Scp160p associates with specific mRNAs in yeast

Scp160p is a multiple KH-domain RNA-binding protein in yeast that has been demonstrated previously to associate with both soluble and membrane-bound polyribosomes as an mRNP component. One key question that has remained unanswered, however, is whether the mRNAs in these mRNP complexes are random or specific. We have addressed this question using microarray analyses of RNAs released from affinity isolated Scp160p-containing complexes, compared with total RNA controls from the same lysates. Our results, confirmed by quantitative RT–PCR analysis, clearly demonstrate that Scp160p associates with specific rather than with random messages, and that among the enriched targets are DHH1, YOR338W, BIK1, YOL155C and NAM8. Furthermore, loss of Scp160p resulted in a significant change in both the abundance and distribution between soluble and membrane-associated fractions for at least one of these messages (YOR338W), and in a subtle yet significant shift from soluble polyribosomes to soluble mRNPs for at least two of these target messages (DHH1 and YOR338W). Together, these data not only identify specific mRNA targets associated with Scp160p in vivo, they demonstrate that the association of Scp160p with these messages is biologically relevant.     

Scp160p is required for translational efficiency of codon-optimized mRNAs in yeast

The budding yeast multi-K homology domain RNA-binding protein Scp160p binds to >1000 messenger RNAs (mRNAs) and polyribosomes, and its mammalian homolog vigilin binds transfer RNAs (tRNAs) and translation elongation factor EF1alpha. Despite its implication in translation, studies on Scp160p''s molecular function are lacking to date. We applied translational profiling approaches and demonstrate that the association of a specific subset of mRNAs with ribosomes or heavy polysomes depends on Scp160p. Interaction of Scp160p with these mRNAs requires the conserved K homology domains 13 and 14. Transfer RNA pairing index analysis of Scp160p target mRNAs indicates a high degree of consecutive use of iso-decoding codons. As shown for one target mRNA encoding the glycoprotein Pry3p, Scp160p depletion results in translational downregulation but increased association with polysomes, suggesting that it is required for efficient translation elongation. Depletion of Scp160p also decreased the relative abundance of ribosome-associated tRNAs whose codons show low potential for autocorrelation on mRNAs. Conversely, tRNAs with highly autocorrelated codons in mRNAs are less impaired. Our data indicate that Scp160p might increase the efficiency of tRNA recharge, or prevent diffusion of discharged tRNAs, both of which were also proposed to be the likely basis for the translational fitness effect of tRNA pairing.     

Both KH and non-KH domain sequences are required for polyribosome association of Scp160p in yeast

Scp160p is a 160 kDa RNA-binding protein in yeast previously demonstrated to associate with specific messages as an mRNP component of both soluble and membrane-bound polyribosomes. Although the vast majority of Scp160p sequence consists of 14 closely spaced KH domains, comparative sequence analyses also demonstrate the presence of a potential nuclear localization sequence located between KH domains 3 and 4, as well as a 110 amino acid non-KH N-terminal region that includes a potential nuclear export sequence (NES). As a step toward investigating the structure/function relationships of Scp160p, we generated two truncated alleles, FLAG.SCP160ΔN1, encoding a protein product that lacks the first 74 amino acids, including the potential NES, and FLAG.SCP160ΔC1, encoding a protein product that lacks the final KH domain (KH14). We report here that the N-truncated protein, expressed as a green fluorescent protein fusion in yeast, remains cytoplasmic, with no apparent nuclear accumulation. Biochemical studies further demonstrate that although the N-truncated protein remains competent to form RNPs, the C-truncated protein does not. Furthermore, polyribosome association is severely compromised for both truncated proteins. Perhaps most important, both truncated alleles appear only marginally functional in vivo, as demonstrated by the inability of each to complement scp160/eap1 synthetic lethality in a tester strain. Together, these data challenge the notion that Scp160p normally shuttles between the nucleus and cytoplasm, and further implicate polyribosome association as an essential component of Scp160p function in vivo. Finally, these data underscore the vital roles of both KH and non-KH domain sequences in Scp160p.     

Scp160p, a multiple KH-domain protein, is a component of mRNP complexes in yeast

Scp160p is a 160 kDa protein in the yeast Saccharomyces cerevisiae that contains 14 repeats of the hnRNP K-homology (KH) domain, and demonstrates significant sequence homology to a family of proteins collectively known as vigilins. As a first step towards defining the function of Scp160p, we have characterized the subcellular distribution and in vivo interactions of this protein. Using sucrose gradient fractionation studies we have demonstrated that Scp160p in cytoplasmic lysates is predominantly associated with polyribosomes. Furthermore, we have found that Scp160p is released from polyribosomes by EDTA in the form of a large complex of ≥1300 kDa that is sensitive both to RNase and NaCl. Using affinity-chromatography to isolate these complexes, we have identified two protein components other than Scp160p: poly(A) binding protein, Pab1p, and Bfr1p. The presence of Pab1p confirms these complexes to be mRNPs. The presence of Bfr1p is intriguing because the null phenotype for this gene is essentially the same as that reported for scp160-null cells: increased cell size and aberrant DNA content. These results demonstrate that Scp160p associates with polyribosome-bound mRNP complexes in vivo, implicating a role for this protein in one or more levels of mRNA metabolism in yeast.     

Responding to attraction: chemotaxis and chemotropism in Dictyostelium and yeast

Polarized growth in response to external signals is essential for both the internal organization of cells and generation of complex multicellular structures during development. Oriented growth or movement requires specific detection of an external cue, reorganization of the cytoskeleton and subsequent growth or movement. Genetic approaches in both the budding yeast Saccharomyces cerevisiae and the social amoeba Dictyostelium discoideum have shed light on the molecular and cellular aspects of growth or movement towards an external signal. This review discusses the mechanisms and signalling pathways that enable yeast and Dictyostelium cells to translate external signals into directed growth and movement, respectively.     

The yeast G protein alpha subunit Gpa1 transmits a signal through an RNA binding effector protein Scp160

In yeast Saccharomyces cerevisiae the G protein betagamma subunits (Ste4/Ste18) have long been known to transmit the signal required for mating. Here we demonstrate that GTPase-deficient mutants of Galpha (Gpa1) directly activate the mating response pathway. We also show that signaling by activated Gpa1 requires direct coupling to an RNA binding protein Scp160. These findings suggest an additional role for Gpa1 and reveal Scp160 as a component of the mating response pathway in yeast.     

The nuclear basket mediates perinuclear mRNA scanning in budding yeast

After synthesis and transit through the nucleus, messenger RNAs (mRNAs) are exported to the cytoplasm through the nuclear pore complex (NPC). At the NPC, messenger ribonucleoproteins (mRNPs) first encounter the nuclear basket where mRNP rearrangements are thought to allow access to the transport channel. Here, we use single mRNA resolution live cell microscopy and subdiffraction particle tracking to follow individual mRNAs on their path toward the cytoplasm. We show that when reaching the nuclear periphery, RNAs are not immediately exported but scan along the nuclear periphery, likely to find a nuclear pore allowing export. Deletion or mutation of the nuclear basket proteins MLP1/2 or the mRNA binding protein Nab2 changes the scanning behavior of mRNPs at the nuclear periphery, shortens residency time at nuclear pores, and results in frequent release of mRNAs back into the nucleoplasm. These observations suggest a role for the nuclear basket in providing an interaction platform that keeps RNAs at the periphery, possibly to allow mRNP rearrangements before export.     

Yeast G-proteins mediate directional sensing and polarization behaviors in response to changes in pheromone gradient direction

Yeast cells polarize by projecting up mating pheromone gradients, a classic cell polarity behavior. However, these chemical gradients may shift direction. We examine how yeast cells sense and respond to a 180^o switch in the direction of microfluidically generated pheromone gradients. We identify two behaviors: at low concentrations of α-factor, the initial projection grows by bending, whereas at high concentrations, cells form a second projection toward the new source. Mutations that increase heterotrimeric G-protein activity expand the bending-growth morphology to high concentrations; mutations that increase Cdc42 activity result in second projections at low concentrations. Gradient-sensing projection bending requires interaction between Gβγ and Cdc24, whereas gradient-nonsensing projection extension is stimulated by Bem1 and hyperactivated Cdc42. Of interest, a mutation in Gα affects both bending and extension. Finally, we find a genetic perturbation that exhibits both behaviors. Overexpression of the formin Bni1, a component of the polarisome, makes both bending-growth projections and second projections at low and high α-factor concentrations, suggesting a role for Bni1 downstream of the heterotrimeric G-protein and Cdc42 during gradient sensing and response. Thus we demonstrate that G-proteins modulate in a ligand-dependent manner two fundamental cell-polarity behaviors in response to gradient directional change.     

Heme sensing and trafficking in fungi

Fungal pathogens cause life-threatening diseases in humans, and the increasing prevalence of these diseases emphasizes the need for new targets for therapeutic intervention. Nutrient acquisition during infection is a promising target, and recent studies highlight the contributions of endomembrane trafficking, mitochondria, and vacuoles in the sensing and acquisition of heme by fungi. These studies have been facilitated by genetically encoded biosensors and other tools to quantitate heme in subcellular compartments and to investigate the dynamics of trafficking in living cells. In particular, the applications of biosensors in fungi have been extended beyond the detection of metabolites, cofactors, pH, and redox status to include the detection of heme. Here, we focus on studies that make use of biosensors to examine mechanisms of heme uptake and degradation, with guidance from the model fungus Saccharomyces cerevisiae and an emphasis on the pathogenic fungi Candida albicans and Cryptococcus neoformans that threaten human health. These studies emphasize a role for endocytosis in heme uptake, and highlight membrane contact sites involving mitochondria, the endoplasmic reticulum and vacuoles as mediators of intracellular iron and heme trafficking.     

The sxa2-dependent inactivation of the P-factor mating pheromone in the fission yeast Schizosaccharomyces pombe

Haploid cells of the fission yeast Schizosaccharomyces pombe exist in one of two mating types, referred to as M and P. Conjugation occurs between cells of opposite mating type and is controlled by the reciprocal action of diffusible pheromones. Loss of function of the sxa2 gene in M cells causes hypersensitivity to the P-factor mating pheromone and a reduction in mating efficiency. Here we demonstrate the secretion of an sxa2-dependent carboxypeptidase that inactivates P-factor by removal of the C-terminal leucine residue.     

Bqt2p is essential for initiating telomere clustering upon pheromone sensing in fission yeast

The telomere bouquet, i.e., telomere clustering on the nuclear envelope (NE) during meiotic prophase, is thought to promote homologous chromosome pairing. Using a visual screen, we identified bqt2/im295, a mutant that disrupts telomere clustering in fission yeast. Bqt2p is required for linking telomeres to the meiotic spindle pole body (SPB) but not for attachment of telomeres or the SPB to the NE. Bqt2p is expressed upon pheromone sensing and colocalizes thereafter to Sad1p, an SPB protein. This localization only depends on Bqt1p, not on other identified proteins required for telomere clustering. Upon pheromone sensing, generation of Sad1p foci next to telomeres depends on Bqt2p. However, depletion of Bqt2p from the SPB is dispensable for dissolving the telomere bouquet at the end of meiotic prophase. Therefore, telomere bouquet formation requires Bqt2p as a linking component and is finely regulated during meiotic progression.     

Morphogen gradient formation and vesicular trafficking

Morphogens are secreted signaling molecules which form spatial concentration gradients while moving away from a restricted source of production. A simple model of gradient formation postulates that the morphogens dilute as they diffuse between cells. In this review we discuss recent data supporting the idea that movement of the morphogen could also occur via vesicular trafficking through the cells. We explore the implications of these results for the control of gradient formation and the determination of the gradient slope which ultimately encodes the coordinates of positional information.     

Internalization and trafficking of fluorescent-labeled phospholipids in yeast

Phospholipid reporter molecules, containing a fluorescent group attached to a short, acyl chain, spontaneously insert into the plasma membrane of yeast cells allowing retrograde trafficking to intracellular organelles as well as their metabolic fates to be monitored. This approach provides the framework for determining the dependence of particular phospholipid trafficking and metabolic steps on a wide range of genes known to be required for related membrane transport functions as well as for developing genetic screens to identify novel genes required for these processes. This review presents an overview of insights gained into phospholipid trafficking and metabolism using this approach.     

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.     

Barrier activity in Candida albicans mediates pheromone degradation and promotes mating

Mating in Candida albicans and Saccharomyces cerevisiae is regulated by the secretion of peptide pheromones that initiate the mating process. An important regulator of pheromone activity in S. cerevisiae is barrier activity, involving an extracellular aspartyl protease encoded by the BAR1 gene that degrades the alpha pheromone. We have characterized an equivalent barrier activity in C. albicans and demonstrate that the loss of C. albicans BAR1 activity results in opaque a cells exhibiting hypersensitivity to alpha pheromone. Hypersensitivity to pheromone is clearly seen in halo assays; in response to alpha pheromone, a lawn of C. albicans Deltabar1 mutant cells produces a marked zone in which cell growth is inhibited, whereas wild-type strains fail to show halo formation. C. albicans mutants lacking BAR1 also exhibit a striking mating defect in a cells, but not in alpha cells, due to overstimulation of the response to alpha pheromone. The block to mating occurs prior to cell fusion, as very few mating zygotes were observed in mixes of Deltabar1 a and alpha cells. Finally, in a barrier assay using a highly pheromone-sensitive strain, we were able to demonstrate that barrier activity in C. albicans is dependent on Bar1p. These studies reveal that a barrier activity to alpha pheromone exists in C. albicans and that the activity is analogous to that caused by Bar1p in S. cerevisiae.     

Developmental variation in Rab11-dependent trafficking in Trypanosoma brucei

In Trypanosoma brucei, endocytosis is developmentally regulated and is substantially more active in the mammalian infective stage, where it likely plays a role in immune evasion. The small GTPase TbRAB11 is highly expressed in the mammalian stage and mediates recycling of glycosylphosphatidylinositol-anchored proteins, including the variant surface glycoprotein (VSG) and the transferrin receptor, plus trafficking of internalized anti-VSG antibody and transferrin. No function has been assigned to TbRAB11 in the procyclic (insect) stage trypanosome. The importance of TbRAB11 to both bloodstream and procyclic form viability was assessed by RNA interference (RNAi). Suppression of TbRAB11 in the bloodstream form was rapidly lethal and led to cells with round morphology and an enlarged flagellar pocket. TbRAB11 RNAi was also lethal in procyclic forms, which also became rounded, but progression to cell death was significantly slower and the flagellar pocket remained normal. In bloodstream forms, silencing of TbRAB11 had no effect on exocytosis of newly synthesized VSG, fluid-phase endocytosis, or transferrin uptake, but export of internalized transferrin was inhibited. Lectin endocytosis assays revealed a block to postendosomal transport mediated by suppressing TbRAB11. By contrast, in procyclic forms, depletion of TbRAB11 blocks both fluid-phase endocytosis and internalization of surface proteins. In normal bloodstream forms, most VSG is recycled, but in procyclics, internalized surface proteins accumulated in the lysosome. These data demonstrate that TbRAB11 controls recycling and is essential in both life stages of T. brucei but that its primary role is subject to developmental variation.