Abstract:
:Background information. Transport of water and small neutral solutes across plasma membranes is facilitated by AQP (aquaporin) and aquaglyceroporin channels, which belong to the MIP (major intrinsic protein) family. So far, more than 800 MIP proteins have been identified on the basis of sequence homology, but only less than 10% of them have been functionally characterized. In most studies, the channel properties of MIP proteins have been determined by using Xenopus oocyte swelling assays or stopped-flow spectrophotometry on proteoliposomes. As both methods sometimes present disadvantages, we developed an alternative method for analysing MIP function.Results. The kinetics of plasmolysis or deplasmolysis of Escherichia coli cells in suspension, in response to osmotic challenges, was analysed by stopped-flow spectrophotometry. Cytoplasmic volume variations were monitored either by GFP (green fluorescent protein) fluorescence quenching or by 90 degrees scattered light. The single exponential response to up-shocks in the impermeant solute mannitol was strongly accelerated when the cells expressed the native E. coli AQP AqpZ (rate constant 37.24 versus 3.05 s(-1) for control cells). The responses to hyperosmotic shocks realized with glycerol were biphasic. First, a light-scattering increase corresponded to cell plasmolysis. Secondly, deplasmolysis occurred when glycerol entered into the cell. Both phases were accelerated when the aquaglyceroporin GlpF was present in cell membranes. We concluded that the behaviour of MIP-expressing bacteria in the stopped-flow system was qualitatively identical with that reported for MIP-expressing oocytes or MIP-containing proteoliposomes. We then used this system to analyse the effects of mutations in the pore constriction of Gla(Llac), the aquaglyceroporin from Lactococcus lactis. In the present study, we show that Gla(Llac) loses its ability to transport glycerol but retains its ability to transport water when Val(223) was replaced by a histidine, the residue at the equivalent position in strict AQPs.Conclusions. These results show that stopped-flow spectrophotometry performed on E. coli cell suspensions is a useful experimental system to analyse the selectivity of wild-type or mutant MIP proteins and that a bifunctional aquaglyceroporin switches to an AQP by a single amino acid mutation in the pore constriction.
journal_name
Biol Celljournal_title
Biology of the cellauthors
Hubert JF,Duchesne L,Delamarche C,Vaysse A,Gueuné H,Raguénès-Nicol Cdoi
10.1042/BC20040125subject
Has Abstractpub_date
2005-09-01 00:00:00pages
675-86issue
9eissn
0248-4900issn
1768-322Xpii
BC20040125journal_volume
97pub_type
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journal_title:Biology of the cell
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journal_title:Biology of the cell
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journal_title:Biology of the cell
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journal_title:Biology of the cell
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journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1111/j.1768-322x.1995.tb00943.x
更新日期:1995-01-01 00:00:00
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journal_title:Biology of the cell
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journal_title:Biology of the cell
pub_type: 杂志文章
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更新日期:1984-01-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
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更新日期:2006-08-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
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更新日期:2004-03-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1111/j.1768-322x.1990.tb00007.x
更新日期:1990-01-01 00:00:00
abstract::Multidrug-resistant (MDR) variants of a human osteosarcoma cell line (U-2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P-glycoprotein. In this work, the morpho...
journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1016/0248-4900(96)89429-9
更新日期:1995-01-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1042/BC20060060
更新日期:2007-02-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
doi:
更新日期:1998-12-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章,评审
doi:10.1111/boc.201600053
更新日期:2017-03-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1016/0248-4900(93)90129-3
更新日期:1993-01-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章,评审
doi:10.1042/BC20050058
更新日期:2006-03-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章,评审
doi:10.1111/j.1768-322x.1994.tb00932.x
更新日期:1994-01-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章,评审
doi:10.1016/j.biolcel.2004.03.008
更新日期:2004-08-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1111/boc.201400063
更新日期:2015-05-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1016/s0248-4900(02)01194-2
更新日期:2002-09-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1042/BC20060012
更新日期:2006-10-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1016/0248-4900(90)90340-9
更新日期:1990-01-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1016/0248-4900(92)90133-l
更新日期:1992-01-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1016/0248-4900(90)90349-8
更新日期:1990-01-01 00:00:00
abstract::The distribution of viral RNA molecules in HeLa cells infected with adenovirus type 5 (Ad5) was determined by in situ hybridization at the ultrastructural level at an intermediate stage of nuclear transformation, when viral DNA synthetic activities were maximal but progeny viruses were still sparse. Transcription site...
journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1016/0248-4900(91)90060-z
更新日期:1991-01-01 00:00:00
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journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1016/s0248-4900(02)01220-0
更新日期:2003-01-01 00:00:00
abstract::Cilia from quail oviduct were isolated with their membrane. The ultrastructural study revealed a good preservation of cilia in the purified fraction. Electrophoresis on SDS-PAGE showed a reproducible pattern of ciliary proteins, the major bands being those of tubulins 57 kDa and dyneins above 250 kDa. Among the minor ...
journal_title:Biology of the cell
pub_type: 杂志文章
doi:10.1111/j.1768-322x.1986.tb00487.x
更新日期:1986-01-01 00:00:00