Effects of amino acid addition on molar fraction of 3-hydroxyvalerate in copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate synthesized by Alcaligenes sp. SH-69

ABSTRACT The eutF locus of Salmonella typhimuriumLT2 was identified as a locus necessary for the utilization of ethanolamine as a sole carbon source. Initial models suggested that EutF was involved in either ethanolamine transport or was a transcriptional regulator of an ethanolamine transporter. Phenotypic characterization of eutF mutants suggested EutF was somehow involved in 1,2-propanediol, propionate, and succinate utilization. Here we provide evidence that two alleles defining the eutFlocus, Δ903 and eutF1115, are partial-loss-of-function tonB alleles. Both mutations were complemented by plasmids containing a wild-type allele of theEscherichia coli tonB gene. Immunoblot analysis using TonB monoclonal antibodies detected a TonB fusion protein in strains carrying eutF alleles. Molecular analysis of the Δ903 allele identified a deletion that resulted in the fusion of the 3′ end of tonB with the 3′ end oftrpA. In-frame translation of the tonB-trpAfusion resulted in the final 9 amino acids of TonB being replaced by a 45-amino-acid addition. We isolated a derivative of a strain carrying allele Δ903 that regained the ability to grow on ethanolamine as a carbon and energy source. The molecular characterization of the mutation that corrected the Eut−phenotype caused by allele Δ903 showed that the new mutation was a deletion of two nucleotides at the tonB-trpAfusion site. This deletion resulted in a frameshift that replaced the 45-amino-acid addition with a 5-amino-acid addition. This change resulted in a TonB protein with sufficient activity to restore growth on ethanolamine and eut operon expression to nearly wild-type levels. It was concluded that the observed EutF phenotypes were due to the partial loss of TonB function, which is proposed to result in reduced cobalamin and ferric siderophore transport in an aerobic environment; thus, the eutF locus does not exist.

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Enhancement of recombinant protein synthesis and stability via coordinated amino acid addition

Biotechnology and Bioengineering ◽

10.1002/bit.260410508 ◽

1993 ◽

Vol 41 (5) ◽

pp. 557-565 ◽

Cited By ~ 44

Author(s):

Delia M. Ramérez ◽

William E. Bentley

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Recombinant Protein ◽

Acid Addition

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Different Ubiquitin Signals Act at the Golgi and Plasma Membrane to Direct GAP1 Trafficking

Molecular Biology of the Cell ◽

10.1091/mbc.e07-06-0627 ◽

2008 ◽

Vol 19 (7) ◽

pp. 2962-2972 ◽

Cited By ~ 39

Author(s):

April L. Risinger ◽

Chris A. Kaiser

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Plasma Membrane ◽

Amino Acid ◽

Protein Sorting ◽

High Capacity ◽

Amino Acid Permease ◽

Acid Addition ◽

Amino Acid Levels ◽

General Amino Acid Permease

The high capacity general amino acid permease, Gap1p, in Saccharomyces cerevisiae is distributed between the plasma membrane and internal compartments according to availability of amino acids. When internal amino acid levels are low, Gap1p is localized to the plasma membrane where it imports available amino acids from the medium. When sufficient amino acids are imported, Gap1p at the plasma membrane is endocytosed and newly synthesized Gap1p is delivered to the vacuole; both sorting steps require Gap1p ubiquitination. Although it has been suggested that identical trans-acting factors and Gap1p ubiquitin acceptor sites are involved in both processes, we define unique requirements for each of the ubiquitin-mediated sorting steps involved in delivery of Gap1p to the vacuole upon amino acid addition. Our finding that distinct ubiquitin-mediated sorting steps employ unique trans-acting factors, ubiquitination sites on Gap1p, and types of ubiquitination demonstrates a previously unrecognized level of specificity in ubiquitin-mediated protein sorting.

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