The PvTIP3;1 gene was subcloned into the pPICZ Pichia
expression vector (20
) and modified to include a carboxy-terminal extension of three glycine and six histidine residues to facilitate purification by immobilized-metal affinity chromatography. The final gene construct (TIP3;1-G3-H6
) was linearized and integrated into the yeast genome by homologous recombination, and recombinant yeast were selected by antibiotic screening. Since gene expression was under the control of the AOX1
alcohol oxidase promoter, protein production was induced by switching to methanol as the sole carbon source in the growth medium. Cells were harvested 25 to 45 h after induction. Immunocytochemistry on ultrathin cryosections of aquaporin-expressing P. pastoris
showed that the expressed TIP3;1-G3
was located in the plasma membrane, as well as in vacuolar and other intracellular membranes (Fig. ). Curiously, the growth rate of yeast expressing the recombinant protein was ~35% of the wild-type rate (data not shown).
FIG. 1. Immunogold labeling of TIP3;1-G3-H6 in cryosections of wild-type KM71H P. pastoris and aquaporin-expressing KM71H P. pastoris. (A) Section of wild-type KM71H P. pastoris. (B) Section of KM71H P. pastoris expressing the TIP3;1-G3-H6 aquaporin. (C) Enlargement (more ...)
In order to verify that the overexpressed protein was functional and therefore properly folded, we developed an osmotic shock assay to measure aquaporin-mediated osmotic water permeability. We reasoned that the rapidity of osmotically induced swelling in aquaporin-expressing yeast would cause the cells to lyse more readily than wild-type cells. In order to test this prediction, the optical absorbances of osmotically shocked TIP3;1-G3-H6-expressing and wild-type P. pastoris cultures were measured and compared (Fig. ). To allow cells to change volume freely, P. pastoris cultures were treated with β-1,3-glucanase to produce spheroplasts lacking a cell wall. Sorbitol was used as an osmotic protectant.
FIG. 2. Pichia in vivo water permeability assay. Comparison of absorbance with osmotic shock gradient (change in M sorbitol) for TIP3;1-G3-H6-expressing yeast and the control, parent yeast strain KM71H. Where indicated, the assay was performed in the presence (more ...)
When subjected to an osmotic shock, wild-type yeast cultures exhibited a change in optical absorbance inversely proportional to the change in external osmolarity (Fig. ). For P. pastoris cultures expressing the TIP3;1-G3-H6 aquaporin, this linear correlation broke down under hypotonic conditions. Compared to wild-type yeast, there was an overall decrease in optical absorbance with hypo-osmotic shocks (Fig. ). Differences in the osmotic shock response between wild-type and recombinant yeast cells were concomitant with cellular accumulation of TIP3;1-G3-H6 (Fig. ). When the cell wall was not enzymatically degraded prior to the osmotic shock, wild-type and aquaporin-expressing yeasts behaved identically (data not shown). Significantly, addition of the aquaporin inhibitor mercury chloride to 3 mM did not affect the osmotic sensitivity of wild-type P. pastoris but restored the linear relationship for TIP3;1-G3-H6 expressing yeast (Fig. ). Concentrations of mercury chloride down to 0.2 mM also produced this effect, to a lesser extent (data not shown).
FIG. 3. Time course of the hypo-osmotic shock response in P. pastoris expressing TIP3;1-G3-H6 and wild-type yeast. Differences in the optical absorbance between recombinant (A) and wild-type (B) yeast spheroplasts resulting from hypo-osmotic shock correspond (more ...)
We assume that the optical absorbance of a P. pastoris spheroplast suspension exposed to a hypo-osmotic shock will either not increase or show a decrease if the cells lyse as a result of the shock. Following a hypotonic shock (1.0 to 0.1 M sorbitol), the optical absorbance (Fig. ) and the cell count (Table ) of untransformed P. pastoris increased by 26% and 20%, respectively. However, for TIP3;1-G3-H6 expressing P. pastoris, the optical absorbance and cell count decreased by 8.5% and 13%, respectively. By comparison, when either untransformed or recombinant yeasts were hypo-osmotically shocked and then plated on solid growth medium, the number of yeast colonies was proportional to the cell count (data not shown).
Cell counts of aquaporin-expressing P. pastoris cultures decrease significantly following hypotonic shock
Untransformed P. pastoris showed a slight increase in optical absorbance and cell number following a hypotonic shock (Table ). This effect may be caused by minor cell aggregation prior to the shock, or following the shock, either by separation of daughter cells from parent yeast or by the production of cellular ghosts, released organelles and resealed vesicles. When cells were not treated with yeast lytic enzyme prior to the osmotic shock, no difference was observed between transformed and untransformed cells (data not shown), indicating that lyticase activity is required for this assay to distinguish between aquaporin-expressing and control P. pastoris cells.
Changes in optical absorbance that occurred with osmotic shock required removal of the cell wall with yeast lytic enzyme. The effects of yeast cell wall-digesting enzymes on P. pastoris
are not well understood, and yeast species can vary widely in their sensitivity to yeast lytic enzyme (24
). Furthermore, Saccharomyces cerevisiae
spheroplasts prepared by lyticase treatment exhibited a resistance to lysis that was dependent on growth conditions (2
) and could show an increase in optical absorbance during lysis (26
). Consequently, in order to reduce variability in yeast lytic enzyme sensitivity and protoplasting efficiency, the same strain of P. pastoris
and the same growth medium were used in all experiments.
It is possible that in vivo overexpression of an integral membrane protein could disrupt the integrity or fluidity of the plasma membrane and cause anomalous responses to an extracellular osmotic shock. This was tested by generating recombinant P. pastoris
that overexpressed the homologous (11
) E. coli
glycerol channel GlpF, which is a weak water channel (35
). The behavior of GlpF-expressing yeast in response to a hypo-osmotic shock was similar to that of wild-type P. pastoris
(Fig. ). Shifts in the range of optical absorbance between the experiments shown in Fig. and represented differences in averaged cell density of the yeast populations used for each set of experiments.
FIG. 4. Comparison of aquaporin-expressing yeast and glycerol channel-expressing yeast using the Pichia in vivo water permeability assay. (A) Relationship of optical absorbance with osmotic shock gradient (change in sorbitol concentration) for TIP3;1-G3-H6-expressing (more ...)
Immunoblot analysis showed that TIP3;1-G3
accumulated over time as a protein of ~25 kDa (Fig. ). The protein was first detected ~4 h after induction, which corresponded to significant differences in the hypo-osmotic shock behavior between wild-type and recombinant yeast spheroplasts (Fig. ). After 24 h of induction, aquaporin-expressing P. pastoris
showed a −14 to −28% decrease in optical absorbance (Fig. ), whereas wild-type yeast showed a slight increase in optical absorbance. This result parallels what is observed in our osmotic shock assay in which absorbance is measured 24 to 40 h after induction (Fig. and Fig. ). It was noted that the osmotic shock response of recombinant yeast was not proportional to the amount of aquaporin present. This observation can be explained by the fact that the water channel activity of PvTIP3;1 is regulated by phosphorylation as well as abundance (34
). Consequently, the P. pastoris
water permeability assay will not show a linear correlation between abundance and activity. Rather, TIP3;1-G3
aquaporin activity will fluctuate according to the level of yeast kinase activity, which is highly dependent on cellular metabolic state and which will vary during the course of incubation (53
). A fraction of the protein expressed in P. pastoris
is appropriately phosphorylated (8
) and hence active, which would account for the observed water channel activity.