scholarly journals N2,N2-dimethylguanosine-specific tRNA methyltransferase contains both nuclear and mitochondrial targeting signals in Saccharomyces cerevisiae.

1989 ◽  
Vol 109 (4) ◽  
pp. 1411-1419 ◽  
Author(s):  
J M Li ◽  
A K Hopper ◽  
N C Martin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Indirect Immunofluorescence ◽  
Trna Modification ◽  
Mitochondrial Targeting ◽  
Nuclear Targeting ◽  
Cytoplasmic Staining ◽  
Immunofluorescence Studies ◽  
Trna Methyltransferase ◽  
Terminal Amino ◽  
Beta Galactosidase

The TRM1 gene of Saccharomyces cerevisiae encodes a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase, which modifies both mitochondrial and cytoplasmic tRNAs. The enzyme is targeted to mitochondria for the modification of mitochondrial tRNAs. Cellular fractionation and indirect immunofluorescence studies reported here demonstrate that this enzyme is also localized to the nucleus. Further, immunofluorescence experiments using strains that overproduce the enzyme show a staining at the periphery of the nucleus suggesting that the enzyme is found in a subnuclear destination near or at the nuclear membrane. There is no obvious cytoplasmic staining in these overproducing strains. Fusion protein technology was used to begin to localize sequences involved in the nuclear targeting of this enzyme. Indirect immunofluorescence studies indicate that sequences between the first 70 and 213 NH2-terminal amino acids of the methyltransferase are sufficient to target Escherichia coli beta-galactosidase to nuclei.

scholarly journals Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei.

1992 ◽  
Vol 12 (12) ◽  
pp. 5652-5658 ◽  
Author(s):  
A M Rose ◽  
P B Joyce ◽  
A K Hopper ◽  
N C Martin
Keyword(s):  
Amino Acids ◽  
Nuclear Periphery ◽  
Immunofluorescent Staining ◽  
Coding Region ◽  
Nuclear Targeting ◽  
Subnuclear Localization ◽  
Amino Terminal ◽  
Linear Sequence ◽  
Trna Methyltransferase ◽  
Terminal Amino

The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.

Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei

1992 ◽  
Vol 12 (12) ◽  
pp. 5652-5658
Author(s):  
A M Rose ◽  
P B Joyce ◽  
A K Hopper ◽  
N C Martin
Keyword(s):  
Amino Acids ◽  
Nuclear Periphery ◽  
Immunofluorescent Staining ◽  
Coding Region ◽  
Nuclear Targeting ◽  
Subnuclear Localization ◽  
Amino Terminal ◽  
Linear Sequence ◽  
Trna Methyltransferase ◽  
Terminal Amino

The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.

scholarly journals Saccharomyces cerevisiae Mod5p-II Contains Sequences Antagonistic for Nuclear and Cytosolic Locations

Genetics ◽  
1999 ◽  
Vol 151 (1) ◽  
pp. 57-75 ◽  
Author(s):  
Leslie H Tolerico ◽  
Ann L Benko ◽  
John P Aris ◽  
David R Stanford ◽  
Nancy C Martin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Translation Initiation ◽  
Nuclear Export ◽  
Fusion Proteins ◽  
Trna Modification ◽  
Mitochondrial Targeting ◽  
Alternative Translation ◽  
Nuclear Localization Sequences ◽  
Passenger Protein

Abstract MOD5 encodes a tRNA modification activity located in three subcellular compartments. Alternative translation initiation generates Mod5p-I, located in the mitochondria and the cytosol, and Mod5p-II, located in the cytosol and nucleus. Here we study the nucleus/cytosol distribution of overexpressed Mod5p-II. Nuclear Mod5p-II appears concentrated in the nucleolus, perhaps indicating that the nuclear pool may have a different biological role than the cytoplasmic and mitochondrial pools. Mod5p contains three motifs resembling bipartite-like nuclear localization sequences (NLSs), but only one is sufficient to locate a passenger protein to the nucleus. Mutations of basic residues of this motif cumulatively contribute to a cytosolic location for the fusion proteins. These alterations also cause decreased nuclear pools of endogenous Mod5p-II. Depletion of nuclear Mod5p-II does not affect tRNATyr function. Despite the NLS, most Mod5p is cytosolic. We assessed whether Mod5p sequences cause a karyophilic reporter to be located in the cytosol. By this assay, Mod5p may contain more than one region that functions as cytoplasmic retention and/or nuclear export sequences. Thus, distribution of Mod5p results from the presence/absence of mitochondrial targeting information and sequences antagonistic for nuclear and cytosolic locations. Mod5p is highly conserved; sequences responsible for subcellular distribution appear to reside in “accessory” motifs missing from prokaryotic counterparts.

scholarly journals Transcriptional regulation of ribosomal proteins during a nutritional upshift in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (7) ◽  
pp. 2429-2435 ◽  
Author(s):  
D M Donovan ◽  
N J Pearson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Protein Gene ◽  
Relative Rate ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Protein Product ◽  
Yeast Genome ◽  
Base Pairs ◽  
Beta Galactosidase

The relative rates of synthesis of Saccharomyces cerevisiae ribosomal proteins increase coordinately during a nutritional upshift. We constructed a gene fusion which contained 528 base pairs of sequence upstream from and including the TATA box of ribosomal protein gene rp55-1 (S16A-1) fused to a CYC1-lacZ fusion. This fusion was integrated in single copy at the rp55-1 locus in the yeast genome. During a nutritional upshift, in which glucose was added to cells growing in an ethanol-based medium, we found that the increase in the relative rate of synthesis of the beta-galactosidase protein product followed the same kinetics as the change in relative rates of synthesis of several ribosomal proteins measured in the same experiment. This demonstrates that the nontranscribed sequences upstream from the rp55-1 gene, which are present in the fusion, are sufficient to mediate the change in rates of synthesis characteristic of ribosomal proteins under these conditions. The results also suggest that a change in transcription rates is mainly responsible for the increase in relative rates of synthesis of ribosomal proteins during a nutritional upshift in S. cerevisiae.

Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein

1992 ◽  
Vol 12 (6) ◽  
pp. 2653-2661
Author(s):  
E Gross ◽  
I Marbach ◽  
D Engelberg ◽  
M Segal ◽  
G Simchen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Adenylyl Cyclase ◽  
Gene Product ◽  
Cyclase Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Terminal Domain ◽  
Yeast Homolog ◽  
Cdc25 Protein ◽  
Beta Galactosidase

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.

Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG

1984 ◽  
Vol 4 (11) ◽  
pp. 2467-2478
Author(s):  
R W West ◽  
R R Yocum ◽  
M Ptashne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Transcription Initiation ◽  
Function Analysis ◽  
Lacz Gene ◽  
Chromosome 2 ◽  
Proximal Half ◽  
Base Pair Deletion ◽  
Upstream Activating Sequence ◽  
Beta Galactosidase

The GAL1 and GAL10 genes, separated by 680 base pairs and divergently transcribed on chromosome 2 of Saccharomyces cerevisiae, were separately fused to the lacZ gene of Escherichia coli so that beta-galactosidase synthesis in S. cerevisiae reflected GAL1 and GAL10 promoter function. Analysis of two sets of deletions defined a 75-base-pair sequence, located ca. midway between the transcription initiation regions of GAL1 and GAL10, that mediates GAL4-dependent induction of both genes. Deletion of various parts of this sequence (called the GAL upstream activating sequence or UASG) reduced GAL1 and GAL10 induction about equally. Sequences in the GAL10-proximal half of UASG in some sequence contexts functioned independently of sequences in the GAL1-proximal half of UASG. A 33-base-pair deletion of the GAL10-proximal half of UASG drastically reduced induction. Deletions between UASG and the GAL1 TATA box caused beta-galactosidase to be synthesized at an unexpectedly high basal level, that is, in the absence of galactose and GAL4 product. Some of these mutations also reduced the repression caused by glucose.

Efficiency and diversity of protein localization by random signal sequences

1990 ◽  
Vol 10 (6) ◽  
pp. 3163-3173
Author(s):  
C A Kaiser ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Protein Localization ◽  
Random Signal ◽  
Perinuclear Region ◽  
Hybrid Protein ◽  
Random Sequences ◽  
Beta Galactosidase

Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.

Trans-acting regulatory mutations that alter transcription of Saccharomyces cerevisiae histone genes

1987 ◽  
Vol 7 (12) ◽  
pp. 4204-4210
Author(s):  
M A Osley ◽  
D Lycan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Fusion Gene ◽  
Chromosome Replication ◽  
Lacz Fusion ◽  
Histone Genes ◽  
Promoter Elements ◽  
Regulatory Mutations ◽  
Cycle Dependent ◽  
Beta Galactosidase

Using a Saccharomyces cerevisiae strain containing an integrated copy of an H2A-lacZ fusion gene, we screened for mutants which overexpressed beta-galactosidase as a way to identify genes which regulate transcription of the histone genes. Five recessive mutants with this phenotype were shown to contain altered regulatory genes because they had lost repression of HTA1 transcription which occurs upon inhibition of chromosome replication (D. E. Lycan, M. A. Osley, and L. Hereford, Mol. Cell. Biol. 7:614-621, 1987). Periodic transcription was affected in the mutants as well, since the HTA1 gene was transcribed during the G1 and G2 phases of the cell cycle, periods in the cell cycle when this gene is normally not expressed. A similar loss of cell cycle-dependent transcription was noted for two of the three remaining histone loci, while the HO and CDC9 genes continued to be expressed periodically. Using isolated promoter elements inserted into a heterologous cycl-lacZ fusion gene, we demonstrated that the mutations fell in genes which acted through a negative site in the TRT1 H2A-H2B promoter.

An essential Saccharomyces cerevisiae gene homologous to SNF2 encodes a helicase-related protein in a new family

1992 ◽  
Vol 12 (4) ◽  
pp. 1893-1902
Author(s):  
B C Laurent ◽  
X Yang ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Nucleoside Triphosphate ◽  
Related Protein ◽  
New Family ◽  
Eukaryotic Proteins ◽  
Beta Galactosidase

The Saccharomyces cerevisiae SNF2 gene affects the expression of many diversely regulated genes and has been implicated in transcriptional activation. We report here the cloning and characterization of STH1, a gene that is homologous to SNF2. STH1 is essential for mitotic growth and is functionally distinct from SNF2. A bifunctional STH1-beta-galactosidase protein is located in the nucleus. The predicted 155,914-Da STH1 protein is 72% identical to SNF2 over 661 amino acids and 46% identical over another stretch of 66 amino acids. Both STH1 and SNF2 contain a putative nucleoside triphosphate-binding site and sequences resembling the consensus helicase motifs. The large region of homology shared by STH1 and SNF2 is conserved among other eukaryotic proteins, and STH1 and SNF2 appear to define a novel family of proteins related to helicases.

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