Abstract:
:In Escherichia coli, the GlpT transporter, a member of the major facilitator superfamily, moves external glycerol 3-phosphate (G3P) into the cytoplasm in exchange for cytoplasmic phosphate. Study of intact cells showed that both GlpT and HisGlpT, a variant with an N-terminal six-histidine tag, are inhibited (50% inhibitory concentration approximately 35 microM) by the hydrophilic thiol-specific agent p-mercurichlorobenzosulfonate (PCMBS) in a substrate-protectable fashion; by contrast, two other thiol-directed probes, N-maleimidylpropionylbiocytin (MPB) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), have no effect. Use of variants in which the HisGlpT native cysteines are replaced individually by serine or glycine implicates Cys-176, on transmembrane helix 5 (TM5), as the major target for PCMBS. The inhibitor sensitivity of purified and reconstituted HisGlpT or its cysteine substitution derivatives was found to be consistent with the findings with intact cells, except that a partial response to PCMBS was found for the C176G mutant, suggesting the presence of a mixed population of both right-side-out (RSO) (resistant) and inside-out (ISO) (sensitive) orientations after reconstitution. To clarify this issue, we studied a derivative (P290C) in which the RSO molecules can be blocked independently due to an MPB-responsive cysteine in an extracellular loop. In this derivative, comparisons of variants with (P290C) and without (P290C/C176G) Cys-176 indicated that this residue shows substrate-protectable inhibition by PCMBS in the ISO orientation in proteoliposomes. Since PCMBS gains access to Cys-176 from both periplasmic and cytoplasmic surfaces of the protein (in intact cells and in a reconstituted ISO orientation, respectively) and since access is unavailable when the substrate is present, we propose that Cys-176 is located on the transport pathway and that TM5 has a role in lining this pathway.
journal_name
J Bacterioljournal_title
Journal of bacteriologyauthors
Fann MC,Busch A,Maloney PCdoi
10.1128/jb.185.13.3863-3870.2003keywords:
subject
Has Abstractpub_date
2003-07-01 00:00:00pages
3863-70issue
13eissn
0021-9193issn
1098-5530journal_volume
185pub_type
杂志文章abstract::The mechanism for the extrusion of Na+ from Mycoplasma gallisepticum cells was examined. Na+ efflux from cells was studied by diluting 22Na+-loaded cells into an isoosmotic NaCl solution and measuring the residual 22Na+ in the cells. Uphill 22Na+ efflux was found to be glucose dependent and linear with time over a 60-...
journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.171.8.4410-4416.1989
更新日期:1989-08-01 00:00:00
abstract::The yeast two-hybrid assay revealed that Bacillus subtilis DnaD, a possible component of the primosome and required for replication initiation, interacted with DnaA and DnaD itself. The mutant DnaD23 was incapable of interacting with DnaA but retained interaction with the wild-type DnaD. These results suggest that int...
journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.183.6.2148-2150.2001
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journal_title:Journal of bacteriology
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doi:10.1128/jb.176.18.5861-5863.1994
更新日期:1994-09-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.175.24.7918-7930.1993
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journal_title:Journal of bacteriology
pub_type: 杂志文章
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doi:10.1128/JB.124.2.843-848.1975
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doi:10.1128/JB.00782-07
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.175.24.8018-8023.1993
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.100.3.1393-1401.1969
更新日期:1969-12-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.178.23.6665-6670.1996
更新日期:1996-12-01 00:00:00
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pub_type: 杂志文章
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更新日期:2004-05-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.164.2.782-796.1985
更新日期:1985-11-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.171.5.2415-2423.1989
更新日期:1989-05-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.172.9.5360-5367.1990
更新日期:1990-09-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.178.12.3447-3456.1996
更新日期:1996-06-01 00:00:00
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pub_type: 杂志文章
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journal_title:Journal of bacteriology
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doi:10.1128/JB.180.9.2387-2394.1998
更新日期:1998-05-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.151.1.48-57.1982
更新日期:1982-07-01 00:00:00
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journal_title:Journal of bacteriology
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pub_type: 杂志文章
doi:10.1128/JB.123.2.400-406.1975
更新日期:1975-08-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.170.6.2763-2769.1988
更新日期:1988-06-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.171.3.1340-1345.1989
更新日期:1989-03-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.102.2.334-340.1970
更新日期:1970-05-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.176.24.7653-7658.1994
更新日期:1994-12-01 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:2001-03-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.176.17.5513-5524.1994
更新日期:1994-09-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/JB.01850-07
更新日期:2008-05-01 00:00:00
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journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.185.12.3527-3537.2003
更新日期:2003-06-01 00:00:00
abstract::Several mycobacterial strains, such as Mycobacterium flavescens, Mycobacterium gastri, Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sol...
journal_title:Journal of bacteriology
pub_type: 杂志文章
doi:10.1128/jb.185.1.142-147.2003
更新日期:2003-01-01 00:00:00