The Ankyrin Repeats and DHHC S-acyl Transferase Domain of AKR1 Act Independently to Regulate Switching from Vegetative to Mating States in Yeast

Signal transduction from G-protein coupled receptors to MAPK cascades through heterotrimeric G-proteins has been described for many eukaryotic systems. One of the best-characterised examples is the yeast pheromone response pathway, which is negatively regulated by AKR1. AKR1-like proteins are present in all eukaryotes and contain a DHHC domain and six ankyrin repeats. Whilst the DHHC domain dependant S-acyl transferase (palmitoyl transferase) function of AKR1 is well documented it is not known whether the ankyrin repeats are also required for this activity. Here we show that the ankyrin repeats of AKR1 are required for full suppression of the yeast pheromone response pathway, by sequestration of the Gβγ dimer, and act independently of AKR1 S-acylation function. Importantly, the functions provided by the AKR1 ankyrin repeats and DHHC domain are not required on the same molecule to fully restore WT phenotypes and function. We also show that AKR1 molecules are S-acylated at locations other than the DHHC cysteine, increasing the abundance of AKR1 in the cell. Our results have important consequences for studies of AKR1 function, including recent attempts to characterise S-acylation enzymology and kinetics. Proteins similar to AKR1 are found in all eukaryotes and our results have broad implications for future work on these proteins and the control of switching between Gβγ regulated pathways

Key Amino Acid Residues of Ankyrin-Sensitive Phosphatidylethanolamine/Phosphatidylcholine-Lipid Binding Site of βI-Spectrin

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that “opening” of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton

Genetic Basis of Hidden Phenotypic Variation Revealed by Increased Translational Readthrough in Yeast

Eukaryotic release factors 1 and 3, encoded by SUP45 and SUP35, respectively, in Saccharomyces cerevisiae, are required for translation termination. Recent studies have shown that, besides these two key factors, several genetic and epigenetic mechanisms modulate the efficiency of translation termination. These mechanisms, through modifying translation termination fidelity, were shown to affect various cellular processes, such as mRNA degradation, and in some cases could confer a beneficial phenotype to the cell. The most studied example of such a mechanism is [PSI+], the prion conformation of Sup35p, which can have pleiotropic effects on growth that vary among different yeast strains. However, genetic loci underlying such readthrough-dependent, background-specific phenotypes have yet to be identified. Here, we used sup35C653R, a partial loss-of-function allele of the SUP35 previously shown to increase readthrough of stop codons and recapitulate some [PSI+]-dependent phenotypes, to study the genetic basis of phenotypes revealed by increased translational readthrough in two divergent yeast strains: BY4724 (a laboratory strain) and RM11_1a (a wine strain). We first identified growth conditions in which increased readthrough of stop codons by sup35C653R resulted in different growth responses between these two strains. We then used a recently developed linkage mapping technique, extreme QTL mapping (X-QTL), to identify readthrough-dependent loci for the observed growth differences. We further showed that variation in SKY1, an SR protein kinase, underlies a readthrough-dependent locus observed for growth on diamide and hydrogen peroxide. We found that the allelic state of SKY1 interacts with readthrough level and the genetic background to determine growth rate in these two conditions

Nitric oxide differentially regulates renal ATP-binding cassette transporters during endotoxemia

Nitric oxide (NO) is an important regulator of renal transport processes. In the present study, we investigated the role of NO, produced by inducible NO synthase (iNOS), in the regulation of renal ATP-binding cassette (ABC) transporters in vivo during endotoxemia. Wistar–Hannover rats were injected with lipopolysaccharide (LPS+) alone or in combination with the iNOS inhibitor, aminoguanidine. Controls received detoxified LPS (LPS−). After LPS+, proximal tubular damage and a reduction in renal function were observed. Furthermore, iNOS mRNA and protein, and the amount of NO metabolites in plasma and urine, increased compared to the LPS− group. Coadministration with aminoguanidine resulted in an attenuation of iNOS induction and reduction of renal damage. Gene expression of 20 ABC transporters was determined. After LPS+, a clear up-regulation in Abca1, Abcb1/P-glycoprotein (P-gp), Abcb11/bile salt export pump (Bsep), and Abcc2/multidrug resistance protein (Mrp2) was found, whereas Abcc8 was down-regulated. Up-regulation of Abcc2/Mrp2 was accompanied by enhanced calcein excretion. Aminoguanidine attenuated the effects on transporter expression. Our data indicate that NO, produced locally by renal iNOS, regulates the expression of ABC transporters in vivo. Furthermore, we showed, for the first time, expression and subcellular localization of Abcb11/Bsep in rat kidney

A Chromosomally Encoded Virulence Factor Protects the Lyme Disease Pathogen against Host-Adaptive Immunity

Borrelia burgdorferi, the bacterial pathogen of Lyme borreliosis, differentially expresses select genes in vivo, likely contributing to microbial persistence and disease. Expression analysis of spirochete genes encoding potential membrane proteins showed that surface-located membrane protein 1 (lmp1) transcripts were expressed at high levels in the infected murine heart, especially during early stages of infection. Mice and humans with diagnosed Lyme borreliosis also developed antibodies against Lmp1. Deletion of lmp1 severely impaired the pathogen's ability to persist in diverse murine tissues including the heart, and to induce disease, which was restored upon chromosomal complementation of the mutant with the lmp1 gene. Lmp1 performs an immune-related rather than a metabolic function, as its deletion did not affect microbial persistence in immunodeficient mice, but significantly decreased spirochete resistance to the borreliacidal effects of anti-B. burgdorferi sera in a complement-independent manner. These data demonstrate the existence of a virulence factor that helps the pathogen evade host-acquired immune defense and establish persistent infection in mammals

AglH, a thermophilic UDP‑<i>N</i>‑acetylglucosamine‑1‑phosphate:dolichyl phosphate GlcNAc‑1‑phosphotransferase initiating protein<i> N</i>‑glycosylation pathway in <i>Sulfolobus acidocaldarius</i>, is capable of complementing the eukaryal Alg7

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D(100)), IV (F(220)) and V (F(264)) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival

Replicative Age Induces Mitotic Recombination in the Ribosomal RNA Gene Cluster of Saccharomyces cerevisiae

Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells

Regulation of Septin Dynamics by the Saccharomyces cerevisiae Lysine Acetyltransferase NuA4

In the budding yeast Saccharomyces cerevisiae, the lysine acetyltransferase NuA4 has been linked to a host of cellular processes through the acetylation of histone and non-histone targets. To discover proteins regulated by NuA4-dependent acetylation, we performed genome-wide synthetic dosage lethal screens to identify genes whose overexpression is toxic to non-essential NuA4 deletion mutants. The resulting genetic network identified a novel link between NuA4 and septin proteins, a group of highly conserved GTP-binding proteins that function in cytokinesis. We show that acetyltransferase-deficient NuA4 mutants have defects in septin collar formation resulting in the development of elongated buds through the Swe1-dependent morphogenesis checkpoint. We have discovered multiple sites of acetylation on four of the five yeast mitotic septins, Cdc3, Cdc10, Cdc12 and Shs1, and determined that NuA4 can acetylate three of the four in vitro. In vivo we find that acetylation levels of both Shs1 and Cdc10 are reduced in a catalytically inactive esa1 mutant. Finally, we determine that cells expressing a Shs1 protein with decreased acetylation in vivo have defects in septin localization that are similar to those observed in NuA4 mutants. These findings provide the first evidence that yeast septin proteins are acetylated and that NuA4 impacts septin dynamics

Spectrin-based skeleton as an actor in cell signaling

This review focuses on the recent advances in functions of spectrins in non-erythroid cells. We discuss new data concerning the commonly known role of the spectrin-based skeleton in control of membrane organization, stability and shape, and tethering protein mosaics to the cellular motors and to all major filament systems. Particular effort has been undertaken to highlight recent advances linking spectrin to cell signaling phenomena and its participation in signal transduction pathways in many cell types