ER Targeting and Insertion of Tail-Anchored Membrane Proteins by the GET Pathway

Hundreds of eukaryotic membrane proteins are anchored to membranes by a single transmembrane domain at their C-terminus. Many of these tail-anchored (TA) proteins are post-translationally targeted to the endoplasmic reticulum (ER) membrane for insertion by the Guided-Entry of TA protein insertion (GET) pathway. In recent years most of the components of this conserved pathway have been biochemically and structurally characterized. Get3 is the pathway targeting factor that utilizes nucleotide-linked conformational changes to mediate the delivery of TA proteins between the GET pre-targeting machinery in the cytosol and the transmembrane pathway components in the ER. Here we focus on the mechanism of the yeast GET pathway and make a speculative analogy between its membrane insertion step and the ATPase-driven cycle of ABC transporters.Molecular and Cellular Biolog

Crystal structures of Rea1-MIDAS bound to its ribosome assembly factor ligands resembling integrin-ligand-type complexes

The Rea1 AAA(+)-ATPase dislodges assembly factors from pre-60S ribosomes upon ATP hydrolysis, thereby driving ribosome biogenesis. Here, we present crystal structures of Rea1-MIDAS, the conserved domain at the tip of the flexible Rea1 tail, alone and in complex with its substrate ligands, the UBL domains of Rsa4 or Ytm1. These complexes have structural similarity to integrin alpha-subunit domains when bound to extracellular matrix ligands, which for integrin biology is a key determinant for force-bearing cell-cell adhesion. However, the presence of additional motifs equips Rea1-MIDAS for its tasks in ribosome maturation. One loop insert cofunctions as an NLS and to activate the mechanochemical Rea1 cycle, whereas an additional beta-hairpin provides an anchor to hold the ligand UBL domains in place. Our data show the versatility of the MIDAS fold for mechanical force transmission in processes as varied as integrin-mediated cell adhesion and mechanochemical removal of assembly factors from pre-ribosomes

Targeting of Drosophila Rhodopsin Requires Helix 8 but Not the Distal C-Terminus

BACKGROUND: The fundamental role of the light receptor rhodopsin in visual function and photoreceptor cell development has been widely studied. Proper trafficking of rhodopsin to the photoreceptor membrane is of great importance. In human, mutations in rhodopsin involving its intracellular mislocalization, are the most frequent cause of autosomal dominant Retinitis Pigmentosa, a degenerative retinal pathology characterized by progressive blindness. Drosophila is widely used as an animal model in visual and retinal degeneration research. So far, little is known about the requirements for proper rhodopsin targeting in Drosophila. METHODOLOGY/PRINCIPAL FINDINGS: Different truncated fly-rhodopsin Rh1 variants were expressed in the eyes of Drosophila and their localization was analyzed in vivo or by immunofluorescence. A mutant lacking the last 23 amino acids was found to properly localize in the rhabdomeres, the light-sensing organelle of the photoreceptor cells. This constitutes a major difference to trafficking in vertebrates, which involves a conserved QVxPA motif at the very C-terminus. Further truncations of Rh1 indicated that proper localization requires the last amino acid residues of a region called helix 8 following directly the last transmembrane domain. Interestingly, the very C-terminus of invertebrate visual rhodopsins is extremely variable but helix 8 shows conserved amino acid residues that are not conserved in vertebrate homologs. CONCLUSIONS/SIGNIFICANCE: Despite impressive similarities in the folding and photoactivation of vertebrate and invertebrate visual rhodopsins, a striking difference exists between mammalian and fly rhodopsins in their requirements for proper targeting. Most importantly, the distal part of helix 8 plays a central role in invertebrates. Since the last amino acid residues of helix 8 are dispensable for rhodopsin folding and function, we propose that this domain participates in the recognition of targeting factors involved in transport to the rhabdomeres

Structural basis for a distinct catalytic mechanism in Trypanosoma brucei tryparedoxin peroxidase

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three cysteine homologues (Px I-III) of classical selenocysteine-containing glutathione peroxidases. The enzymes obtain their reducing equivalents from the unique trypanothione (bis(glutathionyl)spermidine)/tryparedoxin system. During catalysis, these tryparedoxin peroxidases cycle between an oxidized form with an intramolecular disulfide bond between Cys(47) and Cys(95) and the reduced peroxidase with both residues in the thiol state. Here we report on the three-dimensional structures of oxidized T. brucei Px III at 1.4A resolution obtained by x-ray crystallography and of both the oxidized and the reduced protein determined by NMR spectroscopy. Px III is a monomeric protein unlike the homologous poplar thioredoxin peroxidase (TxP). The structures of oxidized and reduced Px III are essentially identical in contrast to what was recently found for TxP. In Px III, Cys(47), Gln(82), and Trp(137) do not form the catalytic triad observed in the selenoenzymes, and related proteins and the latter two residues are unaffected by the redox state of the protein. The mutational analysis of three conserved lysine residues in the vicinity of the catalytic cysteines revealed that exchange of Lys(107) against glutamate abrogates the reduction of hydrogen peroxide, whereas Lys(97) and Lys(99) play a crucial role in the interaction with tryparedoxin

60S ribosome biogenesis requires rotation of the 5S ribonucleoprotein particle

During eukaryotic ribosome biogenesis, nascent ribosomal RNA (rRNA) forms pre-ribosomal particles containing ribosomal proteins and assembly factors. Subsequently, these immature rRNAs are processed and remodelled. Little is known about the premature assembly states of rRNAs and their structural rearrangement during ribosome biogenesis. Using cryo-EM we characterize a pre-60S particle, where the 5S rRNA and its associated ribosomal proteins L18 and L5 (5S ribonucleoprotein (RNP)) are rotated by almost 180 degrees when compared with the mature subunit. Consequently, neighbouring 25S rRNA helices that protrude from the base of the central protuberance are deformed. This altered topology is stabilized by nearby assembly factors (Rsa4 and Nog1),which were identified by fitting their three-dimensional structures into the cryo-EM density. We suggest that the 5S RNP performs a semicircular movement during 60S biogenesis to adopt its final position, fulfilling a chaperone-like function in guiding the flanking 25S rRNA helices of the central protuberance to their final topology

Consistent mutational paths predict eukaryotic thermostability

peer-reviewedBackground: Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published. Results: Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1), we could also characterise the molecular consequences of some of these mutations. Conclusions: The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability

Structural basis for the molecular evolution of SRP-GTPase activation by protein

Small G proteins have key roles in signal transduction pathways. They are switched from the signaling 'on' to the non-signaling 'off' state when GTPase-activating proteins (GAPs) provide a catalytic residue. The ancient signal recognition particle (SRP)-type GTPases form GTP-dependent homo- and heterodimers and deviate from the canonical switch paradigm in that no GAPs have been identified. Here we show that the YlxH protein activates the SRP-GTPase FlhF. The crystal structure of the Bacillus subtilis FlhF–effector complex revealed that the effector does not contribute a catalytic residue but positions the catalytic machinery already present in SRP-GTPases. We provide a general concept that might also apply to the RNA-driven activation of the universally conserved, co-translational protein-targeting machinery comprising the SRP-GTPases Ffh and FtsY. Our study exemplifies the evolutionary transition from RNA- to protein-driven activation in SRP-GTPases and suggests that the current view on SRP-mediated protein targeting is incomplete

Structural basis for 5'-ETS recognition by Utp4 at the early stages of ribosome biogenesis

Eukaryotic ribosome biogenesis begins with the co-transcriptional assembly of the 90S pre-ribosome. The ‘U three protein’ (UTP) complexes and snoRNP particles arrange around the nascent pre-ribosomal RNA chaperoning its folding and further maturation. The earliest event in this hierarchical process is the binding of the UTP-A complex to the 5'-end of the pre-ribosomal RNA (5'-ETS). This oligomeric complex predominantly consists of β-propeller and α-solenoidal proteins. Here we present the structure of the Utp4 subunit from the thermophilic fungus Chaetomium thermophilum at 2.15 Å resolution and analyze its function by UV RNA-crosslinking (CRAC) and in context of a recent cryo-EM structure of the 90S pre-ribosome. Utp4 consists of two orthogonal and highly basic β-propellers that perfectly fit the EM-data. The Utp4 structure highlights an unusual Velcro-closure of its C-terminal β-propeller as relevant for protein integrity and potentially Utp8 recognition in the context of the pre-ribosome. We provide a first model of the 5'-ETS RNA from the internally hidden 5'-end up to the region that hybridizes to the 3'-hinge sequence of U3 snoRNA and validate a specific Utp4/5'-ETS interaction by CRAC analysis.This work was supported by Deutsche Forschungsgemeinschaft (DFG) (SFB638, Z4 to I. S. and HU363/15-1 to E.H. and the Leibniz programme to I.S.); Cluster of Excellence CellNetworks (EcTOP1 to I.S. and E.H.); Funding for open access charge: DFG [Leibniz Programme]. M.K. was funded by a Kekule Fellowship (VCI)

Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33

GTPases act as molecular switches to control many cellular processes, including signalling, protein translation and targeting. Switch activity can be regulated by external effector proteins or intrinsic properties, such as dimerization. The recognition and translocation of pre-proteins into chloroplasts [via the TOC/TIC (translocator at the outer envelope membrane of chloroplasts/inner envelope membrane of chloroplasts)] is controlled by two homologous receptor GTPases, Toc33 and Toc159, whose reversible dimerization is proposed to regulate translocation of incoming proteins in a GTP-dependent manner. Toc33 is a homodimerizing GTPase. Functional analysis suggests that homodimerization is a key step in the translocation process, the molecular functions of which, as well as the elements regulating this event, are largely unknown. In the present study, we show that homodimerization reduces the rate of nucleotide exchange, which is consistent with the observed orientation of the monomers in the crystal structure. Pre-protein binding induces a dissociation of the Toc33 homodimer and results in the exchange of GDP for GTP. Thus homodimerization does not serve to activate the GTPase activity as discussed many times previously, but to control the nucleotide-loading state. We discuss this novel regulatory mode and its impact on the current models of protein import into the chloroplast

Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site

During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1–RpL5-N–RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1–RpL5–RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP