Yeast Nucleus Research Articles

SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat.:

Abstract Mutations in the SPT5 gene of Saccharomyces cerevisiae were isolated previously as suppressors of delta insertion mutations at HIS4 and LYS2. In this study we have shown that spt5 mutations suppress the his4-912 delta and lys2-128 delta alleles by altering transcription. We cloned the SPT5 gene and found that either an increase or a decrease in the copy number of the wild-type SPT5 gene caused an Spt- phenotype. Construction and analysis of an spt5 null mutation demonstrated that SPT5 is essential for growth, suggesting that SPT5 may be required for normal transcription of a large number of genes. The SPT5 DNA sequence was determined; it predicted a 116-kDa protein with an extremely acidic amino terminus and a novel six-amino-acid repeat at the carboxy terminus (consensus = S-T/A-W-G-G-A/Q). By indirect immunofluorescence microscopy we showed that a bifunctional SPT5-beta-galactosidase protein was located in the yeast nucleus. This molecular analysis of the SPT5 gene revealed a number of interesting similarities to the previously characterized SPT6 gene of S. cerevisiae. These results suggest that SPT5 and SPT6 act in a related fashion to influence essential transcriptional processes in S. cerevisiae.

SPT5, an Essential Gene Important for Normal Transcription in Saccharomyces cerevisiae, Encodes an Acidic Nuclear Protein with a Carboxy-Terminal Repeat

Mutations in the SPT5 gene of Saccharomyces cerevisiae were isolated previously as suppressors of delta insertion mutations at HIS4 and LYS2. In this study we have shown that spt5 mutations suppress the his4-912 delta and lys2-128 delta alleles by altering transcription. We cloned the SPT5 gene and found that either an increase or a decrease in the copy number of the wild-type SPT5 gene caused an Spt- phenotype. Construction and analysis of an spt5 null mutation demonstrated that SPT5 is essential for growth, suggesting that SPT5 may be required for normal transcription of a large number of genes. The SPT5 DNA sequence was determined; it predicted a 116-kDa protein with an extremely acidic amino terminus and a novel six-amino-acid repeat at the carboxy terminus (consensus = S-T/A-W-G-G-A/Q). By indirect immunofluorescence microscopy we showed that a bifunctional SPT5-beta-galactosidase protein was located in the yeast nucleus. This molecular analysis of the SPT5 gene revealed a number of interesting similarities to the previously characterized SPT6 gene of S. cerevisiae. These results suggest that SPT5 and SPT6 act in a related fashion to influence essential transcriptional processes in S. cerevisiae.

SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat.

Mutations in the SPT5 gene of Saccharomyces cerevisiae were isolated previously as suppressors of delta insertion mutations at HIS4 and LYS2. In this study we have shown that spt5 mutations suppress the his4-912 delta and lys2-128 delta alleles by altering transcription. We cloned the SPT5 gene and found that either an increase or a decrease in the copy number of the wild-type SPT5 gene caused an Spt- phenotype. Construction and analysis of an spt5 null mutation demonstrated that SPT5 is essential for growth, suggesting that SPT5 may be required for normal transcription of a large number of genes. The SPT5 DNA sequence was determined; it predicted a 116-kDa protein with an extremely acidic amino terminus and a novel six-amino-acid repeat at the carboxy terminus (consensus = S-T/A-W-G-G-A/Q). By indirect immunofluorescence microscopy we showed that a bifunctional SPT5-beta-galactosidase protein was located in the yeast nucleus. This molecular analysis of the SPT5 gene revealed a number of interesting similarities to the previously characterized SPT6 gene of S. cerevisiae. These results suggest that SPT5 and SPT6 act in a related fashion to influence essential transcriptional processes in S. cerevisiae.

Double-strand-break repair and recombination catalyzed by a nuclear extract of Saccharomyces cerevisiae.

An in vitro system for double-strand-break repair and recombination of plasmid substrates catalyzed by extracts prepared from yeast nuclei has been developed. Recombination events that generate crossover products were detected amongst reaction products by Southern blot hybridization, or by the polymerase chain reaction (PCR). The recombination reaction was found to be stimulated by a double-strand break within homologous sequences and proceeded by a mechanism that involved branched DNA intermediates. In addition to pairing events that generate crossovers, the formation of inverted repeats (head-to-head and tail-to-tail joined products) was also detected. Two models are presented which propose that the formation of crossover products and inverted repeats occur by similar mechanisms.

Yeast cell-free nuclear protein import requires ATP hydrolysis

Saccharomyces cerevisiae nuclear proteins are shown to localize specifically to isolated yeast nuclei under conditions selective for nuclear proteins. Nuclear association is time- and temperature-dependent, requires ATP hydrolysis, and is abolished by protease pretreatment of nuclei. The nucleus-localized protein is translocated across the nuclear envelope as determined by inaccessibility to externally added immobilized protease. This cell-free system, consisting of components from an organism amenable to genetic analysis, will facilitate the study of the poorly understood mechanism of nuclear protein localization. The finding that ATP hydrolysis is required for nuclear import is the most direct evidence that nuclear localization is energy-dependent.

Expression in Saccharomyces cerevisiae of a gene associated with cytoplasmic male sterility from maize: Respiratory dysfunction and uncoupling of yeast mitochondria

We asked whether the mitochondrial T-urf13 gene, associated with the male sterility phenotype of T cytoplasm in maize, can be expressed in Saccharomyces cerevisiae and whether this expression can mimic the effects observed in maize. We introduced the universal code equivalent of the T-urf13 gene into the S. cerevisiae nucleus by transformation and directed its translation product into mitochondria by means of a fusion with the targeting presequence from Neurospora crassa AT-Pase subunit 9. We show that expression of the universal code equivalent of the T-urf13 gene in the yeast nucleus does indeed mimic its effects in maize: respiratory growth of yeast is inhibited, respiration-deficient cytoplasmic mutants accumulate and NADH oxidation of isolated mitochondria is uncoupled. All these effects are observed only if the mitochondrial targeting peptide and methomyl or HmT toxin are present.

Escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae

The migration of genetic information from ancestral prokaryotic endosymbionts into eukaryotic nuclei is thought to have had an important role in the evolution of mitochondria and chloroplasts. Here we describe an assay for the detection of movement of DNA between mitochondria and the nucleus in yeast. Because recombinant plasmid DNA replicates after transformation into mitochondria of yeast strains lacking endogenous mitochondrial DNA we were able to propagate the nuclear genetic marker URA3 in mitochondria. As expected, the wild-type URA3 gene in mitochondria failed to complement the uracil auxotrophy (Ura-) caused by a nuclear ura3 mutation. But selection of Ura+ prototrophs from a Ura- strain carrying URA3 on a plasmid in its mitochondria enabled us to detect plasmid movement to the nucleus. Conversely, as the plasmid used also contained the mitochondrial gene COX2 required for respiratory growth, we were able to set up corresponding selections to detect migration of DNA from the nucleus to the mitochondria. Our results show that, in yeast, DNA escapes from mitochondria and appears in the nucleus at a surprisingly high frequency (approximately 2 x 10(-5) per cell per generation). But the rate at which DNA makes the journey in the opposite direction--nucleus to mitochondria--is apparently at least 100,000 times less.

Heavy Ion Effects on Yeast: Inhibition of Ribosomal RNA Synthesis

Diploid wild-type yeast cells were exposed to beams of heavy ions covering a wide range of linear energy transfer (LET) (43-13,700 keV/microns). Synthesis of ribosomal RNA (rRNA) was assessed as a functional measure of damage produced by particle radiation. An exponential decrease of relative rRNA synthesis with particle fluence was demonstrated in all cases. The inactivation cross sections derived were found to increase with LET over the entire range of LET studied. The corresponding values for relative biological effectiveness were slightly less than unity. Maximum cross sections measured were close to 1 micron 2, implying that some larger structure within the yeast nucleus (e.g., the nucleolus) might represent the target for an impairment of synthetic activity by very heavy ions rather than the genes coding for rRNA. Where tested, an oxygen effect for rRNA synthesis could not be demonstrated.

Differential distribution of factors involved in pre-mRNA processing in the yeast cell nucleus.

The yeast cell nucleus has previously been shown to be divided into two regions by a variety of microscopic approaches. We used antibodies specific for the 2,2,7-trimethylguanosine cap structure of small nuclear ribonucleic acids (snRNAs) and for a protein component of small nuclear ribonucleoprotein particles to identify the distribution of small nuclear ribonucleoprotein particles within the yeast cell nucleus. These studies were performed with the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae. By using immunofluorescence microscopy and immunoelectron microscopy, most of the abundant snRNAs were localized to the portion of the nucleus which has heretofore been referred to as the nucleolus. This distribution of snRNAs is different from that found in mammalian cells and suggests that the nucleolar portion of the yeast nucleus contains functional domains in addition to those associated with RNA polymerase I activity.

Differential Distribution of Factors Involved in Pre-mRNA Processing in the Yeast Cell Nucleus

The yeast cell nucleus has previously been shown to be divided into two regions by a variety of microscopic approaches. We used antibodies specific for the 2,2,7-trimethylguanosine cap structure of small nuclear ribonucleic acids (snRNAs) and for a protein component of small nuclear ribonucleoprotein particles to identify the distribution of small nuclear ribonucleoprotein particles within the yeast cell nucleus. These studies were performed with the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae. By using immunofluorescence microscopy and immunoelectron microscopy, most of the abundant snRNAs were localized to the portion of the nucleus which has heretofore been referred to as the nucleolus. This distribution of snRNAs is different from that found in mammalian cells and suggests that the nucleolar portion of the yeast nucleus contains functional domains in addition to those associated with RNA polymerase I activity.

Characterization of nuclear localizing sequences derived from yeast ribosomal protein L29.

Two particular seven-amino-acid segments from yeast ribosomal protein L29 caused a non-nuclear reporter protein to associate almost exclusively with the yeast nucleus. The two L29-derived nuclear localizing sequences were identical in five of the seven residues, many of which were basic amino acids. Generally, localization of the reporter protein was most impaired by replacement of the basic residues. A particular Arg residue was unique; substitution by any amino acid including Lys diminished nuclear localization of the reporter protein. In L29 the corresponding Arg 25----Lys substitution within the nuclear localizing sequence distal to the N-terminus was without effect, as evidence by normal rates of ribosome assembly and cell growth. However, the analogous Arg 8----Lys substitution within the localizing sequence proximal to the N-terminus led to greatly reduced rates of ribosome assembly and cell growth. Finally, when both localizing sequences contained the Arg----Lys substitution a still greater decrease in ribosome assembly and cell growth was observed. These results were as expected if the two short peptide sequences functioned in nuclear localization and/or assembly of yeast ribosomal protein L29.

In vitro translocation through the yeast nuclear envelope: Signal-dependent transport requires ATP and calcium

Abstract Nuclei rapidly purified from yeast Saccharomyces cerevisiae using a cytochalasin B enucleation procedure are substantially free of cell wall, secretory vesicle, plasma membrane vacuolar, and cytoplasmic and mitochondrial contamination. Nuclei obtained in this manner in high yield retain transport properties comparable to nuclei in situ. An in vitro nuclear import assay system has been developed using isolated nuclei and radiolabeled proteins prepared by a coupled in vitro transcription/translation system. Both wild-type SV40 large T-antigen and nucleoplasmin are imported into isolated yeast nuclei, whereas a missense cytoplasmic mutant of the SV40 large T-antigen (Lys128----Thr) and cytoplasmic dihydrofolate reductase are not imported. Association and import of these proteins in a time- and signal-dependent manner resulted in their protection from trypsin that was tethered to agarose beads. Greater than 70% of the labeled protein harboring a karyophilic signal was imported in a reaction that could be blocked by prior treatment of nuclei with trypsin-agarose. Nuclear accumulation of SV40 large T-antigen and nucleoplasmin was unidirectional, ATP and Ca2+ dependent, and was not inhibited by a vast excess of exogenous nonnuclear competitor protein. This system provides an important new tool in combination with powerful yeast genetic methods for analysis of the mechanism and the apparatus for transport at the nuclear envelope.

Higher order structure is present in the yeast nucleus: autoantibody probes demonstrate that the nucleolus lies opposite the spindle pole body.

A panel of sera from 892 autoimmune patients was screened by indirect immunofluorescence on mammalian cells. Seventy-three sera were identified that recognize the nucleolus. Three of these sera appear to stain the nucleolus in yeast, suggesting that they recognize highly conserved antigens. These three sera also immunoprecipitate mammalian U3 snRNA-containing particles, which reside in the nucleolus and have been implicated in rRNA processing. Double immunofluorescence experiments with anti-nucleolus and anti-tubulin antibodies revealed a novel form of non-random nuclear organization in yeast. The spindle pole body and the nucleolus-both of which are associated with the nuclear envelope-preferentially localize at opposite ends of the nucleus. Organization of these and other components into specific regions of the nucleus may be important for optimizing their proper function.

Identification of a wheat germ agglutinin-sensitive ATPase in yeast nuclei

We have found that wheat germ agglutinin (WGA), a lectin that specifically binds to N-acetylglucosamine residues inhibits the in vitro transport of plasmid DNA, pJDB219, into yeast nuclei. Histochemical staining of the isolated nuclei with biotinylated WGA and streptavidin-biotinylated peroxidase complex revealed the presence of WGA-binding materials around the nuclear pore under an electron microscope. Using WGA-agarose column chromatography of yeast nuclear extracts, a novel Mg 2+-dependent ATPase was isolated. Its activity was highly sensitive to WGA and stimulated by Nonidet P-40 or phosphatidylserine. We suggest that the WGA-sensitive ATPase plays a role in yeast nuclear transport of DNA.

Functional domains of a negative regulatory protein, GAL80, of Saccharomyces cerevisiae.

To study the functional domains of a transcriptional repressor encoded by the GAL80 gene of Saccharomyces cerevisiae, we constructed various deletion and insertion mutations in the GAL80 coding region and determined the ability of these mutations to repress synthesis of galactose-metabolizing enzymes as well as the capacity of the mutant proteins to respond to the inducer. Two regions, from amino acids 1 to 321 and from amino acids 341 to 423, in the total sequence of 435 amino acids were required for repression. The internal region from amino acids 321 to 340 played a role in the response to the inducer. The 12 amino acids at the carboxy terminus were dispensable for normal functioning of the GAL80 protein. Using indirect immunofluorescence and subcellular fractionation techniques, we also found that two distinct regions (amino acids 1 to 109 and 342 to 405) within the putative repression domain were capable of directing cytoplasmically synthesized Escherichia coli beta-galactosidase to the yeast nucleus. In addition, three gal80 mutations were mapped at amino acid residues 183, 298, and 310 in the domain required for repression. On the basis of these results, we suggest that the GAL80 protein consists of a repression domain located in two separate regions (amino acid residues 1 to 321 and 341 to 423) that are interrupted by an inducer interaction domain (residues 322 to 340) and two nuclear localization domains (1 to 109 and 342 to 405) that overlap the repression domains.

Functional Domains of a Negative Regulatory Protein, GAL80, of Saccharomyces cerevisiae

To study the functional domains of a transcriptional repressor encoded by the GAL80 gene of Saccharomyces cerevisiae, we constructed various deletion and insertion mutations in the GAL80 coding region and determined the ability of these mutations to repress synthesis of galactose-metabolizing enzymes as well as the capacity of the mutant proteins to respond to the inducer. Two regions, from amino acids 1 to 321 and from amino acids 341 to 423, in the total sequence of 435 amino acids were required for repression. The internal region from amino acids 321 to 340 played a role in the response to the inducer. The 12 amino acids at the carboxy terminus were dispensable for normal functioning of the GAL80 protein. Using indirect immunofluorescence and subcellular fractionation techniques, we also found that two distinct regions (amino acids 1 to 109 and 342 to 405) within the putative repression domain were capable of directing cytoplasmically synthesized Escherichia coli beta-galactosidase to the yeast nucleus. In addition, three gal80 mutations were mapped at amino acid residues 183, 298, and 310 in the domain required for repression. On the basis of these results, we suggest that the GAL80 protein consists of a repression domain located in two separate regions (amino acid residues 1 to 321 and 341 to 423) that are interrupted by an inducer interaction domain (residues 322 to 340) and two nuclear localization domains (1 to 109 and 342 to 405) that overlap the repression domains.

Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins.

We have used a monoclonal antibody raised against rat liver nuclear proteins to study two cross-reactive proteins in the yeast nucleus. In rat liver, this monoclonal antibody, mAb 414, binds to nuclear pore complex proteins, including one of molecular weight 62,000 (Davis, L. I., and G. Blobel. 1987. Proc. Natl. Acad. Sci. USA. 84:7552-7556). In yeast, mAb 414 cross reacts by immunoblotting with two proteins that have apparent molecular weights of 110,000 and 95,000, and are termed p110 and p95, respectively. Examination of subcellular fractions by immunoblotting shows that both p110 and p95 are located exclusively in the nuclear fraction. The mAb 414 immunoprecipitates several proteins from a crude yeast cell extract, including p110, p95, and a approximately 55-kD protein. Immunoprecipitation from subcellular fractions yields only p110 and p95 from purified nuclei, whereas the approximately 55-kD protein is immunoprecipitated from the soluble fraction. Digestion of purified nuclei with DNase to produce nuclear envelopes releases some of p110, but the majority of p110 is solubilized only after treatment of envelopes with 1 M NaCl. Immunofluorescence localization using yeast cells and isolated nuclei shows a punctate and patchy staining pattern of the nucleus. Confocal laser scanning immunofluorescence microscopy resolves the punctate and patchy staining pattern better and shows regions of fluorescence at the nuclear envelope. Postembedding immunogold electron microscopy using purified nuclei and mAb 414 shows colloidal gold decoration of the yeast nuclear envelope, but resolves pore complexes too poorly to achieve further ultrastructural localization. Immunogold labeling of nuclei followed by embedding suggests decoration of pore complexes. Thus, p110 and/or p95 are localized to the nuclear envelope in yeast, and may be components of the nuclear pore complex.

Increased amounts of HMG-CoA reductase induce "karmellae": a proliferation of stacked membrane pairs surrounding the yeast nucleus.

Overproduction of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in yeast resulted in striking morphological effects on the structure of intracellular membranes. Specifically, stacks of paired membranes closely associated with the nuclear envelope were observed in strains that over-produced the HMG1 isozyme, one of two isozymes for HMG-CoA reductase in yeast. These nuclear-associated, paired membranes have been named "karmellae." In strains that overproduced the HMG1 isozyme, HMG-CoA reductase was present in the karmellar layers. At mitosis, karmellae were asymmetrically segregated: the mother cells inherited all of the karmellae and the daughter cells inherited none. A membranous structure of different morphology was occasionally found in cells that overproduced the HMG2 isozyme. These observations further establish the existence of cellular mechanisms that monitor the levels of membrane proteins and compensate for changes in these levels by inducing synthesis of particular types of membrane.

Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein.

We have produced monoclonal antibodies against purified nuclei from the yeast Saccharomyces cerevisiae and have characterized three different antibodies that recognize a protein with an apparent molecular weight of 38,000, termed p38. Subcellular fractionation shows that virtually all of p38 occurs in the nuclear fraction. High concentrations of salt (1 M) or urea (6 M) effectively solubilize p38 from a nuclear envelope fraction prepared by digestion of nuclei with DNase. Indirect immunofluorescence demonstrates a crescent shaped distribution of p38 at the inner periphery of the nucleus, with p38 extending between dividing pairs of cells during (closed) mitosis. Postembedding immunogold electron microscopy shows decoration of the densely stained "crescent" region of the yeast nucleus, confirming the localization of p38 to the nucleolus. One of the monoclonals, D77, cross reacts on immunoblots with a single protein of molecular weight 37,000 from purified rat liver nuclei. Indirect immunofluorescence localizes this protein to the nucleolus, and shows that it is dispersed throughout the cell during mitosis. The yeast and rat liver nucleolar proteins behave similarly when electrophoresed in two dimensions, and appear to have basic pI values. Analysis of immunological cross-reactivity using D77, and antibodies specific for nucleolar proteins from other sources, suggests that the rat liver protein is fibrillarin, and demonstrates that p38 shares epitopes with fibrillarin, as well as with other vertebrate nucleolar proteins.

RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.