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.

Novel characteristics of the biological properties of the yeast Saccharomyces cerevisiae eukaryotic initiation factor 2A

Eukaryotic initiation factor 2A (eIF2A) has been shown to direct binding of the initiator methionyl-tRNA (Met-tRNA(i)) to 40 S ribosomal subunits in a codon-dependent manner, in contrast to eIF2, which requires GTP but not the AUG codon to bind initiator tRNA to 40 S subunits. We show here that yeast eIF2A genetically interacts with initiation factor eIF4E, suggesting that both proteins function in the same pathway. The double eIF2A/eIF4E-ts mutant strain displays a severe slow growth phenotype, which correlated with the accumulation of 85% of the double mutant cells arrested at the G(2)/M border. These cells also exhibited a disorganized actin cytoskeleton and elevated actin levels, suggesting that eIF2A might be involved in controlling the expression of genes involved in morphogenic processes. Further insights into eIF2A function were gained from the studies of eIF2A distribution in ribosomal fractions obtained from either an eIF5BDelta (fun12Delta) strain or a eIF3b-ts (prt1-1) strain. It was found that the binding of eIF2A to 40 and 80 S ribosomes was not impaired in either strain. We also found that eIF2A functions as a suppressor of Ure2p internal ribosome entry site-mediated translation in yeast cells. The regulation of expression from the URE2 internal ribosome entry site appears to be through the levels of eIF2A protein, which has been found to be inherently unstable with a half-life of approximately 17 min. It was hypothesized that this instability allows for translational control through the level of eIF2A protein in yeast cells.

Apodisation of photo-induced waveguide gratings using double-exposure with complementary duty cycles

We present a novel apodisation scheme for photo-induced waveguide gratings. The apodisation is implemented with double exposures that have reversely varying duty cycles. We have successfully applied the proposed scheme to remove the sidelobes of long period gratings (LPGs). We also observed for the first time super strong sidelobes in LPGs when creating them with only a single varying-duty-cycle exposure. The strong sidelobes can be well explained with a Mach-Zehnder interference model.

Apodisation of photo-induced waveguide gratings using double-exposure with complementary duty cycles

We present a novel apodisation scheme for photo-induced waveguide gratings. The apodisation is implemented with double exposures that have reversely varying duty cycles. We have successfully applied the proposed scheme to remove the sidelobes of long period gratings (LPGs). We also observed for the first time super strong sidelobes in LPGs when creating them with only a single varying-duty-cycle exposure. The strong sidelobes can be well explained with a Mach-Zehnder interference model.

Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression

Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid derived from the modification of lysine by spermidine. Two genes, TIF51A and TIF51B, encode eIF5A in the yeast Saccharomyces cerevisiae. In an effort to understand the structure-function relationship of eIF5A, we have generated yeast mutants by introducing plasmid-borne tif51A into a double null strain where both TIF51A and TIF51B have been disrupted. One of the mutants, tsL102A strain (tif51A L102A tif51aDelta tif51bDelta) exhibits a strong temperature-sensitive growth phenotype. At the restrictive temperature, tsL102A strain also exhibits a cell shape change, a lack of volume change in response to temperature increase and becomes more sensitive to ethanol, a hallmark of defects in the PKC/WSC cell wall integrity pathway. In addition, a striking change in actin dynamics and a complete cell cycle arrest at G1 phase occur in tsL102A cells at restrictive temperature. The temperature-sensitivity of tsL102A strain is due to a rapid loss of mutant eIF5A with the half-life reduced from 6 h at permissive temperature to 20 min at restrictive temperature. Phenylmethyl sulfonylfluoride (PMSF), an irreversible inhibitor of serine protease, inhibited the degradation of mutant eIF5A and suppressed the temperature-sensitive growth arrest. Sorbitol, an osmotic stabilizer that complement defects in PKC/WSC pathways, stabilizes the mutant eIF5A and suppresses all the observed temperature-sensitive phenotypes.