Dependent on phosphate availability the yeast Saccharomyces cerevisiae expresses either low or high affinity phosphate transporters. In the presence of phosphate yeast cells still express low... Show moreDependent on phosphate availability the yeast Saccharomyces cerevisiae expresses either low or high affinity phosphate transporters. In the presence of phosphate yeast cells still express low levels of the high affinity phosphate transporter Pho84. The regulator Spl2 is expressed in approximately 90% of the cells, and is not expressed in the remaining cells. Here we report that deletion of RRP6, encoding an exonuclease degrading non-coding RNA, or BMH1, encoding the major 14-3-3 isoform, resulted in less cells expressing SPL2 and in increased levels of RNA transcribed from sequences upstream of the SPL2 coding region. SPL2 stimulates its own expression and that of PHO84 ensuing a positive feedback. Upon deletion of the region responsible for upstream SPL2 transcription almost all cells express SPL2. These results indicate that the cell-to-cell variation in PHO84 and SPL2 expression is dependent on a specific part of the SPL2 promoter and is controlled by Bmh1 and Spl2. Show less
Agrobacterium tumefaciens is the etiological agent of plant crown gall disease, which is induced by the delivery of a set of oncogenic genes into plant cells from its tumor-inducing (Ti) plasmid.... Show moreAgrobacterium tumefaciens is the etiological agent of plant crown gall disease, which is induced by the delivery of a set of oncogenic genes into plant cells from its tumor-inducing (Ti) plasmid. Here we present the first complete sequence of a succinamopine-type Ti-plasmid. Plasmid pTiEU6 is comprised of 176,375 bp with an overall GC content of 56.1% and 195 putative protein-coding sequences could be identified. This Ti-plasmid is most closely related to nopaline-type Ti-plasmids. It contains a single T-region which is somewhat smaller than that of the nopaline-type Ti-plasmids and in which the gene for nopaline synthesis is replaced by a gene (sus) for succinamopine synthesis. Also in pTiEU6 the nopaline catabolic genes are replaced by genes for succinamopine catabolism. In order to trace the evolutionary origin of pTiEU6, we sequenced six nopaline Ti-plasmids to enlarge the scope for comparison to this class of plasmids. Average nucleotide identity analysis revealed that pTiEU6 was most closely related to nopaline Ti-plasmids pTiT37 and pTiSAKURA. In line with this traces of several transposable elements were present in all the nopaline Ti plasmids and in pTiEU6, but one specific transposable element insertion, that of a copy of IS1182, was present at the same site only in pTiEU6, pTiT37, and pTiSAKURA, but not in the other Ti plasmids. This suggests that pTiEU6 evolved after diversification of nopaline Ti-plasmids by DNA recombination between a pTiT37-like nopaline Ti-plasmid and another plasmid, thus introducing amongst others new catabolic genes matching a new opine synthase gene for succinamopine synthesis. Show less
Agrobacterium tumefaciens can genetically transform plants by translocating a piece of oncogenic DNA, called T‐DNA, into host cells. Transfer is mediated by a type IV secretion system (T4SS).... Show moreAgrobacterium tumefaciens can genetically transform plants by translocating a piece of oncogenic DNA, called T‐DNA, into host cells. Transfer is mediated by a type IV secretion system (T4SS). Besides the T‐DNA, which is transferred in a single‐stranded form and at its 5′ end covalently bound to VirD2, several other effector proteins (VirE2, VirE3, VirD5, and VirF) are translocated into the host cells. The fate and function of the translocated proteins inside the host cell are only partly known. Therefore, several studies were conducted to visualize the translocation of the VirE2 protein. As GFP‐tagged effector proteins are unable to pass the T4SS, other approaches like the split GFP system were used, but these require specific transgenic recipient cells expressing the complementary part of GFP. Here, we investigated whether use can be made of the photostable variant of LOV, phiLOV2.1, to visualize effector protein translocation from Agrobacterium to non‐transgenic yeast and plant cells. We were able to visualize the translocation of all five effector proteins, both to yeast cells, and to cells in Nicotiana tabacum leaves and Arabidopsis thaliana roots. Clear signals were obtained that are easily distinguishable from the background, even in cases in which by comparison the split GFP system did not generate a signal. Show less
Ion homeostasis is an essential property of living organisms. The yeast Saccharomyces cerevisiae is an ideal model organism to investigate ion homeostasis at all levels. In this yeast genes... Show moreIon homeostasis is an essential property of living organisms. The yeast Saccharomyces cerevisiae is an ideal model organism to investigate ion homeostasis at all levels. In this yeast genes involved in high-affinity phosphate uptake (PHO genes) are strongly induced during both phosphate and potassium starvation, indicating a link between phosphate and potassium homeostasis. However, the signal transduction processes involved are not completely understood. As 14-3-3 proteins are key regulators of signal transduction processes, we investigated the effect of deletion of the 14-3-3 genes BMH1 or BMH2 on gene expression during potassium starvation and focused especially on the expression of genes involved in phosphate uptake. Genome-wide analysis of the effect of disruption of either BMH1 or BMH2 revealed that the mRNA levels of the PHO genes PHO84 and SPL2 are greatly reduced in the mutant strains compared to the levels in wild type strains. This was especially apparent at standard potassium and phosphate concentrations. Furthermore the promoter of these genes is less active after deletion of BMH1. Microscopic and flow cytometric analysis of cells with GFP-tagged SPL2 showed that disruption of BMH1 resulted in two populations of genetically identical cells, cells expressing the protein and the majority of cells with no detectible expression. Heterogeneity was also observed for the expression of GFP under control of the PHO84 promoter. Upon deletion of PHO80 encoding a regulator of the transcription factor Pho4, the effect of the BMH1 deletion on SPL2 and PHO84 promoter was lost, suggesting that the BMH1 deletion mainly influences processes upstream of the Pho4 transcription factor. Our data indicate that that yeast cells can be in either of two states, expressing or not expressing genes required for high-affinity phosphate uptake and that 14-3-3 proteins are involved in the process(es) that establish the activation state of the PHO regulon. BACKGROUND RESULTS CONCLUSION Show less
Cell viability requires adaptation to changing environmental conditions. Ubiquitin-mediated endocytosis plays a crucial role in this process, because it provides a mechanism to remove transport... Show moreCell viability requires adaptation to changing environmental conditions. Ubiquitin-mediated endocytosis plays a crucial role in this process, because it provides a mechanism to remove transport proteins from the membrane. Arrestin-related trafficking proteins are important regulators of the endocytic pathway in yeast, facilitating selective ubiquitylation of target proteins by the E3 ubiquitin ligase, Rsp5. Specifically, Rod1 (Art4) has been reported to regulate the endocytosis of both the Hxt1, Hxt3, and Hxt6 glucose transporters and the Jen1 lactate transporter. Also, the AMP kinase homologue, Snf1, and 14-3-3 proteins have been shown to regulate Jen1 via Rod1. Here, we further characterized the role of Rod1, Snf1, and 14-3-3 in the signal transduction route involved in the endocytic regulation of the Hxt6 high affinity glucose transporter by showing that Snf1 interacts specifically with Rod1 and Rog3 (Art7), that the interaction between the Bmh2 and several arrestin-related trafficking proteins may be modulated by carbon source, and that both the 14-3-3 protein Bmh2 and the Snf1 regulatory domain interact with the arrestin-like domain containing the N-terminal half of Rod1 (amino acids 1–395). Finally, using both co-immunoprecipitation and bimolecular fluorescence complementation, we demonstrated the interaction of Rod1 with Hxt6 and showed that the localization of the Rod1-Hxt6 complex at the plasma membrane is affected by carbon source and is reduced upon overexpression of SNF1 and BMH2. Show less
During Agrobacterium tumefaciens-mediated transformation of plant cells a part of the tumour-inducing plasmid, T-DNA, is integrated into the host genome. In addition, a number of virulence proteins... Show moreDuring Agrobacterium tumefaciens-mediated transformation of plant cells a part of the tumour-inducing plasmid, T-DNA, is integrated into the host genome. In addition, a number of virulence proteins are translocated into the host cell. The virulence protein VirE3 binds to the Arabidopsis thaliana pBrp protein, a plant-specific general transcription factor of the TFIIB family. To study a possible role for VirE3 in transcriptional regulation, we stably expressed virE3 in A. thaliana under control of a tamoxifen-inducible promoter. By RNA sequencing we showed that upon expression of virE3 the RNA levels of 607 genes were increased more than three-fold and those of 132 genes decreased more than three-fold. One of the strongly activated genes was that encoding VBF (At1G56250), an F-box protein that may affect the levels of the VirE2 and VIP1 proteins. Using Arabidopsis cell suspension protoplasts we showed that VirE3 stimulates the VBF promoter, especially when co-expressed with pBrp. Although pBrp is localized at the external surface of plastids, co-expression of VirE3 and pBrp in Arabidopsis cell suspension protoplasts resulted in the accumulation of pBrp in the nucleus. Our results suggest that VirE3 affects the transcriptional machinery of the host cell to favour the transformation process. Show less
Agrobacterium tumefaciens is a Gram-negative soil bacterium that genetically transforms plants and, under laboratory conditions, also transforms non-plant organisms, such as fungi and yeasts.... Show moreAgrobacterium tumefaciens is a Gram-negative soil bacterium that genetically transforms plants and, under laboratory conditions, also transforms non-plant organisms, such as fungi and yeasts. During the transformation process a piece of ssDNA (T-strand) is transferred into the host cells via a type IV secretion system. The VirD2 relaxase protein, which is covalently attached at the 5' end of the T-strand through Tyr29, mediates nuclear entry as it contains a nuclear localization sequence. How the T-strand reaches the chromatin and becomes integrated in the chromosomal DNA is still far from clear. Here, we investigated whether VirD2 binds to histone proteins in the yeast Saccharomyces cerevisiae. Using immobilized GFP-VirD2 and in vitro synthesized His6-tagged S. cerevisiae proteins, interactions between VirD2 and the histones H2A, H2B, H3 and H4 were revealed. In vivo, these interactions were confirmed by bimolecular fluorescence complementation experiments. After co-cultivation of Agrobacterium strains expressing VirD2 tagged with a fragment of the yellow fluorescent protein analogue Venus with yeast strains expressing histone H2A or H2B tagged with the complementary part of Venus, fluorescence was detected in dot-shaped structures in the recipient yeast cells. The results indicated that VirD2 was transferred from Agrobacterium to yeast cells and that it interacted with histones in the host cell, and thus may help direct the T-DNA (transferred DNA) to the chromatin as a prelude to integration into the host chromosomal DNA. Show less
The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion... Show moreThe FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of ≈10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells. Show less
