Protein Kinase A Negatively Regulates the Acetic Acid Stress Response in S. cerevisiae.

Bioethanol fermentation from lignocellulosic hydrolysates is negatively affected by the presence of acetic acid. The budding yeast S. cerevisiae adapts to acetic acid stress partly by activating the transcription factor, Haa1. Haa1 induces the expression of many genes, which are responsible for increased fitness in the presence of acetic acid. Here, we show that protein kinase A (PKA) is a negative regulator of Haa1-dependent gene expression under both basal and acetic acid stress conditions. Deletions of RAS2, encoding a positive regulator of PKA, and PDE2, encoding a negative regulator of PKA, lead to an increased and decreased expression of Haa1-regulated genes, respectively. Importantly, the deletion of HAA1 largely reverses the effects of ras2∆. Additionally, the expression of a dominant, hyperactive RAS2A18V19 mutant allele also reduces the expression of Haa1-regulated genes. We found that both pde2Δ and RAS2A18V19 reduce cell fitness in response to acetic acid stress, while ras2Δ increases cellular adaptation. There are three PKA catalytic subunits in yeast, encoded by TPK1, TPK2, and TPK3. We show that single mutations in TPK1 and TPK3 lead to the increased expression of Haa1-regulated genes, while tpk2Δ reduces their expression. Among tpk double mutations, tpk1Δ tpk3Δ greatly increases the expression of Haa1-regulated genes. We found that acetic acid stress in a tpk1Δ tpk3Δ double mutant induces a flocculation phenotype, which is reversed by haa1Δ. Our findings reveal PKA to be a negative regulator of the acetic acid stress response and may help engineer yeast strains with increased efficiency of bioethanol fermentation.

Vibrio cholerae Alkalizes Its Environment via Citrate Metabolism to Inhibit Enteric Growth In Vitro.

Vibrio cholerae is a Gram-negative pathogen, living in constant competition with other bacteria in marine environments and during human infection. One competitive advantage of V. cholerae is the ability to metabolize diverse carbon sources, such as chitin and citrate. We observed that when some V. cholerae strains were grown on a medium with citrate, the medium's chemical composition turned into a hostile alkaline environment for Gram-negative bacteria, such as Escherichia coli and Shigella flexneri. We found that although the ability to exclude competing bacteria was not contingent on exogenous citrate, V. cholerae C6706 citrate metabolism mutants ΔoadA-1, ΔcitE, and ΔcitF were not able to inhibit S. flexneri or E. coli growth. Lastly, we demonstrated that while the V. cholerae C6706-mediated increased medium pH was necessary for the enteric exclusion phenotype, secondary metabolites, such as bicarbonate (protonated to carbonate in the raised pH) from the metabolism of citrate, enhanced the ability to inhibit the growth of E. coli. These data provide a novel example of how V. cholerae outcompetes other Gram-negative bacteria. IMPORTANCE Vibrio cholerae must compete with other bacteria in order to cause disease. Here, we show that V. cholerae creates an alkaline environment, which is able to inhibit the growth of other enteric bacteria. We demonstrate that V. cholerae environmental alkalization is linked to the capacity of the bacteria to metabolize citrate. This behavior could potentially contribute to V. cholerae's ability to colonize the human intestine.

A single 2'-O-methylation of ribosomal RNA gates assembly of a functional ribosome.

RNA modifications are widespread in biology and abundant in ribosomal RNA. However, the importance of these modifications is not well understood. We show that methylation of a single nucleotide, in the catalytic center of the large subunit, gates ribosome assembly. Massively parallel mutational scanning of the essential nuclear GTPase Nog2 identified important interactions with rRNA, particularly with the 2'-O-methylated A-site base Gm2922. We found that methylation of G2922 is needed for assembly and efficient nuclear export of the large subunit. Critically, we identified single amino acid changes in Nog2 that completely bypass dependence on G2922 methylation and used cryoelectron microscopy to directly visualize how methylation flips Gm2922 into the active site channel of Nog2. This work demonstrates that a single RNA modification is a critical checkpoint in ribosome biogenesis, suggesting that such modifications can play an important role in regulation and assembly of macromolecular machines.

Release of the ribosome biogenesis factor Bud23 from small subunit precursors in yeast.

The two subunits of the eukaryotic ribosome are produced through quasi-independent pathways involving the hierarchical actions of numerous trans-acting biogenesis factors and the incorporation of ribosomal proteins. The factors work together to shape the nascent subunits through a series of intermediate states into their functional architectures. One of the earliest intermediates of the small subunit (SSU or 40S) is the SSU processome which is subsequently transformed into the pre-40S intermediate. This transformation is, in part, facilitated by the binding of the methyltransferase Bud23. How Bud23 is released from the resultant pre-40S is not known. The ribosomal proteins Rps0, Rps2, and Rps21, termed the Rps0-cluster proteins, and several biogenesis factors bind the pre-40S around the time that Bud23 is released, suggesting that one or more of these factors could induce Bud23 release. Here, we systematically examined the requirement of these factors for the release of Bud23 from pre-40S particles. We found that the Rps0-cluster proteins are needed but not sufficient for Bud23 release. The atypical kinase/ATPase Rio2 shares a binding site with Bud23 and is thought to be recruited to pre-40S after the Rps0-cluster proteins. Depletion of Rio2 prevented the release of Bud23 from the pre-40S. More importantly, the addition of recombinant Rio2 to pre-40S particles affinity-purified from Rio2-depleted cells was sufficient for Bud23 release in vitro. The ability of Rio2 to displace Bud23 was independent of nucleotide hydrolysis. We propose a novel role for Rio2 in which its binding to the pre-40S actively displaces Bud23 from the pre-40S.

Genetics animates structure: leveraging genetic interactions to study the dynamics of ribosome biogenesis.

The assembly of eukaryotic ribosomes follows an assembly line-like pathway in which numerous trans-acting biogenesis factors act on discrete pre-ribosomal intermediates to progressively shape the nascent subunits into their final functional architecture. Recent advances in cryo-electron microscopy have led to high-resolution structures of many pre-ribosomal intermediates; however, these static snapshots do not capture the dynamic transitions between these intermediates. To this end, molecular genetics can be leveraged to reveal how the biogenesis factors drive these dynamic transitions. Here, we briefly review how we recently used the deletion of BUD23 (bud23∆) to understand its role in the assembly of the ribosomal small subunit. The strong growth defect of bud23∆ mutants places a selective pressure on yeast cells for the occurrence of extragenic suppressors that define a network of functional interactions among biogenesis factors. Mapping these suppressing mutations to recently published structures of pre-ribosomal complexes allowed us to contextualize these suppressing mutations and derive a detailed model in which Bud23 promotes a critical transition event to facilitate folding of the central pseudoknot of the small subunit. This mini-review highlights how genetics can be used to understand the dynamics of complex structures, such as the maturing ribosome.

Bud23 promotes the final disassembly of the small subunit Processome in Saccharomyces cerevisiae.

The first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is thought to represent an early checkpoint in the assembly pathway. Transition of the SSU Processome towards continued maturation requires the removal of the U3 snoRNA and biogenesis factors as well as ribosomal RNA processing. While the factors that drive these events are largely known, how they do so is not. The methyltransferase Bud23 has a role during this transition, but its function, beyond the nonessential methylation of ribosomal RNA, is not characterized. Here, we have carried out a comprehensive genetic screen to understand Bud23 function. We identified 67 unique extragenic bud23Δ-suppressing mutations that mapped to genes encoding the SSU Processome factors DHR1, IMP4, UTP2 (NOP14), BMS1 and the SSU protein RPS28A. These factors form a physical interaction network that links the binding site of Bud23 to the U3 snoRNA and many of the amino acid substitutions weaken protein-protein and protein-RNA interactions. Importantly, this network links Bud23 to the essential GTPase Bms1, which acts late in the disassembly pathway, and the RNA helicase Dhr1, which catalyzes U3 snoRNA removal. Moreover, particles isolated from cells lacking Bud23 accumulated late SSU Processome factors and ribosomal RNA processing defects. We propose a model in which Bud23 dissociates factors surrounding its binding site to promote SSU Processome progression.

The L1 stalk is required for efficient export of nascent large ribosomal subunits in yeast.

The ribosomal protein Rpl1 (uL1 in universal nomenclature) is essential in yeast and constitutes part of the L1 stalk which interacts with E site ligands on the ribosome. Structural studies of nascent pre-60S complexes in yeast have shown that a domain of the Crm1-dependent nuclear export adapter Nmd3, binds in the E site and interacts with Rpl1, inducing closure of the L1 stalk. Based on this observation, we decided to reinvestigate the role of the L1 stalk in nuclear export of pre-60S subunits despite previous work showing that Rpl1-deficient ribosomes are exported from the nucleus and engage in translation. Large cargoes, such as ribosomal subunits, require multiple export factors to facilitate their transport through the nuclear pore complex. Here, we show that pre-60S subunits lacking Rpl1 or truncated for the RNA of the L1 stalk are exported inefficiently. Surprisingly, this is not due to a measurable defect in the recruitment of Nmd3 but appears to result from inefficient recruitment of the Mex67-Mtr2 heterodimer.

Tsr4 Is a Cytoplasmic Chaperone for the Ribosomal Protein Rps2 in Saccharomyces cerevisiae.

Eukaryotic ribosome biogenesis requires the action of approximately 200 trans-acting factors and the incorporation of 79 ribosomal proteins (RPs). The delivery of RPs to preribosomes is a major challenge for the cell because RPs are often highly basic and contain intrinsically disordered regions prone to nonspecific interactions and aggregation. To counteract this, eukaryotes developed dedicated chaperones for certain RPs that promote their solubility and expression, often by binding eukaryote-specific extensions of the RPs. Rps2 (uS5) is a universally conserved RP that assembles into nuclear pre-40S subunits. However, a chaperone for Rps2 had not been identified. Our laboratory previously characterized Tsr4 as a 40S biogenesis factor of unknown function. Here, we report that Tsr4 cotranslationally associates with Rps2. Rps2 harbors a eukaryote-specific N-terminal extension that is critical for its interaction with Tsr4. Moreover, Tsr4 perturbation resulted in decreased Rps2 levels and phenocopied Rps2 depletion. Despite Rps2 joining nuclear pre-40S particles, Tsr4 appears to be restricted to the cytoplasm. Thus, we conclude that Tsr4 is a cytoplasmic chaperone dedicated to Rps2.

Utp14 interaction with the small subunit processome.

The SSU processome (sometimes referred to as 90S) is an early stable intermediate in the small ribosomal subunit biogenesis pathway of eukaryotes. Progression of the SSU processome to a pre-40S particle requires a large-scale compaction of the RNA and release of many biogenesis factors. The U3 snoRNA is a primary component of the SSU processome and hybridizes to the rRNA at multiple locations to organize the structure of the SSU processome. Thus, release of U3 is a prerequisite for the transition to pre-40S. Our laboratory proposed that the RNA helicase Dhr1 plays a crucial role in the transition by unwinding U3 and that this activity is controlled by the SSU processome protein Utp14. How Utp14 times the activation of Dhr1 is an open question. Despite being highly conserved, Utp14 contains no recognizable domains, and how Utp14 interacts with the SSU processome is not well characterized. Here, we used UV crosslinking and analysis of cDNA (CRAC) and yeast two-hybrid interaction to characterize how Utp14 interacts with the preribosome. Moreover, proteomic analysis of SSU particles lacking Utp14 revealed that the presence of Utp14 is needed for efficient recruitment of the RNA exosome. Our analysis positions Utp14 to be uniquely poised to communicate the status of assembly of the SSU processome to Dhr1 and possibly to the exosome as well.

Casein Kinase I Isoform Hrr25 Is a Negative Regulator of Haa1 in the Weak Acid Stress Response Pathway in Saccharomyces cerevisiae.

Haa1 is a transcription factor that adapts Saccharomyces cerevisiae cells to weak organic acid stresses by activating the expression of various genes. Many of these genes encode membrane proteins, such as TPO2 and YRO2 How Haa1 is activated by weak acids is not clear. Here, we show that casein kinase I isoform Hrr25 is an important negative regulator of Haa1. Haa1 is known to be multiply phosphorylated. We found that mutations in HRR25 lead to reduced Haa1 phosphorylation and increased expression of Haa1 target genes and that Hrr25 interacts with Haa1. The other three casein kinase I isoforms, Yck1, Yck2, and Yck3, do not seem to play critical roles in Haa1 regulation. Hrr25 has a 200-residue C-terminal region, including a proline- and glutamine-rich domain. Our data suggest that the C-terminal region of Hrr25 is required for normal inhibition of expression of Haa1 target genes TPO2 and YRO2 and is important for cell growth but is not required for cell morphogenesis. We propose that Hrr25 is an important regulator of cellular adaptation to weak acid stress by inhibiting Haa1 through phosphorylation.IMPORTANCE Our study has revealed the casein kinase I protein Hrr25 to be a negative regulator of Haa1, a transcription factor mediating the cellular response to stresses caused by weak acids. Many studies have focused on the target genes of Haa1 and their roles in weak acid stress responses, but little has been reported on the regulatory mechanism of Haa1. Weak acids, such as acetic acid, have long been used for food preservation by slowing down the growth of fungal species, including S. cerevisiae In the biofuel industry, acetic acid in the lignocellulosic hydrolysates limits the production of ethanol, which is undesirable. By understanding how Haa1 is regulated, we can make advances in the field of food sciences to better preserve food and engineer acetic acid-resistant strains that will increase productivity in the biofuel industry.