In Vivo Functional Assay of a Recombinant Aquaporin inPichia pastoris

Abstract

The water channel protein PvTIP3;1 (␣-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.The first water channel gene was cloned serendipitously during the characterization of human blood group antigens and was found to encode a putative channel in the red blood cell plasma membrane. When the protein was expressed in Xenopus laevis oocytes, its function as a water channel was suggested by a concomitant increase in the swelling rate of osmotically shocked cells (41). This demonstration of water channel activity ended decades of speculation that proteins were responsible for the high water permeability observed in certain biological membranes.Over the last decade many organisms have been shown to possess a class of protein channels, termed "aquaporins," which are specialized to facilitate the transcellular movement of water. Aquaporins are members of the major intrinsic protein (MIP) superfamily found in all organisms, from archaebacteria to animals (27). The family progenitor is thought to have arisen from the tandem duplication of a three-transmembrane spanning domain protein with an Asn-Pro-Ala consensus sequence duplicated between the two halves of the protein (45). Generally, aquaporins facilitate the movement of water across cell membranes in response to osmotic gradients, functioning in cellular and organismal osmoregulation and solute transport (5, 32).MIP family proteins are small (ϳ28 kDa) and are usually found as homotetramers. Recent high-resolution structures determined by electron crystallogra...

Two Distinct Aquaporin 0s Required for Development and Transparency of the Zebrafish Lens

“…This protocol was modified from the Pichia pastoris assay developed by Daniels et al 45 Open reading frames of Aqp0a and Aqp0b were cloned into the BamHI or BamHI-HindII site of the pYES2 expression vector (Invitrogen, Carlsbad, CA), respectively. The resultant constructs, as well as an empty pYES2 vector control, were transformed by the lithium acetate method 46 into the W303-1A strain of Saccharomyces cerevisiae (ATCC 208352).…”

“…Changes in cell shape and volume can be determined by photometry, 60 and studies on wild-type P. pastoris spheroplasts showed that changes in optical absorbance were inversely proportional to the changes in external osmolarity. 45 In our assay, spheroplast shrinking in response to hyper-osmotic shock was monitored by measuring ⌬OD 600 between T 0 and T 20sec . Control spheroplasts and spheroplasts expressing nonfunctional water channels shrink significantly over the course of the 20-second experiment (Fig.…”

Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis

“…Overexpression of the recombinant TgPIP2s caused a rapid drop in the OD 600 after osmotic shock, which indicated increasing water influx and bursting of spheroplasts (9). Figure 2 further shows that the treatment of spheroplasts with HgCl 2 , an inhibitor of AQPs, decreased the hypo-osmotic sensitivity of TgPIP2;1-G 3 -H 6 -and TgPIP2;2-G 3 -H 6 -expressing spheroplasts while it did not affect the WCA of the pPICZ-B-containing spheroplasts.…”

“…This might be due to abnormal localization within the PM of oocytes or other undetermined reasons. In addition to the analysis of WCA (9), this study further shows that the factors affecting or regulating the channel activity can be monitored using the P. pastoris expression system. For example, regulatory mechanisms, in particular the pH gating observed only in PIPs (34), could be observed using the spheroplastbursting assay as well as the growth assay ( Fig.…”

Aquaporin Biomimetic Membranes

“…Alternatively, the methylotrophic yeast P. pastoris has been effectively used to produce a high number of distinct aquaporins. The potentially produced selection includes all 13 human aquaporins [40] and a wide range of active plant aquaporins [41][42][43][44][45][46][47]. Large-scale expressions of various functional recombinant aquaporins have been obtained with the aid of a baculovirus/insect cell system [48][49][50][51][52][53][54][55].…”