We have used epitope tag addition to analyze the transmembrane topology of the Na,K-ATPase catalytic (alpha) subunit. An antigenic peptide derived from the hemagglutinin (HA) of influenza virus was inserted at 15 different positions within the rat Na,K-ATPase alpha 1 subunit isoform. The functional integrity of the tagged proteins was tested by their capacity to confer ouabain resistance upon human HEK 293 cells. Constructs with the tag at aa positions 119, 173, 318, 815, 881, 953, 987, and 1023 conferred ouabain resistance, and the mutant proteins were detectable in the plasma membrane of transfected cells. In contrast, alpha 1 subunits with insertions at aa positions 338, 797, 805, 868, 895, 910, and 921 were unable to confer drug resistance. Immunofluorescence analysis of permeabilized and intact cells using a monoclonal antibody specific for the HA epitope showed that double tags at positions 119 and 318 were located extracellularly, whereas single or double tags at positions 173, 815, 881, 987, and 1023 were cytoplasmically disposed. These results are consistent with an eight transmembrane domain arrangement for the alpha subunit. Epitope insertion within TM4, and the region linking transmembrane segments TM6-TM7, caused the loss of alpha subunit function, suggesting that the integrity of these domains is essential for the proper biosynthesis and/or maturation of the alpha subunit.
The yeast RNAI gene encodes a cytosolic protein that affects pre-tRNA splicing, pre-rRNA processing, the production of mRNA, and the export of RNA from the nucleus to the cytosol. In an attempt to understand how the RNA1 protein affects such a diverse set of processes, we sought second-site suppressors of a mutation, rnal-1, of the RNA] locus. Mutations in a single complementation group were obtained. These lesions proved to be in the same gene, SRNI, identified previously in a search for second-site suppressors of mutations that affect the removal of intervening sequences from pre-mRNAs. The SRNI gene was mapped, cloned, and sequenced. DNA sequence analysis and the phenotype of disruption mutations showed that, surprisingly, SRNI is identical to HEX2IREGI, a gene that negatively regulates glucose-repressible genes. Interestingly, SRNI is not a negative regulator ofRNAI at the transcriptional, translational, or protein stability level. However, SRNI does regulate the level of two newly discovered antigens, p43 and p70, one of which is not glucose repressible. These studies for the first time link RNA processing and carbon catabolite repression.Primary transcription products are often trimmed and modified to yield mature species via a series of processing steps. These processing steps may include 5' and 3' nucleotide alterations and splicing of intervening sequences, as well as addition and modification of nucleotides. In Saccharomyces cerevisiae, genes encoding some of the products necessary for RNA processing have been identified. The primary effect of mutations in these genes is usually at a specific step in the production of a single class of RNA. For example, mutations of the LOS1, SEN1, SEN2, TPD1, PTA1, and STPJ genes result in defects in the removal of intervening sequences from pre-tRNAs (for reviews, see references 5 and 11), whereas mutations in TRMI, MOD5, TRM2, and ML41 affect single nucleotide modifications of tRNAs (for a review, see reference 11). Mutations of the PRP genes affect the removal of intervening sequences from pre-mRNAs, causing a depletion of ribosomal proteins which in turn affects the accumulation of mature rRNAs (for a review, see reference 42).In contrast to the mutations in the genes described above, the mnal-1 mutation of the RNAJ gene (10) pleiotropically affects the processing of all three major classes of RNAs at the nonpermissive temperature. Processing of tRNA in rnal-1 cells is defective at the step of removal of the intervening sequence (12, 18), and rRNA is affected at the step of processing the 35S primary pre-rRNA (12). The defect in the production of mRNA in mal-1 cells is not clearly defined, but one report described elongated 3' ends (39) and another report described alterations in poly(A) lengths (33). Cells with the mal-1 allele are apparently not defective in the removal of intervening sequences from pre-mRNA (34). The transport of RNA from the nucleus to the cytosol is also affected by the mal-I mutation (15, 37).Paradoxically, it has been found that the RNA1 pr...
Chinese hamster ovary cells (CHO) were co-transfected with pSV2neo and sheared DNA from either a human cell line (HT29) expressing high levels of O6-alkylguanine-DNA alkyltransferase (AGT) or from a cell line (BE) deficient in this activity. Cells expressing the selectable marker were obtained by exposure to G418 and colonies resistant to alkylation damage isolated by growth in the presence of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). The number of colonies of cells expressing AGT activity arising after transfection with DNA from BE cells was similar to the number arising from cells exposed to HT29 DNA. Although the amount of AGT repair protein expressed in the transfectant colonies from this experiment was relatively low, these results indicate that repair of alkylation damage can be restored in AGT-deficient cells by transfection of human DNA from both repair-deficient and proficient cells. A separate transfection of CHOMG cells [a mutant of CHO cells resistant to the drug, methylglyoxal bis(guanylhydrazone) (MGBG)] with HT29 DNA and pSV2neo followed by selection of G418 and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in three colonies with high AGT levels. These transfectants had different growth rates and expressed levels of the AGT protein between 230 and 300 fmol/mg protein. The transfectants were as resistant to the cytotoxic effects of BCNU, Clomesone, methylnitrosourea (MNU) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) as HT29 cells which were much more resistant than the parental CHOMG cells. Pretreatment of transfectant cells with 0.4 mM O6-methylguanine for 24 h reduced AGT activity to 14% basal levels, which upon removal of the base increased to approximately 74% basal level within 8 h. The sensitivity to the cytotoxic effects of both the chloroethylating and methylating agents was enhanced by treatment with O6-methylguanine. In the same manner, the number of BCNU-induced DNA interstrand cross-links increased in transfectant cells pretreated with O6-methylguanine. These results provide further evidence that the formation of methyl or chloroethyl adducts at the O6-position contribute significantly to cell lethality.
The yeast RNA1 gene encodes a cytosolic protein that affects pre-tRNA splicing, pre-rRNA processing, the production of mRNA, and the export of RNA from the nucleus to the cytosol. In an attempt to understand how the RNA1 protein affects such a diverse set of processes, we sought second-site suppressors of a mutation, rna1-1, of the RNA1 locus. Mutations in a single complementation group were obtained. These lesions proved to be in the same gene, SRN1, identified previously in a search for second-site suppressors of mutations that affect the removal of intervening sequences from pre-mRNAs. The SRN1 gene was mapped, cloned, and sequenced. DNA sequence analysis and the phenotype of disruption mutations showed that, surprisingly, SRN1 is identical to HEX2/REG1, a gene that negatively regulates glucose-repressible genes. Interestingly, SRN1 is not a negative regulator of RNA1 at the transcriptional, translational, or protein stability level. However, SRN1 does regulate the level of two newly discovered antigens, p43 and p70, one of which is not glucose repressible. These studies for the first time link RNA processing and carbon catabolite repression.
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