Properties of permease dimer, a fusion protein containing two lactose permease molecules from Escherichia coli

Cys into either the first or the second half of the dimer results in a 50% decrease in activity, whereas introduction of the mutation into both halves of the dimer abolishes transport. Lactose transport by permease dimer is completely inactivated by N-ethylmaleimide; however, 40-45% activity is retained after N-ethylmaleimide treatment when either the first or the second half of the dimer is replaced with a mutant devoid of cysteine residues. The observations demonstrate that both halves of the fusion protein are equally active and suggest that each half may function independently. To test the possibility that oligomerization between dimers might account for the findings, a permease dimer was constructed that contains two different deletion mutants that complement functionally when expressed as untethered molecules. Because this construct does not catalyze lactose transport to any extent whatsoever, it is unlikely that the two halves of the dimer interact or that there is an oligomeric interaction between dimers. The approach is consistent with the contention that the functional unit of lactose permease is a monomer. -->

ISSN:	1091-6490 0027-8424
DOI:	10.1073/pnas.91.12.5421
Přístupová URL adresa:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::fccbd1059800959c1aa2721b2b96e9ef https://doi.org/10.1073/pnas.91.12.5421
Rights:	OPEN
Přírůstkové číslo:	edsair.doi.dedup.....fccbd1059800959c1aa2721b2b96e9ef
Autor:	M C Lawrence, Miklós Sahin-Tóth, H R Kaback
Rok vydání:	1994
Předmět:	Lactose permease Monosaccharide Transport Proteins Recombinant Fusion Proteins Dimer Molecular Sequence Data Mutant Biological Transport Active Lactose Lactose transport Structure-Activity Relationship chemistry.chemical_compound Bacterial Proteins Escherichia coli Amino Acid Sequence Multidisciplinary Base Sequence Symporters biology Permease Membrane transport protein Escherichia coli Proteins Membrane Proteins Membrane Transport Proteins Fusion protein chemistry Biochemistry Mutagenesis Site-Directed biology.protein Research Article Cysteine
Zdroj:	Proceedings of the National Academy of Sciences. 91:5421-5425
ISSN:	1091-6490 0027-8424
DOI:	10.1073/pnas.91.12.5421
Popis:	An engineered fusion protein containing two tandem lactose permease molecules (permease dimer) exhibits high transport activity and is used to test the phenomenon of negative dominance. Introduction of the mutation Glu-325-->Cys into either the first or the second half of the dimer results in a 50% decrease in activity, whereas introduction of the mutation into both halves of the dimer abolishes transport. Lactose transport by permease dimer is completely inactivated by N-ethylmaleimide; however, 40-45% activity is retained after N-ethylmaleimide treatment when either the first or the second half of the dimer is replaced with a mutant devoid of cysteine residues. The observations demonstrate that both halves of the fusion protein are equally active and suggest that each half may function independently. To test the possibility that oligomerization between dimers might account for the findings, a permease dimer was constructed that contains two different deletion mutants that complement functionally when expressed as untethered molecules. Because this construct does not catalyze lactose transport to any extent whatsoever, it is unlikely that the two halves of the dimer interact or that there is an oligomeric interaction between dimers. The approach is consistent with the contention that the functional unit of lactose permease is a monomer.
Databáze:	OpenAIRE
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