Proteome-Wide Structural Probing of Low-Abundant Protein Interactions by Cross-Linking Mass Spectrometry.

Proteome-wide cross-linking studies have spurred great interest as they facilitate structural probing of protein interactions in living cells and organisms. However, current studies have a bias for high-abundant proteins. In this study we demonstrate both experimentally and by a kinetic model that this bias is also caused by the propensity of cross-links to preferentially form on high abundant proteins and not by the inability to detect cross-links due to limitations in current technology. We further show, by using both an in vitro mimic of a crowded cellular environment and eukaryotic cell lysates, that parameters optimized toward a pseudo first order kinetics model result in a significant increase in the detection of lower-abundant proteins on a proteome-wide scale. Our study therefore explains the cause of a major limitation in current proteome-wide cross-linking studies and demonstrates how to address a larger part of the proteome by cross-linking.

Early Scanning of Nascent Polypeptides inside the Ribosomal Tunnel by NAC.

Cotranslational processing of newly synthesized proteins is fundamental for correct protein maturation. Protein biogenesis factors are thought to bind nascent polypeptides not before they exit the ribosomal tunnel. Here, we identify a nascent chain recognition mechanism deep inside the ribosomal tunnel by an essential eukaryotic cytosolic chaperone. The nascent polypeptide-associated complex (NAC) inserts the N-terminal tail of its ß subunit (N-ßNAC) into the ribosomal tunnel to sense substrates directly upon synthesis close to the peptidyl-transferase center. N-ßNAC escorts the growing polypeptide to the cytosol and relocates to an alternate binding site on the ribosomal surface. Using C. elegans as an in vivo model, we demonstrate that the tunnel-probing activity of NAC is essential for organismal viability and critical to regulate endoplasmic reticulum (ER) protein transport by controlling ribosome-Sec61 translocon interactions. Thus, eukaryotic protein maturation relies on the early sampling of nascent chains inside the ribosomal tunnel.

Chaperone Interactions at the Ribosome.

The continuous refreshment of the proteome is critical to maintain protein homeostasis and to adapt cells to changing conditions. Thus, de novo protein biogenesis by ribosomes is vitally important to every cellular system. This process is delicate and error-prone and requires, besides cytosolic chaperones, the guidance by a specialized set of molecular chaperones that bind transiently to the translation machinery and the nascent protein to support early folding events and to regulate cotranslational protein transport. These chaperones include the bacterial trigger factor (TF), the archaeal and eukaryotic nascent polypeptide-associated complex (NAC), and the eukaryotic ribosome-associated complex (RAC). This review focuses on the structures, functions, and substrates of these ribosome-associated chaperones and highlights the most recent findings about their potential mechanisms of action.

Neomycin-dependent hammerhead ribozymes for the direct control of gene expression in Saccharomyces cerevisiae.

Hammerhead ribozyme-based RNA switches have been proven to be powerful tools for conditional gene regulation in various organisms. We present neomycin-dependent hammerhead ribozymes (HHR) that influence gene expression in a ligand- and dose-dependent manner in S. cerevisiae. We utilized a novel design of fusing the aptamer domain to the HHR enabling for the first time the identification of genetic ON- and OFF-switches within the same library. For this purpose a neomycin aptamer was fused to stem 1 of a type 3 hammerhead ribozyme via an addressable three-way junction that shows high flexibility at the connection site. An in vivo screening approach identified sequences that allow to induce or repress gene expression 2- to 3-fold in response to neomycin addition. The ribozyme switches operate at neomycin concentrations that show no toxic effect on cell growth and pose powerful genetic tools to study and modulate cellular function in yeast.

Distinct adaptive mechanisms drive recovery from aneuploidy caused by loss of the Ulp2 SUMO protease.

In response to acute loss of the Ulp2 SUMO-specific protease, yeast become disomic for chromosome I (ChrI) and ChrXII. Here we report that ChrI disomy, which creates an adaptive advantage in part by increasing the dosage of the Ccr4 deadenylase, was eliminated by extended passaging. Loss of aneuploidy is often accompanied by mutations in essential SUMO-ligating enzymes, which reduced polySUMO-conjugate accumulation. The mRNA levels for almost all ribosomal proteins increase transiently upon initial loss of Ulp2, but elevated Ccr4 levels limit excess ribosome formation. Notably, extended passaging leads to increased levels of many small nucleolar RNAs (snoRNAs) involved in ribosome biogenesis, and higher dosage of three linked ChrXII snoRNA genes suppressed ChrXII disomy in ulp2Δ cells. Our data reveal that aneuploidy allows rapid adaptation to Ulp2 loss, but long-term adaptation restores euploidy. Cellular evolution restores homeostasis through countervailing mutations in SUMO-modification pathways and regulatory shifts in ribosome biogenesis.

Vms1: A Cytosolic CAT-Tailing Antagonist to Protect Mitochondria.

In eukaryotes, a cytosolic ribosome quality control complex recycles erroneously stalled ribosomes and modifies faulty nascent chains by ubiquitination and by C-terminal Ala- and Thr-extension (CAT-tailing). Reported recently in Cell, Izawa et al. identify cytosolic Vms1 (VCP/Cdc48-associated mitochondrial stress-responsive 1) as an inhibitor of CAT-tailing, which prevents mitochondrial dysfunction caused by imported CAT-tailed polypeptides.

Assessing climate change-robustness of protected area management plans-The case of Germany.

Protected areas are arguably the most important instrument of biodiversity conservation. To keep them fit under climate change, their management needs to be adapted to address related direct and indirect changes. In our study we focus on the adaptation of conservation management planning, evaluating management plans of 60 protected areas throughout Germany with regard to their climate change-robustness. First, climate change-robust conservation management was defined using 11 principles and 44 criteria, which followed an approach similar to sustainability standards. We then evaluated the performance of individual management plans concerning the climate change-robustness framework. We found that climate change-robustness of protected areas hardly exceeded 50 percent of the potential performance, with most plans ranking in the lower quarter. Most Natura 2000 protected areas, established under conservation legislation of the European Union, belong to the sites with especially poor performance, with lower values in smaller areas. In general, the individual principles showed very different rates of accordance with our principles, but similarly low intensity. Principles with generally higher performance values included holistic knowledge management, public accountability and acceptance as well as systemic and strategic coherence. Deficiencies were connected to dealing with the future and uncertainty. Lastly, we recommended the presented principles and criteria as essential guideposts that can be used as a checklist for working towards more climate change-robust planning.

A global map of roadless areas and their conservation status.

Roads fragment landscapes and trigger human colonization and degradation of ecosystems, to the detriment of biodiversity and ecosystem functions. The planet's remaining large and ecologically important tracts of roadless areas sustain key refugia for biodiversity and provide globally relevant ecosystem services. Applying a 1-kilometer buffer to all roads, we present a global map of roadless areas and an assessment of their status, quality, and extent of coverage by protected areas. About 80% of Earth's terrestrial surface remains roadless, but this area is fragmented into ~600,000 patches, more than half of which are <1 square kilometer and only 7% of which are larger than 100 square kilometers. Global protection of ecologically valuable roadless areas is inadequate. International recognition and protection of roadless areas is urgently needed to halt their continued loss.

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation.

Specific proteins are modified by ubiquitin at the endoplasmic reticulum (ER) and are degraded by the proteasome, a process referred to as ER-associated protein degradation. In Saccharomyces cerevisiae, two principal ER-associated protein degradation ubiquitin ligases (E3s) reside in the ER membrane, Doa10 and Hrd1. The membrane-embedded Doa10 functions in the degradation of substrates in the ER membrane, nuclear envelope, cytoplasm, and nucleoplasm. How most E3 ligases, including Doa10, recognize their protein substrates remains poorly understood. Here we describe a previously unappreciated but highly conserved C-terminal element (CTE) in Doa10; this cytosolically disposed 16-residue motif follows the final transmembrane helix. A conserved CTE asparagine residue is required for ubiquitylation and degradation of a subset of Doa10 substrates. Such selectivity suggests that the Doa10 CTE is involved in substrate discrimination and not general ligase function. Functional conservation of the CTE was investigated in the human ortholog of Doa10, MARCH6 (TEB4), by analyzing MARCH6 autoregulation of its own degradation. Mutation of the conserved Asn residue (N890A) in the MARCH6 CTE stabilized the normally short lived enzyme to the same degree as a catalytically inactivating mutation (C9A). We also report the localization of endogenous MARCH6 to the ER using epitope tagging of the genomic MARCH6 locus by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. These localization and CTE analyses support the inference that MARCH6 and Doa10 are functionally similar. Moreover, our results with the yeast enzyme suggest that the CTE is involved in the recognition and/or ubiquitylation of specific protein substrates.

Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins.

Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3'-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane.

The yeast ERAD-C ubiquitin ligase Doa10 recognizes an intramembrane degron.

Aberrant endoplasmic reticulum (ER) proteins are eliminated by ER-associated degradation (ERAD). This process involves protein retrotranslocation into the cytosol, ubiquitylation, and proteasomal degradation. ERAD substrates are classified into three categories based on the location of their degradation signal/degron: ERAD-L (lumen), ERAD-M (membrane), and ERAD-C (cytosol) substrates. In Saccharomyces cerevisiae, the membrane proteins Hrd1 and Doa10 are the predominant ERAD ubiquitin-protein ligases (E3s). The current notion is that ERAD-L and ERAD-M substrates are exclusively handled by Hrd1, whereas ERAD-C substrates are recognized by Doa10. In this paper, we identify the transmembrane (TM) protein Sec61 ß-subunit homologue 2 (Sbh2) as a Doa10 substrate. Sbh2 is part of the trimeric Ssh1 complex involved in protein translocation. Unassembled Sbh2 is rapidly degraded in a Doa10-dependent manner. Intriguingly, the degron maps to the Sbh2 TM region. Thus, in contrast to the prevailing view, Doa10 (and presumably its human orthologue) has the capacity for recognizing intramembrane degrons, expanding its spectrum of substrates.

Scaffold function of long non-coding RNA HOTAIR in protein ubiquitination.

Although mammalian long non-coding (lnc)RNAs are best known for modulating transcription, their post-transcriptional influence on mRNA splicing, stability and translation is emerging. Here we report a post-translational function for the lncRNA HOTAIR as an inducer of ubiquitin-mediated proteolysis. HOTAIR associates with E3 ubiquitin ligases bearing RNA-binding domains, Dzip3 and Mex3b, as well as with their respective ubiquitination substrates, Ataxin-1 and Snurportin-1. In this manner, HOTAIR facilitates the ubiquitination of Ataxin-1 by Dzip3 and Snurportin-1 by Mex3b in cells and in vitro, and accelerates their degradation. HOTAIR levels are highly upregulated in senescent cells, causing rapid decay of targets Ataxin-1 and Snurportin-1, and preventing premature senescence. These results uncover a role for a lncRNA, HOTAIR, as a platform for protein ubiquitination.

Roles of larval sea urchin spicule SM50 domains in organic matrix self-assembly and calcium carbonate mineralization.

The larval spicule matrix protein SM50 is the most abundant occluded matrix protein present in the mineralized larval sea urchin spicule. Recent evidence implicates SM50 in the stabilization of amorphous calcium carbonate (ACC). Here, we investigate the molecular interactions of SM50 and CaCO3 by investigating the function of three major domains of SM50 as small ubiquitin-like modifier (SUMO) fusion proteins - a C-type lectin domain (CTL), a glycine rich region (GRR) and a proline rich region (PRR). Under various mineralization conditions, we find that SUMO-CTL is monomeric and influences CaCO3 mineralization, SUMO-GRR aggregates into large protein superstructures and SUMO-PRR modifies the early CaCO3 mineralization stages as well as growth. The combination of these mineralization and self-assembly properties of the major domains synergistically enable the full-length SM50 to fulfill functions of constructing the organic spicule matrix as well as performing necessary mineralization activities such as Ca(2+) ion recruitment and organization to allow for proper growth and development of the mineralized larval sea urchin spicule.

Split-Doa10: a naturally split polytopic eukaryotic membrane protein generated by fission of a nuclear gene.

Large polytopic membrane proteins often derive from duplication and fusion of genes for smaller proteins. The reverse process, splitting of a membrane protein by gene fission, is rare and has been studied mainly with artificially split proteins. Fragments of a split membrane protein may associate and reconstitute the function of the larger protein. Most examples of naturally split membrane proteins are from bacteria or eukaryotic organelles, and their exact history is usually poorly understood. Here, we describe a nuclear-encoded split membrane protein, split-Doa10, in the yeast Kluyveromyces lactis. In most species, Doa10 is encoded as a single polypeptide with 12-16 transmembrane helices (TMs), but split-KlDoa10 is encoded as two fragments, with the split occurring between TM2 and TM3. The two fragments assemble into an active ubiquitin-protein ligase. The K. lactis DOA10 locus has two ORFs separated by a 508-bp intervening sequence (IVS). A promoter within the IVS drives expression of the C-terminal KlDoa10 fragment. At least four additional Kluyveromyces species contain an IVS in the DOA10 locus, in contrast to even closely related genera, allowing dating of the fission event to the base of the genus. The upstream Kluyveromyces Doa10 fragment with its N-terminal RING-CH and two TMs resembles many metazoan MARCH (Membrane-Associated RING-CH) and related viral RING-CH proteins, suggesting that gene splitting may have contributed to MARCH enzyme diversification. Split-Doa10 is the first unequivocal case of a split membrane protein where fission occurred in a nuclear-encoded gene. Such a split may allow divergent functions for the individual protein segments.

Aberrant substrate engagement of the ER translocon triggers degradation by the Hrd1 ubiquitin ligase.

Little is known about quality control of proteins that aberrantly or persistently engage the endoplasmic reticulum (ER)-localized translocon en route to membrane localization or the secretory pathway. Hrd1 and Doa10, the primary ubiquitin ligases that function in ER-associated degradation (ERAD) in yeast, target distinct subsets of misfolded or otherwise abnormal proteins based primarily on degradation signal (degron) location. We report the surprising observation that fusing Deg1, a cytoplasmic degron normally recognized by Doa10, to the Sec62 membrane protein rendered the protein a Hrd1 substrate. Hrd1-dependent degradation occurred when Deg1-Sec62 aberrantly engaged the Sec61 translocon channel and underwent topological rearrangement. Mutations that prevent translocon engagement caused a reversion to Doa10-dependent degradation. Similarly, a variant of apolipoprotein B, a protein known to be cotranslocationally targeted for proteasomal degradation, was also a Hrd1 substrate. Hrd1 therefore likely plays a general role in targeting proteins that persistently associate with and potentially obstruct the translocon.

FAT10 and NUB1L bind to the VWA domain of Rpn10 and Rpn1 to enable proteasome-mediated proteolysis.

FAT10 is the only ubiquitin-like modifier that can target proteins for degradation by the proteasome in a ubiquitin-independent manner. The degradation of FAT10-linked proteins by the proteasome is strongly accelerated by the ubiquitin-like-ubiquitin-associated protein NEDD8 ultimate buster-1 long (NUB1L). Here we show how FAT10 and NUB1L dock with the 26S proteasome to initiate proteolysis. We identify the 26S proteasome subunit hRpn10/S5a as the receptor for FAT10, whereas NUB1L can bind to both Rpn10 and Rpn1/S2. Unexpectedly, FAT10 and NUB1L both interact with hRpn10 via the VWA domain. FAT10 degradation in yeast shows that human Rpn10 can functionally reconstitute Rpn10-deficient yeast and that the VWA domain of hRpn10 suffices to enable FAT10 degradation. Depletion of hRpn10 causes an accumulation of FAT10-conjugates also in human cells. In conclusion, we identify the VWA domain of hRpn10 as a receptor for ubiquitin-like proteins within the 26S proteasome and elucidate how FAT10 mediates efficient proteolysis by the proteasome.

Roadless and Low-Traffic Areas as Conservation Targets in Europe.

With increasing road encroachment, habitat fragmentation by transport infrastructures has been a serious threat for European biodiversity. Areas with no roads or little traffic ("roadless and low-traffic areas") represent relatively undisturbed natural habitats and functioning ecosystems. They provide many benefits for biodiversity and human societies (e.g., landscape connectivity, barrier against pests and invasions, ecosystem services). Roadless and low-traffic areas, with a lower level of anthropogenic disturbances, are of special relevance in Europe because of their rarity and, in the context of climate change, because of their contribution to higher resilience and buffering capacity within landscape ecosystems. An analysis of European legal instruments illustrates that, although most laws aimed at protecting targets which are inherent to fragmentation, like connectivity, ecosystem processes or integrity, roadless areas are widely neglected as a legal target. A case study in Germany underlines this finding. Although the Natura 2000 network covers a significant proportion of the country (16%), Natura 2000 sites are highly fragmented and most low-traffic areas (75%) lie unprotected outside this network. This proportion is even higher for the old Federal States (western Germany), where only 20% of the low-traffic areas are protected. We propose that the few remaining roadless and low-traffic areas in Europe should be an important focus of conservation efforts; they should be urgently inventoried, included more explicitly in the law and accounted for in transport and urban planning. Considering them as complementary conservation targets would represent a concrete step towards the strengthening and adaptation of the Natura 2000 network to climate change.