A short regulatory domain restricts glycerol transport through yeast Fps1p

The controlled export of solutes is crucial for cellular adaptation to hypotonic conditions. In the yeast Saccharomyces cerevisiae glycerol export is mediated by Fpslp, a member of the major intrinsic protein (MIP) family ]of channel proteins. Here we describe a short regulatory domain that restricts glycerol transport through Fpslp. This domain is required for retention of cellular glycerol under hypertonic stress and hence acquisition of osmotolerance. It is located in the N-terminal cytoplasmic extension close to the first transmembrane domain. Several residues within that domain and its precise position are critical for channel control while the proximal residues 13-215 of the N-terminal extension are not required. The sequence of the regulatory domain and its position are perfectly conserved in orthologs from other yeast species. The regulatory domain has an amphiphilic character, and structural predictions indicate that it could fold back into the membrane bilayer. Remarkably, this domain has structural similarity to the channel forming loops B and E of Fpslp and other glycerol facilitators. Intragenic second-site suppressor mutations of the sensitivity to high osmolarity conferred by truncation of the regulatory domain caused diminished glycerol transport, confirming that elevated channel activity is the cause of the osmosensitive phenotype

Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p

Fps1p is a glycerol efflux channel from Saccharomyces cerevisiae. In this atypical major intrinsic protein neither of the signature NPA motifs of the family, which are part of the pore, is preserved. To understand the functional consequences of this feature, we analyzed the pseudo-NPA motifs of Fps1p by site-directed mutagenesis and assayed the resultant mutant proteins in vivo. In addition, we took advantage of the fact that the closest bacterial homolog of Fps1p, Escherichia coli GlpF, can be functionally expressed in yeast, thus enabling the analysis in yeast cells of mutations that make this typical major intrinsic protein more similar to Fps1p. We observed that mutations made in Fps1p to "restore" the signature NPA motifs did not substantially affect channel function. In contrast, when GlpF was mutated to resemble Fps1p, all mutants had reduced activity compared with wild type. We rationalized these data by constructing models of one GlpF mutant and of the transmembrane core of Fps1p. Our model predicts that the pore of Fps1p is more flexible than that of GlpF. We discuss the fact that this may accommodate the divergent NPA motifs of Fps1p and that the different pore structures of Fps1p and GlpF may reflect the physiological roles of the two glycerol facilitators

Scalable In Situ Hybridization on Tissue Arrays for Validation of Novel Cancer and Tissue-Specific Biomarkers

Tissue localization of gene expression is increasingly important for accurate interpretation of large scale datasets from expression and mutational analyses. To this end, we have (1) developed a robust and scalable procedure for generation of mRNA hybridization probes, providing >95% first-pass success rate in probe generation to any human target gene and (2) adopted an automated staining procedure for analyses of formalin-fixed paraffin-embedded tissues and tissue microarrays. The in situ mRNA and protein expression patterns for genes with known as well as unknown tissue expression patterns were analyzed in normal and malignant tissues to assess procedure specificity and whether in situ hybridization can be used for validating novel antibodies. We demonstrate concordance between in situ transcript and protein expression patterns of the well-known pathology biomarkers KRT17, CHGA, MKI67, PECAM1 and VIL1, and provide independent validation for novel antibodies to the biomarkers BRD1, EZH2, JUP and SATB2. The present study provides a foundation for comprehensive in situ gene set or transcriptome analyses of human normal and tumor tissues

Using yeast to study transport and structure-function relationship in aquaglyceroporins

Aquaporins are small membrane proteins that transport waterwhile the closely related aquaglyceroporins also can be permeableto polyols, urea and even arsenic. These substances can pass thepore in both directions by facilitated diffusion. Aquaporins arerepresented in organisms ranging from archaea to human, andtheir discovery was awarded the Nobel Prize in chemistry in2003. Eleven different aquaporins (0–10) have been identified inmammals. Of these, AQP3, 7, 9 and 10 are aquaglyceroporins.They are expressed in a tissue-specific manner and play key rolesAbstracts196in the regulation of water balance. Examples for relevant applicationsare transpiration, water retention in the kidneys and glyceroltransport following fat metabolism. Aquaglyceroporins areprobably also an entry point for arsenic in the liver. To study thefunction of aquaglyceroporins, we have developed a test systemin Saccharomyces cerevisiae. When exposed to a hyperosmoticstress, yeast cells uses glycerol as a compatible solute to regainthe turgor pressure decreased by water loss. When aquaglyceroporinsare expressed in such cells, they cause sensitivity tohyperosmotic stress, due to glycerol loss through the aquaglyceroporins.When expressed in a strain deficient in glycerol production,the sensitivity of that strain to high levels of certain polyolsis suppressed because the polyol can be taken up by the cellthrough the aquaglyceroporin. We have employed this system ofconditional osmotic shock to design a genetic screen which hasmade it possible to identify residues responsible for the regulationof the yeast aquaglyceroporin Fps1. The genetic screen is beingfurther developed to unravel key residues in channel specificity aswell as the mode of action of potential inhibitors. We are alsousing this system to study mammalian aquaglyceroporins

Turgor restoration: the critical event in signal cessation of the HOG pathway

Men of the Hour as told in cartoon and verse in the Lewiston Journal 1907.https://digitalmaine.com/men_of_the_hour/1031/thumbnail.jp