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Applied and Environmental Microbiology, February 2006, p. 1507-1514, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1507-1514.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vivo Functional Assay of a Recombinant Aquaporin in Pichia pastoris

Mark J. Daniels,1 Malcolm R. Wood,1 and Mark Yeager1,2*

Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037,1 Division of Cardiovascular Diseases, Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, California 920372

Received 20 July 2005/ Accepted 23 October 2005

The water channel protein PvTIP3;1 ({alpha}-TIP) is a member of the major intrinsic protein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeast Pichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-type P. pastoris displayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts of P. pastoris expressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of the Escherichia coli glycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity.

* Corresponding author. Mailing address: Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 784-8584. Fax: (858) 784-2504. E-mail: yeager{at}scripps.edu.

Applied and Environmental Microbiology, February 2006, p. 1507-1514, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1507-1514.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Azad, A. K., Sawa, Y., Ishikawa, T., Shibata, H. (2009). Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis. Appl. Environ. Microbiol. 75: 2792-2797 [Abstract] [Full Text]  
  • Azad, A. K., Katsuhara, M., Sawa, Y., Ishikawa, T., Shibata, H. (2008). Characterization of Four Plasma Membrane Aquaporins in Tulip Petals: A Putative Homolog is Regulated by Phosphorylation. Plant Cell Physiol 49: 1196-1208 [Abstract] [Full Text]  


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