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FIC proteins regulate molecular processes from bacteria to humans by catalyzing post-translational modifications (PTM), the most frequent being the addition of AMP or AMPylation. In many AMPylating FIC proteins, a structurally conserved glutamate represses AMPylation and, in mammalian FICD, also supports deAMPylation of BiP/GRP78, a key chaperone of the unfolded protein response. Currently, a direct signal regulating these FIC proteins has not been identified. Here, we use X-ray crystallography and in vitro PTM assays to address this question. We discover that Enterococcus faecalis FIC (EfFIC) catalyzes both AMPylation and deAMPylation and that the glutamate implements a multi-position metal switch whereby Mg 2+ and Ca 2+ control AMPylation and deAMPylation differentially without a conformational change. Remarkably, Ca 2+ concentration also tunes deAMPylation of BiP by human FICD. Our results suggest that the conserved glutamate is a signature of AMPylation/deAMPylation FIC bifunctionality and identify metal ions as diffusible signals that regulate such FIC proteins directly.

Small GTPases regulate many aspects of cell logistics by alternating between an inactive, GDP-bound form and an active, GTP-bound form. This nucleotide switch is coupled to a cytosol/membrane cycle, such that GTP-bound small GTPases carry out their functions at the periphery of endomembranes. A global understanding of the molecular determinants of the interaction of small GTPases with membranes and of the resulting supramolecular organization is beginning to emerge from studies of model systems. Recent studies highlighted that small GTPases establish multiple interactions with membranes involving their lipid anchor, their lipididated hypervariable region and elements in their GTPase domain, which combine to determine the strength, specificity and orientation of their association with lipids. Thereby, membrane association potentiates small GTPase interactions with GEFs, GAPs and effectors through colocalization and positional matching. Furthermore, it leads to small GTPase nanoclustering and to lipid demixing, which drives the assembly of molecular platforms in which proteins and lipids co-operate in producing high-fidelity signals through feedback and feedforward loops. Although still fragmentary, these observations point to an integrated model of signaling by membrane-attached small GTPases that involves a diversity of direct and indirect interactions, which can inspire new therapeutic strategies to block their activities in diseases., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

During the last decade, FIC proteins have emerged as a large family comprised of a variety of bacterial enzymes and a single member in animals. The air de famille of FIC proteins stems from a domain of conserved structure, which catalyzes the post-translational modification of proteins (PTM) by a phosphate-containing compound. In bacteria, examples of FIC proteins include the toxin component of toxin/antitoxin modules, such as Doc-Phd and VbhT-VbhA, toxins secreted by pathogenic bacteria to divert host cell processes, such as VopS, IbpA and AnkX, and a vast majority of proteins of unknown functions. FIC proteins catalyze primarily the transfer of AMP (AMPylation), but they are not restricted to this PTM and also carry out other modifications, for example by phosphocholine or phosphate. In a recent twist, animal FICD/HYPE was shown to catalyze both AMPylation and de-AMPylation of the endoplasmic reticulum BIP chaperone to regulate the unfolded protein response. FICD shares structural features with some bacterial FIC proteins, raising the possibility that bacteria also encode such dual activities. In this review, we discuss how structural, biochemical and cellular approaches have fertilized each other to understand the mechanism, regulation and function of FIC proteins from bacterial pathogens to humans.

Active, GTP-bound small GTPases need to be attached to membranes by post-translational lipid modifications in order to process and propagate information in cells. However, generating and manipulating lipidated GTPases has remained difficult, which has limited our quantitative understanding of their activation by guanine nucleotide exchange factors (GEFs) and their termination by GTPase-activating proteins. Here, we replaced the lipid modification by a histidine tag in 11 full-length, human small GTPases belonging to the Arf, Rho and Rab families, which allowed to tether them to nickel-lipid-containing membranes and characterize the kinetics of their activation by GEFs. Remarkably, this strategy uncovered large effects of membranes on the efficiency and/or specificity in all systems studied. Notably, it recapitulated the release of autoinhibition of Arf1, Arf3, Arf4, Arf5 and Arf6 GTPases by membranes and revealed that all isoforms are efficiently activated by two GEFs with different regulatory regimes, ARNO and Brag2. It demonstrated that membranes stimulate the GEF activity of Trio toward RhoG by ∼30 fold and Rac1 by ∼10 fold, and uncovered a previously unknown broader specificity toward RhoA and Cdc42 that was undetectable in solution. Finally, it demonstrated that the exceptional affinity of the bacterial RabGEF DrrA for the phosphoinositide PI(4)P delimits the activation of Rab1 to the immediate vicinity of the membrane-bound GEF. Our study thus validates the histidine-tag strategy as a potent and simple means to mimic small GTPase lipidation, which opens a variety of applications to uncover regulations brought about by membranes., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H(2)Se/HSe(-/)Se(2-)). Among the genes whose deletion caused hypersensitivity, homologous recombination and DNA damage checkpoint genes were over-represented, suggesting that DNA double-strand breaks are a dominant cause of hydrogen selenide toxicity. Consistent with this hypothesis, treatment of S. cerevisiae cells with sodium selenide triggered G2/M checkpoint activation and induced in vivo chromosome fragmentation. In vitro, sodium selenide directly induced DNA phosphodiester-bond breaks via an O(2)-dependent reaction. The reaction was inhibited by mannitol, a hydroxyl radical quencher, but not by superoxide dismutase or catalase, strongly suggesting the involvement of hydroxyl radicals and ruling out participations of superoxide anions or hydrogen peroxide. The (•)OH signature could indeed be detected by electron spin resonance upon exposure of a solution of sodium selenide to O(2). Finally we showed that, in vivo, toxicity strictly depended on the presence of O(2). Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O(2)-dependent radical-based mechanism.

An Rpa43/Rpa14 stalk protrudes from RNA polymerase I (RNAPI), with homology to Rpb7/Rpb4 (RNAPII), Rpc25/Rpc17 (RNAPIII) and RpoE/RpoF (archaea). In fungi and vertebrates, Rpa43 contains hydrophilic domains forming about half of its size, but these domains lack in Schizosaccharomyces pombe and most other eukaryote lineages. In Saccharomyces cerevisiae, they can be lost with little or no growth effect, as shown by deletion mapping and by domain swapping with fission yeast, but genetically interact with rpa12Δ, rpa34Δ or rpa49Δ, lacking non-essential subunits important for transcript elongation. Two-hybrid data and other genetic evidence suggest that Rpa43 directly bind Spt5, an RNAPI elongation factor also acting in RNAPII-dependent transcription, and may also interact with the nucleosomal chaperone Spt6., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

A43, an essential subunit of yeast RNA polymerase I (pol I), interacts with Rrn3, a class I general transcription factor required for rDNA transcription. The pol I-Rrn3 complex is the only form of enzyme competent for promoter-dependent transcription initiation. In this paper, using biochemical and genetic approaches, we demonstrate that the A43 polypeptide forms a stable heterodimer with the A14 pol I subunit and interacts with the common ABC23 subunit, the yeast counterpart of the omega subunit of bacterial RNA polymerase. We show by immunoelectronic microscopy that A43, ABC23, and A14 colocalize in the three-dimensional structure of the pol I, and we demonstrate that the presence of A43 is required for the stabilization of both A14 and ABC23 within the pol I. Because the N-terminal half of A43 is clearly related to the pol II Rpb7 subunit, we propose that the A43-A14 pair is likely the pol I counterpart of the Rpb7-Rpb4 heterodimer, although A14 distinguishes from Rpb4 by specific sequence and structure features. This hypothesis, combined with our structural data, suggests a new localization of Rpb7-Rpb4 subunits in the three-dimensional structure of yeast pol II.

Regulation of rDNA transcription depends on the formation and dissociation of a functional complex between RNA polymerase I (pol I) and transcription initiation factor Rrn3p. We analyzed whether phosphorylation is involved in this molecular switch. Rrn3p is a phosphoprotein that is predominantly phosphorylated in vivo when it is not bound to pol I. In vitro, Rrn3p is able both to associate with pol I and to enter the transcription cycle in its nonphosphorylated form. By contrast, phosphorylation of pol I is required to form a stable pol I-Rrn3p complex for efficient transcription initiation. Furthermore, association of pol I with Rrn3p correlates with a change in the phosphorylation state of pol I in vivo. We suggest that phosphorylation at specific sites of pol I is a prerequisite for proper transcription initiation and that phosphorylation/dephosphorylation of pol I is one possibility to modulate cellular rDNA transcription activity.

RNA polymerase I (Pol I) is dedicated to transcription of the large ribosomal DNA (rDNA). The mechanism of Pol I recruitment onto rDNA promoters is poorly understood. Here we present evidence that subunit A43 of Pol I interacts with transcription factor Rrn3: conditional mutations in A43 were found to disrupt the transcriptionally competent Pol I-Rrn3 complex, the two proteins formed a stable complex when co-expressed in Escherichia coli, overexpression of Rrn3 suppressed the mutant phenotype, and A43 and Rrn3 mutants showed synthetic lethality. Consistently, immunoelectron microscopy data showed that A43 and Rrn3 co-localize within the Pol I-Rrn3 complex. Rrn3 has several protein partners: a two-hybrid screen identified the C-terminus of subunit Rrn6 of the core factor as a Rrn3 contact, an interaction supported in vitro by affinity chromatography. Our results suggest that Rrn3 plays a central role in Pol I recruitment to rDNA promoters by bridging the enzyme to the core factor. The existence of mammalian orthologues of A43 and Rrn3 suggests evolutionary conservation of the molecular mechanisms underlying rDNA transcription in eukaryotes.

There is limited information on how eukaryotic RNA polymerases (Pol) recognize their cognate preinitiation complex. We have characterized a polypeptide copurifying with yeast Pol III. This protein, C17, was found to be homologous to a mammalian protein described as a hormone receptor. Deletion of the corresponding gene, RPC17, was lethal and its regulated extinction caused a selective defect in transcription of class III genes in vivo. Two-hybrid and coimmunoprecipitation experiments indicated that C17 interacts with two Pol III subunits, one of which, C31, is important for the initiation reaction. C17 also interacted with TFIIIB70, the TFIIB-related component of TFIIIB. The interaction domain was found to be in the N-terminal, TFIIB-like half of TFIIIB70, downstream of the zinc ribbon and first imperfect repeat. Although Pol II similarly interacts with TFIIB, it is notable that C17 has no similarity to any Pol II subunit. The data indicate that C17 is a novel specific subunit of Pol III which participates together with C34 in the recruitment of Pol III by the preinitiation complex.