Publications by authors named "Xin-Gang Guan"

2 Publications

  • Page 1 of 1

NPA motifs play a key role in plasma membrane targeting of aquaporin-4.

IUBMB Life 2010 Mar;62(3):222-6

Northeast Normal University, Changchun, China.

The two highly conserved NPA motifs (asparagine-proline-alanine, NPA) are the most important structural domains that play a crucial role in water-selective permeation in aquaporin water channels. However, the functions of NPA motifs in aquaporin (AQP) biogenesis remain largely unknown. Few AQP members with variations in NPA motifs such as AQP11 and AQP12 do not express in the plasma membrane, suggesting an important role of NPA motifs in AQP plasma membrane targeting. In this study, we examined the role of the two NPA motifs in AQP4 plasma membrane targeting by mutagenesis. We constructed a series of AQP4 mutants with NPA deletions or single amino acid substitutions in AQP4-M1 and AQP4-M23 isoforms and analyzed their expression patterns in transiently transfected FRT and COS-7 cells. Western blot analysis showed similar protein bands of all the AQP4 mutants and the wild-type AQP4. AQP4 immunofluorescence indicated that deletion of one or both NPA motifs resulted in defective plasma membrane targeting, with apparent retention in endoplasmic reticulum (ER). The A99T mutant mimicking AQP12 results in ER retention, whereas the A99C mutant mimicking AQP11 expresses normally in plasma membrane. Furthermore, the AQP4-M1 but not the M23 isoform with P98A substitution in the first NPA motif can target to the plasma membrane, indicating an interaction of N-terminal sequence of AQP4-M1 with the first NPA motif. These results suggest that NPA motifs play a key role in plasma membrane expression of AQP4 but are not involved in AQP4 protein synthesis and degradation. The NPA motifs may interact with other structural domains in the regulation of membrane trafficking during aquaporin biogenesis.
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http://dx.doi.org/10.1002/iub.311DOI Listing
March 2010

[Expression of glutathione S-transferase-aquaporin 1 carboxyl terminal domain fusion protein in Escherchia coli].

Fen Zi Xi Bao Sheng Wu Xue Bao 2008 Feb;41(1):81-5

Membrane Channel Research Laboratory, School of Life Sciences, Northeast Normal University, Changchun 130024.

We constructed a recombinant plasmid of water channel protein Aquaporin 1 (AQP1) carboxyl terminal domain (DNA sequence from 700bp-801bp) in pGEX-4T-1 vector and express the carboxyl terminal hydrophilic peptide AQP1 in E. coli. In this study, the DNA sequence of AQP1 hydrophilic peptide was amplified by PCR and was cloned into pGEX-4T-1 expression vector. After identified by restriction enzyme digestion and sequencing, the recombinant clone was transformed into the competent expression cells of E. coli BL21. The GST-AQP1 fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain a fusion protein with molecular weight of 30KD. So the fusion protein of AQP1 C-terminal hydrophilic peptide combined with GST was successfully expressed and purified. We set up important bases for the further research in AQP1 gene function.
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February 2008
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