Expression of heterologous aquaporins for functional analysis in Saccharomyces cerevisiae

In this study the yeast Saccharomyces cerevisiae, which is a genetically tractable model for analysis of osmoregulation, has been used for analysis of heterologous aquaporins. Aquaporin water channels play important roles in the control of water homeostasis in individual cells and multicellular organisms. We have investigated the effects of functional expression of the mammalian aquaporins AQP1 and AQP5 and the aquaglyceroporins AQP3 and AQP9. Expression of aquaporins caused moderate growth inhibition under hyperosmotic stress, while expression of aquaglyceroporins mediated strong growth inhibition due to glycerol loss. Water transport was monitored in protoplasts, where the kinetics of bursting was influenced by presence of aquaporins but not aquaglyceroporins. We observed glycerol transport through aquaglyceroporins, but not aquaporins, in a yeast strain deficient in glycerol production, whose growth depends on glycerol inflow. In addition, a gene reporter assay allowed to indirectly monitor the effect of AQP9-mediated enhanced glycerol loss on osmoadaptation. Transport activity of certain aqua(glycero)porins was diminished by low pH or CuSO 4, suggesting that yeast can potentially be used for screening of putative aquaporin inhibitors. We conclude that yeast is a versatile system for functional studies of aquaporins, and it can be developed to screen for compounds of potential pharmacological use. © Springer-Verlag 2006.

Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen

Aquaporins and aquaglyceroporins mediate the transport of water and solutes across biological membranes. Saccharomyces cerevisiae Fps1 is an aquaglyceroporin that mediates controlled glycerol export during osmoregulation. The transport function of Fps1 is rapidly regulated by osmotic changes in an apparently unique way and distinct regions within the long N- and C-terminal extensions are needed for this regulation. In order to learn more about the mechanisms that control Fps1 we have set up a genetic screen for hyperactive Fps1 and isolated mutations in 14 distinct residues, all facing the inside of the cell. Five of the residues lie within the previously characterized N-terminal regulatory domain and two mutations are located within the approach to the first transmembrane domain. Three mutations cause truncation of the C-terminus, confirming previous studies on the importance of this region for channel control. Furthermore, the novel mutations identify two conserved residues in the channel-forming B-loop as critical for channel control. Structural modelling-based rationalization of the observed mutations supports the notion that the N-terminal regulatory domain and the B-loop could interact in channel control. Our findings provide a framework for further genetic and structural analysis to better understand the mechanism that controls Fps1 function by osmotic changes.

Polyol transport in yeast: studying transport proteins and manipulating osmotic responses

The Saccharomyces cerevisiae MIP channel Fps1p plays an important role in yeast osmoregulation by exporting glycerol. Glycerol accumulates in the cell as a compatible osmolyte during hyperosmotic conditions and is exported once conditions become hypotonic. A gpd1 gpd2 mutant is unable to produce glycerol and is therefore very sensitive to high concentrations of polyols in the growth medium. The sensitivity to C3, C4 and C5, but not C6 polyols, is suppressed by expression of truncated, hyperactive Fps1p. This is because the polyols can then equilibrate over the membrane and hence the concentration gradient collapses. This experiments reveals the substrate spectrum of Fps1p. The system can be used in different ways. For instance, growth assays on different polyols elucidate the substrate range of heterologous channels such as that of the rat aquaglyceroporin AQP9. In addition, the same system is used to search for novel hyperactive mutants of Fps1p, which provide additional information on the mechanism underlying channel regulation. Finally we illustrate that the gpd1 gpd2 double mutant expressing hyperactive Fps1p can be used to manipulate activation and deactivation of the HOG pathway, contributing to our understanding of the control of this osmoregulatory system.

The V-ATPase in Paramecium:functional specialization by multiple gene isoforms

The vacuolar H(+)-ATPase (V-ATPase), a multisubunit, adenosine triphosphate (ATP)-driven proton pump, is essential for numerous cellular processes in all eukaryotes investigated so far. While structure and catalytic mechanism are similar to the evolutionarily related F-type ATPases, the V-ATPase's main function is to establish an electrochemical proton potential across membranes using ATP hydrolysis. The holoenzyme is formed by two subcomplexes, the transmembraneous V(0) and the cytoplasmic V(1) complexes. Sequencing of the whole genome of the ciliate Paramecium tetraurelia enabled the identification of virtually all the genes encoding V-ATPase subunits in this organism and the studying of the localization of the enzyme and roles in membrane trafficking and osmoregulation. Surprisingly, the number of V-ATPase genes in this free-living protozoan is strikingly higher than in any other species previously studied. Especially abundant are V(0)-a-subunits with as many as 17 encoding genes. This abundance creates the possibility of forming a large number of different V-ATPase holoenzymes by combination and has functional consequences by differential targeting to various organelles.

The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles in Paramecium

The vacuolar proton-ATPase (V-ATPase) is a multisubunit enzyme complex that is able to transfer protons over membranes against an electrochemical potential under ATP hydrolysis. The enzyme consists of two subcomplexes: V0, which is membrane embedded; and V1, which is cytosolic. V0 was also reported to be involved in fusion of vacuoles in yeast. We identified six genes encoding c-subunits (proteolipids) of V0 and two genes encoding F-subunits of V1 and studied the role of the V-ATPase in trafficking in Paramecium. Green fluorescent protein (GFP) fusion proteins allowed a clear subcellular localization of c- and F-subunits in the contractile vacuole complex of the osmoregulatory system and in food vacuoles. Several other organelles were also detected, in particular dense core secretory granules (trichocysts). The functional significance of the V-ATPase in Paramecium was investigated by RNA interference (RNAi), using a recently developed feeding method. A novel strategy was used to block the expression of all six c- or both F-subunits simultaneously. The V-ATPase was found to be crucial for osmoregulation, the phagocytotic pathway and the biogenesis of dense core secretory granules. No evidence was found supporting participation of V0 in membrane fusion.