Abstract:
:A superfamily of transport proteins, which includes the lactose permease of Escherichia coli, contains a highly conserved motif, G-X-X-X-D/E-R/K-X-G-R/K-R/K, in the loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. Previous analysis of this motif in the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995) has shown that the conserved glycine residue found at the first position in the motif (i.e., Gly-64) is important for transport function. Every substitution at this site, with the exception of alanine, greatly diminished lactose transport activity. In this study, three mutants in which glycine-64 was changed to cysteine, serine, and valine were used as parental strains to isolate 64 independent suppressor mutations that restored transport function. Of these 64 isolates, 39 were first-site revertants to glycine or alanine, while 25 were second-site mutations that restored transport activity yet retained a cysteine, serine, or valine at position 64. The second-site mutations were found to be located at several sites within the lactose permease (Pro-28 --> Ser, Leu, or Thr; Phe-29 --> Ser; Ala-50 --> Thr, Cys-154 --> Gly; Cys-234 --> Phe; Gln-241 --> Leu; Phe-261 --> Val; Thr-266 --> Iso; Val-367 --> Glu; and Ala-369 --> Pro). A kinetic analysis was conducted which compared lactose uptake in the three parental strains and several suppressor strains. The apparent Km values of the Cys-64, Ser-64, and Val-64 parental strains were 0.8 mM, 0.7 mM, and 4.6 mM, respectively, which was similar to the apparent Km of the wild-type permease (1.4 mM). In contrast, the Vmax values of the Cys-64, Ser-64, and Val-64 strains were sharply reduced (3.9, 10.1, and 13.2 nmol of lactose/min x mg of protein, respectively) compared with the wild-type strain (676 nmol of lactose/min x mg of protein). The primary effect of the second-site suppressor mutations was to restore the maximal rate of lactose transport to levels that were similar to the wild-type strains. Taken together, these results support the notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes that are necessary for lactose translocation across the membrane. According to our tertiary model, this site is at an interface between the two halves of the protein.
journal_name
J Bacterioljournal_title
Journal of bacteriologyauthors
Jessen-Marshall AE,Parker NJ,Brooker RJdoi
10.1128/jb.179.8.2616-2622.1997subject
Has Abstractpub_date
1997-04-01 00:00:00pages
2616-22issue
8eissn
0021-9193issn
1098-5530journal_volume
179pub_type
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更新日期:2004-10-01 00:00:00
abstract::Gaines, Sidney (Walter Reed Army Institute of Research, Washington, D.C.), Julius A. Currie, and Joseph G. Tully. Detection and persistence of Vi antigen in tissues of actively immunized mice. J. Bacteriol. 89:776-781. 1965.-The presence, distribution, and persistence of Vi antigen in mouse tissue was determined by me...
journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.89.3.776-781.1965
更新日期:1965-03-01 00:00:00
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更新日期:1966-01-01 00:00:00
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doi:10.1128/jb.170.2.693-699.1988
更新日期:1988-02-01 00:00:00
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更新日期:2013-08-01 00:00:00
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更新日期:2019-11-20 00:00:00
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pub_type: 杂志文章
doi:10.1128/JB.180.14.3715-3718.1998
更新日期:1998-07-01 00:00:00
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更新日期:2007-03-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.97.3.1305-1309.1969
更新日期:1969-03-01 00:00:00
abstract::We present the genomic sequence of the human pathogen Legionella pneumophila serogroup 12 strain 570-CO-H (ATCC 43290), a clinical isolate from the Colorado Department of Health, Denver, CO. This is the first example of a genome sequence of L. pneumophila from a serogroup other than serogroup 1. We highlight the simil...
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pub_type: 杂志文章
doi:10.1128/jb.177.9.2475-2480.1995
更新日期:1995-05-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.173.20.6383-6389.1991
更新日期:1991-10-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.121.3.933-941.1975
更新日期:1975-03-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.94.1.184-191.1967
更新日期:1967-07-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
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更新日期:1996-08-01 00:00:00
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更新日期:1975-01-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
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更新日期:1967-12-01 00:00:00
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pub_type: 杂志文章
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更新日期:2002-11-01 00:00:00
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更新日期:2003-02-01 00:00:00
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更新日期:2009-04-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.87.4.828-831.1964
更新日期:1964-04-01 00:00:00
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更新日期:2007-12-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.86.5.904-910.1963
更新日期:1963-11-01 00:00:00
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更新日期:2000-05-01 00:00:00
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journal_title:Journal of bacteriology
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更新日期:2002-07-01 00:00:00
abstract::Organic compounds exhibit various levels of toxicity toward living organisms based upon their ability to insert into biological membranes and disrupt normal membrane function. The primary mechanism responsible for organic solvent tolerance in many bacteria is energy-dependent extrusion via efflux pumps. One such bacte...
journal_title:Journal of bacteriology
pub_type: 杂志文章
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更新日期:2011-06-01 00:00:00