scholarly journals The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal.

1986 ◽  
Vol 102 (2) ◽  
pp. 523-533 ◽  
Author(s):  
S D Emr ◽  
A Vassarotti ◽  
J Garrett ◽  
B L Geller ◽  
M Takeda ◽  
...  
Keyword(s):  
Beta Subunit ◽  
Lacz Gene ◽  
Gene Fusions ◽  
Yeast Mitochondria ◽  
Subunit Protein ◽  
F1 Atpase ◽  
Amino Terminal ◽  
Hybrid Proteins ◽  
Suc2 Gene

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells.

scholarly journals Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (11) ◽  
pp. 2347-2355 ◽  
Author(s):  
S D Emr ◽  
I Schauer ◽  
W Hansen ◽  
P Esmon ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Lacz Gene ◽  
Gene Fusions ◽  
Glucose Medium ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Hybrid Proteins ◽  
Suc2 Gene ◽  
Beta Galactosidase

The yeast SUC2 gene codes for the secreted enzyme invertase. A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase. Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae. The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase. Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium. Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted. Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER). The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex. Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer. beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles. In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I. Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells. In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.

Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae

1984 ◽  
Vol 4 (11) ◽  
pp. 2347-2355
Author(s):  
S D Emr ◽  
I Schauer ◽  
W Hansen ◽  
P Esmon ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Lacz Gene ◽  
Gene Fusions ◽  
Glucose Medium ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Hybrid Proteins ◽  
Suc2 Gene ◽  
Beta Galactosidase

The yeast SUC2 gene codes for the secreted enzyme invertase. A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase. Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae. The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase. Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium. Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted. Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER). The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex. Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer. beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles. In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I. Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells. In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.

scholarly journals Random mutagenesis of the gene for the β-subunit of F1-ATPase from Escherichia coli

1989 ◽  
Vol 259 (2) ◽  
pp. 421-426 ◽  
Author(s):  
F A Kironde ◽  
D Parsonage ◽  
A E Senior
Keyword(s):  
Escherichia Coli ◽  
Double Mutant ◽  
Catalytic Mechanism ◽  
Random Mutagenesis ◽  
Proton Pumping ◽  
Beta Subunit ◽  
Phenotypic Screening ◽  
Haploid Strain ◽  
F1 Atpase

ATP synthesis by oxidative phosphorylation in Escherichia coli occurs in catalytic sites on the beta-subunits of F1-ATPase. Random mutagenesis of the beta-subunit combined with phenotypic screening is potentially important for studies of the catalytic mechanism. However, when applied to haploid strains, this approach is hampered by a preponderance of mutants in which assembly of F1-ATPase in vivo is defective, precluding enzyme purification. Here we mutagenized plasmids carrying the uncD (beta-subunit) gene with hydroxylamine or N-methyl-N'-nitro-N-nitrosoguanidine and isolated, by phenotypic screening and complementation tests, six plasmids carrying mutant uncD alleles. When the mutant plasmids were used to transform a suitable uncD- strain, assembly of F1-ATPase in vivo occurred in each case. Moreover, in one case (beta Gly-223----Asp) F1-ATPase assembly proceeded although it had previously been reported that this mutation, when present on the chromosome of a haploid strain, prevented assembly of the enzyme in vivo. Therefore, this work demonstrates an improved approach for random mutagenesis of the F1-beta-subunit. Six new mutant uncD alleles were identified: beta Cys-137----Tyr; beta Gly-142----Asp; beta Gly-146----Ser; beta Gly-207----Asp; beta-Gly-223----Asp; and a double mutant beta Pro-403----Ser,Gly-415----Asp which we could not separate. The first five of these lie within or very close to the predicted catalytic nucleotide-binding domain of the beta-subunit. The double mutant lies outside this domain; we speculate that the region around residues beta 403-415 is part of an alpha-beta intersubunit contact surface. Membrane ATPase and ATP-driven proton pumping activities were impaired by all six mutations. Purified F1-ATPase was obtained from each mutant and shown to have impaired specific ATPase activity.

scholarly journals Structure-Function Analysis of IntDOT

2009 ◽  
Vol 192 (2) ◽  
pp. 575-586 ◽  
Author(s):  
Seyeun Kim ◽  
Brian M. Swalla ◽  
Jeffrey F. Gardner
Keyword(s):  
Homology Modeling ◽  
Protein Interactions ◽  
Active Site ◽  
Dna Cleavage ◽  
Function Analysis ◽  
Indicator Strain ◽  
Amino Terminal ◽  
Dna Ligation ◽  
Polypeptide Backbone

ABSTRACT CTnDOT integrase (IntDOT) is a member of the tyrosine family of site-specific DNA recombinases. IntDOT is unusual in that it catalyzes recombination between nonidentical sequences. Previous mutational analyses centered on mutants with substitutions of conserved residues in the catalytic (CAT) domain or residues predicted by homology modeling to be close to DNA in the core-binding (CB) domain. That work suggested that a conserved active-site residue (Arg I) of the CAT domain is missing and that some residues in the CB domain are involved in catalysis. Here we used a genetic approach and constructed an Escherichia coli indicator strain to screen for random mutations in IntDOT that disrupt integrative recombination in vivo. Twenty-five IntDOT mutants were isolated and characterized for DNA binding, DNA cleavage, and DNA ligation activities. We found that mutants with substitutions in the amino-terminal (N) domain were catalytically active but defective in forming nucleoprotein complexes, suggesting that they have altered protein-protein interactions or altered interactions with DNA. Replacement of Ala-352 of the CAT domain disrupted DNA cleavage but not DNA ligation, suggesting that Ala-352 may be important for positioning the catalytic tyrosine (Tyr-381) during cleavage. Interestingly, our biochemical data and homology modeling of the CAT domain suggest that Arg-285 is the missing Arg I residue of IntDOT. The predicted position of Arg-285 shows it entering the active site from a position on the polypeptide backbone that is not utilized in other tyrosine recombinases. IntDOT may therefore employ a novel active-site architecture to catalyze recombination.

scholarly journals Lysine 155 in beta-subunit is a catalytic residue of Escherichia coli F1 ATPase.

1993 ◽  
Vol 268 (10) ◽  
pp. 6989-6994
Author(s):  
A.E. Senior ◽  
S. Wilke-Mounts ◽  
M.K. al-Shawi
Keyword(s):  
Escherichia Coli ◽  
Beta Subunit ◽  
Catalytic Residue ◽  
F1 Atpase
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