2 was repressed by heme. The effect of heme on HMG1 expression was mediated by the HAP1 transcriptional regulator and was independent of HAP2. Thus, the genes encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase isozymes join a growing list of gene pairs that are regulated by heme in opposite ways.
Amino Acid Sequence↗
Evidence for Drosophila P element transposase activity in mammalian cells and yeast.
Drosophila P element transposase expression is limited to the germline by tissue-specific splicing of one of its three introns. Removal of this intron by mutagenesis in vitro has allowed both P element excision and transposition to be detected in Drosophila somatic tissues. In order to determine if P element transposase can function in other organisms, we have expressed modified P elements either lacking one intron or lacking all three introns in mammalian cells and yeast, respectively. Using an assay for P element excision, we have detected apparent excision events in cultured monkey cells. Furthermore, expression of the complete P element cDNA is lethal to Saccharomyces cerevisiae cells carrying a mutation in the RAD52 gene, indicating that double-stranded DNA breaks are generated, presumably by transposase action.
Animals↗
Roles of two DNA-binding factors in replication, segregation and transcriptional repression mediated by a yeast silencer.
The HMR E silencer is required for SIR-dependent transcriptional repression of the silent mating-type locus, HMR. The silencer also behaves as an origin of replication (ARS element) and allows plasmids to replicate autonomously in yeast. The replication and segregation properties of these plasmids are also dependent on the four SIR genes. We have previously characterized two DNA-binding factors in yeast extracts that recognize specific sequences at the HMR E silencer. These proteins, called ABFI (ARS-Binding Factor) and GRFI (General Regulatory Factor), are not encoded by any of the SIR genes. To investigate the biological roles of these factors, single-base-pair mutations were constructed in both binding sites at the HMR E silencer that were no longer recognized by the corresponding proteins in vitro. Our results indicate that the GRFI-binding site is required for the efficient segregation of plasmids replicated by the HMR E silencer. SIR-dependent transcriptional repression requires either an intact ABFI-binding site or GRFI-binding site, although the GRFI-binding site appears to be more important. A double-mutant silencer that binds neither ABFI nor GRFI does not mediate transcriptional repression of HMR. The replacement of HMR E with a chromosomal origin of replication (ARS1) allows partial SIR-dependent transcriptional repression of HMR, indicating a role for replication in silencer function. Together, these results suggest that the SIR proteins influence the properties of the HMR E silencer through interactions with other DNA-binding proteins.
Base Sequence↗