Functions and molecular mechanisms of eukaryotic ribosome-associated chaperones
D ISSERTATION
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften doctor rerum naturalium (Dr. rer. nat.) vorgelegt von
M ARIE A NNE H ANEBUTH
an der Mathematisch-Naturwissenschaftlichen Sektion, Fachbereich Biologie, Molekulare Mikrobiologie der
Tag der mündlichen Prüfung 05.08.2016 1. Referentin: Elke Deuerling 2. Referent: Martin Scheffner 3. Referent: Matthias P. Mayer
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-355592
Functions and molecular mechanisms of eukaryotic ribosome-associated chaperones
D ISSERTATION
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften doctor rerum naturalium (Dr. rer. nat.) vorgelegt von
M ARIE A NNE H ANEBUTH
an der Mathematisch-Naturwissenschaftlichen Sektion, Fachbereich Biologie, Molekulare Mikrobiologie der
Tag der mündlichen Prüfung 05.08.2016
1. Referentin: Elke Deuerling
2. Referent: Martin Scheffner
3. Referent: Matthias P. Mayer
“Wichtig ist dass man nicht aufhört zu fragen!”
Albert Einstein
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Summary (English version)
De novo synthesis of proteins by the ribosome is a highly complex and error-prone process that involves a plethora of accessory factors. Newly synthesized polypeptides must fold into defined three-dimensional structures to become biologically active. Others need to be targeted to different cellular compartments or to be integrated into large macromolecular assemblies. In all kingdoms of life various ribosome-associated factors directly interact with nascent chains and guide their folding, modification, targeting, or degradation.
A specialized group of these factors are molecular chaperones that represent central elements of the cellular proteostasis network. Eukaryotes evolved two highly conserved assemblies at the ribosomal exit site, an Hsp70- and J-protein-based system forming the stable ribosome-associated complex (RAC) and the heterodimeric nascent polypeptide-associated complex (NAC). In the yeast Saccharomyces cerevisiae RAC and an additional ribosome-bound Hsp70 chaperone, Ssb, together form a functional triad. Many aspects concerning the precise mode of action or molecular mechanism of these ribosome-associated chaperone networks are still unknown. Therefore, the major aim of this study was to investigate the cellular functions and mechanisms of yeast Ssb-RAC and nematode NAC in maintaining protein homeostasis. The scientific contribution of this work is summarized in the following section.
(A) Multivalent contacts of Ssb position this Hsp70 chaperone on ribosomes
In the yeast S. cerevisiae different Hsp70 family members are functionally distinct and only Ssb directly interacts with ribosomes. How an Hsp70 chaperone can be positioned at the translation machinery was unclear so far. This work elucidates the mechanism and molecular details underlying the ribosomal interaction of the specialized Hsp70 Ssb to support nascent polypeptide folding. Besides the contact with its ribosome-associated cofactor RAC the interaction of Ssb with the ribosome is multilayered involving direct interactions with the translation machinery via two Ssb specific regions characterized by positively charged amino acid side chains.
The key contact to ribosomes is mediated by 13 amino acid residues of the C-terminal lid, a second contact with lower affinity is provided by a KRR motif within the substrate-binding domain. Strikingly, ribosome binding of Ssb is not essential for its functionality, as a ribosome-binding mutant completely substitutes for wild type Ssb.
Autonomous interaction of Ssb with the ribosome only becomes necessary if the cofactor RAC is absent, suggesting a RAC-mediated nascent chain interaction of cytosolic Ssb. Another important result of this study is the in vitro interaction of Ssb with several kinds of model peptide substrates observed for the first time. This interaction is characterized by a very low affinity, at least in the absence of cofactors, but a similar substrate specificity if compared to other Hsp70 chaperones. Ribosome binding of Ssb via the two specific motifs in combination with its interplay with RAC allows positioning of the Hsp70 in an optimal orientation at the ribosomal tunnel exit that guarantees a productive interaction with the nascent chain. The high concentration of nascent polypeptides at the ribosomal tunnel exit allows efficient substrate binding of Ssb even with low affinity.
Hanebuth MA, Kityk R, Jain A, Fries S, Frickey T, Peter C, Mayer PM, Frydman J, Deuerling E. Submitted.
Summary
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(B) An additional role of the yeast Hsp70 Ssb during nuclear maturation of ribosomal particles
The Hsp70/40 chaperone system Ssb-RAC of the yeast S. cerevisiae is transiently connected to ribosomes to assist co-translational protein folding. In this part of the thesis it could be shown that the Hsp70 Ssb fulfills an important additional role during assembly of new ribosomal particles, both in the cytosol and at multiple steps in the nucleus. Deletion of Ssb leads to several phenotypes similar to that of cells lacking canonic ribosome biogenesis factors and the combined deletion of Ssb and some of these factors leads to synthetic growth defects.
In line with the aggregation of ribosomal proteins in the absence of Ssb it was demonstrated that the Hsp70 interacts with these aggregates ex vivo as well as with ribosomal peptide substrates in vitro. In addition, the processing of ribosomal rRNA in the nucleolus is blocked early if Ssb is missing. TAP-pulldown experiments to analyze the Ssb interactome revealed a significant interaction of Ssb with factors involved in ribosomal assembly, architecture and function. By purifying yeast nuclei it was shown that nuclear Ssb interacts significantly with ribogenesis factors and binds a large set of ribosomal proteins. It could be demonstrated that Ssb physically interacts with several stages of ribosomal precursors in the nucleus as well as in the cytosol.
Crosslinking experiments revealed a direct interaction of Ssb with diverse ribosomal proteins within a pre-ribosomal complex, most significantly with eL31 close to the ribosomal tunnel exit. Importantly, in a situation where Ssb is depleted from cytosolic translating ribosomes cells display defects in growth and translation, but fully complement the aggregation of ribosomal proteins and show normal levels of mature ribosomal particles. This proves an important nuclear function of Ssb in addition to its task as a ribosome-bound chaperone. In summary, data from this part of the study highlight a crucial function of the Hsp70 Ssb during nuclear assembly of ribosomal subunits besides its general chaperoning function on nascent polypeptides at mature ribosomes.
Hanebuth MA, Fries S, Stengel F, Deuerling E. To be submitted.
(C) The principle of antagonism ensures protein targeting specificity at the endoplasmic reticulum
Sorting of newly synthesized polypeptides to the correct cellular compartments is a fundamental process that involves ribosome-associated factors. In this study it could be demonstrated that the highly conserved nascent polypeptide-associated complex (NAC) functions as a negative regulator of ER targeting in the nematode model organism Caenorhabditis elegans. NAC does not affect the correct targeting of ribosomes to the Sec61 translocon of the ER membrane which is dependent on a positive regulator, the signal recognition particle (SRP).
The complex rather inhibits unintended interactions of ribosomes and the translocon pore by blocking their autonomous binding affinity. The contribution of this work was to investigate the interaction between ribosomes and the ER membrane in dependency of NAC by different in vitro analyses. Wild type and ribosome-binding deficient C. elegans NAC protein was used to demonstrate that NAC prevents the interaction of ribosomes and the ER membrane. Furthermore, it could be shown that NAC enhances the release of ribosomes from the ER membrane upon translational termination. These in vitro results strongly support a set of comprehensive in vivo analyses performed in C. elegans that were addressed in a close collaboration (M. Gamerdinger) and that highlight the importance of NAC as an antagonistic targeting factor to preserve protein targeting specificity.
Gamerdinger M, Hanebuth MA, Frickey T, Deuerling E (2015) Science.
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Zusammenfassung (deutsche Version)
Die ribosomale de novo Synthese von Proteinen ist ein hoch komplexer und fehleranfälliger Prozess, an dem eine Fülle von Faktoren beteiligt ist. Neu synthetisierte Proteine müssen sich in ihre dreidimensionale Struktur falten, um biologisch funktional zu sein. Andere Proteine müssen zu zellulären Kompartimenten transportiert oder in makromolekulare Komplexe eingebaut werden. In allen Domänen des Lebens interagieren verschiedenste ribosomengebundene Faktoren direkt mit der naszierenden Kette, um deren Faltung, Modifikation, Transport oder Abbau zu unterstützen. Eine spezialisierte Gruppe dieser Faktoren sind die molekularen Chaperone, welche ein zentrales Element des zellulären Proteostasenetzwerkes darstellen.
Eukaryonten haben zwei hoch konservierte Systeme evolviert, die am ribosomalen Tunnelausgang lokalisiert sind: ein Hsp70- und J-Protein basiertes System, das den stabilen ribosomenassoziierten Komplex RAC (ribosome-associated complex) bildet, und den heterodimeren Komplex NAC (nascent polypeptide-associated complex). In der Hefe Saccharomyces cerevisiae wird RAC von einem zusätzlichen ribosomengebundenen Hsp70-Chaperon (Ssb) unterstützt und zusammen bilden sie eine funktionelle Triade. Viele Aspekte bezüglich der präzisen Aufgaben dieses ribosomengebundenen Chaperonnetzwerkes oder seines molekularen Mechanismus’ sind bisher nicht aufgeklärt. Hauptziel dieser Arbeit war daher, die zelluläre Funktion von Ssb-RAC in Hefe und von NAC in Nematoden zu untersuchen sowie ihren Mechanismus bei der Aufrechterhaltung der zellulären Homöostase zu verstehen. Der wissenschaftliche Beitrag dieser Doktorarbeit ist im Folgenden zusammengefasst.
(A) Multivalente Kontakte positionieren das Hsp70-Chaperon Ssb am Ribosom
In Hefe erfüllen Mitglieder der Hsp70-Chaperonfamilie unterschiedlichste Funktionen, wobei nur Ssb direkt mit dem Ribosom interagiert. Wie genau ein Hsp70 funktionell am Ribosom positioniert werden kann ist jedoch unklar. Ergebnisse dieser Arbeit erläutern den Mechanismus sowie die molekularen Details, welche der Ribosomenbindung des spezialisierten Hsp70 Ssb zugrunde liegen. Zusätzlich zum Kontakt mit seinem Cofaktor RAC ist die Wechselwirkung von Ssb mit dem Ribosom mehrschichtig. Konservierte Regionen von Ssb mit positiv geladenen Aminosäureketten sind als Kontaktpunkte involviert. Der Schlüsselkontakt wird durch 13 Aminosäuren der C-terminalen Domäne vermittelt, eine zweite Bindestelle mit niedrigerer Affinität liegt in einem KRR Motiv innerhalb der Substratbindedomäne. Bemerkenswerterweise ist Ribosomenbindung für die Funktion von Ssb nicht essentiell, da eine Bindemutante als Ssb Ersatz fungiert. Die autonome Interaktion von Ssb mit dem Ribosom wird nur in Abwesenheit seines Cofaktors RAC notwendig, was eine RAC-vermittelte Wechselwirkung von cytosolischem Ssb mit der naszierenden Kette vermuten lässt. Ein weiteres wichtiges Resultat dieser Studie ist die erstmals beobachtete in vitro Interaktion von Ssb mit verschiedensten Peptid- Modellsubstraten. Diese Bindung ist durch eine niedrige Affinität in Abwesenheit von Cofaktoren, aber der typischen Hsp70-Substratspezifität charakterisiert. Die Ribosomenbindung von Ssb über zwei konservierte Bindemotive und in Kombination mit RAC erlaubt seine optimale Orientierung am ribosomalen Tunnelausgang und dadurch eine effiziente Interaktion mit der naszierenden Kette. Die hohe Konzentration an naszierenden Polypeptiden am Tunnelausgang ermöglicht Bindung durch Ssb auch mit niedriger Substrataffinität.
Hanebuth MA, Kityk R, Jain A, Fries S, Frickey T, Peter C, Mayer PM, Frydman J, Deuerling E. Eingereichtes Manuskript
Zusammenfassung
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(B) Eine zusätzliche Rolle des Hsp70 Ssb während der Reifung von ribosomalen Partikeln im Zellkern
In Hefe ist Ssb-RAC vorübergehend mit dem Ribosom verbunden, um die co-translationale Proteinfaltung zu unterstützen. In diesem Teil der Arbeit konnte gezeigt werden, dass Ssb eine zusätzliche Rolle bei der Assemblierung von neuen ribosomalen Partikeln im Cytosol und im Kern spielt. Die Deletion von Ssb führt zu verschiedensten Phänotypen, welche für Zellen denen klassische Ribosomenbiogenesefaktoren fehlen, typisch sind, und die kombinierte Deletion von Ssb und mancher dieser Faktoren führt zu synthetischen Wachstumsdefekten. Passend dazu, dass ribosomale Proteine in Abwesenheit von Ssb aggregieren, interagiert das Hsp70 ex vivo mit diesen Aggregaten und in vitro mit ribosomalen Peptiden. Zusätzlich ist in ssb!-Zellen auch die Prozessierung der ribosomalen rRNA im Nucleolus blockiert. TAP-pulldown Experimente, welche durchgeführt wurden um das Ssb-Interaktom zu analysieren, zeigen eine signifikante Interaktion mit Faktoren, die an der Assemblierung, Architektur und Funktion des Ribosomes beteiligt sind. Zellkernaufreinigungen zeigten, dass Ssb im Kern signifikant mit einer großen Anzahl an ribosomalen Proteinen sowie Ribosomenbiogenesefaktoren interagiert. Des Weiteren wurde Ssb in verschiedensten Stadien ribosomaler Vorläufer im Kern sowie im Cytosol nachgewiesen. Crosslinkexperimente deckten auf, dass Ssb mit unterschiedlichen Proteinen innerhalb eines prä-ribosomalen Komplexes interagiert, signifikant mit eL31, welches nahe am Tunnelausgang liegt. Interessanterweise zeigen Zellen in welchen Ssb vom Cytosol depletiert ist Defekte in Wachstum und Translation, sie komplementieren jedoch vollständig die Aggregation von ribosomalen Proteinen sowie die Menge an ribosomalen Untereinheiten. Dies verdeutlicht eine wichtige Kernfunktion von Ssb, in Ergänzung zu seiner Aufgabe als ribosomengebundenes Chaperon. Zusammenfassend heben die Daten eine entscheidende Rolle von Ssb während der Ribosomenassemblierung im Kern hervor zusätzlich zu seiner Funktion an translatierenden Ribosomen.
Hanebuth MA, Fries S, Stengel F, Deuerling E. Fertiges Manuskript
(C) Das Prinzip des Antagonismus gewährleistet Proteintargetingspezifität am endoplasmatischen Retikulum Das Sortieren von neu synthetisierten Polypeptiden zu den korrekten zellulären Kompartimenten ist ein fundamentaler Prozess, an dem ribosomenassoziierte Faktoren beteiligt sind. In dieser Studie konnte im Nematoden-Modellorganismus C. elegans gezeigt werden, dass der hoch konservierte Komplex NAC als negativer ER-targeting Regulator fungiert. Dabei beeinflusst NAC nicht das korrekte Targeting von Ribosomen an die ER-Membran und zum Sec61-Translokon welches vom positiven Regulator SRP (signal recognition particle) abhängig ist. Eher inhibiert NAC unbeabsichtigte Interaktionen zwischen Ribosom und Translokon, indem es deren autonome Bindungsaffinität blockiert. Der Beitrag dieser Arbeit war, die NAC-abhängige Interaktion zwischen Ribosomen und der ER-Membran in verschiedenen in vitro Analysen zu untersuchen.
Wildtypisches und ribosomenbindungsdefizientes NAC wurden rekombinant gereinigt und genutzt um zu zeigen, dass NAC die Interaktion von gereinigten Ribosomen mit der ER-Membran verhindert. Außerdem konnte gezeigt werden, dass NAC die Ribosomenloslösung von der ER-Membran nach der Termination der Translation verstärkt. Diese in vitro Ergebnisse unterstützen eine Reihe von umfassenden in vivo Analysen, welche in C. elegans in enger Zusammenarbeit (M. Gamerdinger) durchgeführt wurden, und unterstreichen die Bedeutung von NAC als antagonistischer Targetingfaktor zur Erhaltung der Targetingspezifität.
Gamerdinger M, Hanebuth MA, Frickey T, Deuerling E (2015) Science.
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Table of contents
Summary (English version) ...I Zusammenfassung (deutsche Version) ... III Table of contents ... V
1 Introduction ... 1
1.1 Cellular protein homeostasis ... 1
1.2 Ribosomes and protein synthesis ... 2
1.2.1 Architecture and function of ribosomes ... 2
- Discovery ... 2
- General features ... 3
- Subunits and translation ... 4
- Eukaryotic ribosomes ... 5
- The ribosomal tunnel ... 5
1.2.2 Modification, folding and targeting of nascent polypeptides ... 7
- The tunnel exit site ... 7
- Nascent chain modifications ... 7
- Nascent chain targeting by SRP ... 9
- De novo protein folding ... 10
1.2.3 Eukaryotic assembly of new ribosomal particles ... 10
- Synthesis and assembly of rRNA and r-proteins ... 11
- Ribosomal precursor particles ... 13
- Cytoplasmic quality control ... 13
- Chaperoning ribosomal proteins ... 14
- Control of ribosome assembly & degradation ... 14
1.3 Protein folding and aggregation ... 15
1.3.1 General principle of protein folding ... 15
1.3.2 De novo protein folding ... 16
1.3.3 Cellular strategies to support protein folding ... 18
1.3.4 Protein misfolding and aggregation ... 19
- The principle of aggregation ... 19
- Cellular stress responses ... 19
- Aggregate deposition and sequestration ... 21
- The role of chaperones ... 22
1.4 Molecular chaperones ... 23
1.4.1 Classes of molecular chaperones ... 23
- General classifications ... 23
- Heat shock protein families ... 24
1.4.2 The Hsp70/40 chaperone machinery ... 25
- Structure and function of Hsp70 chaperones ... 25
- The J-domain Hsp40 co-chaperones ... 29
- Nucleotide exchange factors ... 30
1.4.3 Ribosome-associated chaperones ... 30
1.4.4 The nascent polypeptide-associated complex NAC ... 32
1.4.5 The Ssb-RAC triad of Saccharomyces cerevisiae ... 34
2 Aims of this thesis ... 40
Table of contents
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3 Results, discussion and outlook (part A) ... 42
Multivalent contacts of the Hsp70 chaperone Ssb contribute to its architecture on ribosomes and nascent polypeptide interaction ... 42
3.1 Results (part A) ... 42
3.1.1 The Hsp70 Ssb possesses two non conserved basic regions within its substrate binding domain and the C-terminal lid ... 42
3.1.2 Length and charge of the C-terminus of Ssb is crucial for ribosome binding ... 43
3.1.3 The specific KRR motif in the substrate-binding domain of Ssb is not essential but contributes to ribosome binding ... 47
3.1.4 Ssb lacking ribosome-binding ability complements translation, protein folding and ribogenesis defects of ssb1,2! cells ... 48
3.1.5 The activity of ribosome-binding deficient Ssb critically depends on RAC ... 50
3.1.6 Ssb shows low affinity interaction with classic Hsp70 substrate peptides in vitro ... 54
3.2 Discussion and Outlook (part A) ... 59
4 Results, discussion and outlook (part B) ... 63
An important role of the yeast Hsp70 Ssb during nuclear maturation of ribosomal particles ... 63
4.1 Results (part B) ... 63
4.1.1 Ssb recognizes ssb1,2! aggregates ex vivo and interacts with ribosomal peptide substrates in vitro ... 63
4.1.2 Loss of Ssb blocks efficient 35S rRNA processing in the nucleolus ... 66
4.1.3 Ssb1,2! cells have phenotypes similar to yeast strains lacking ribosome biogenesis factors ... 67
4.1.4 Ssb1 interacts with factors predominantly involved in early nuclear ribogenesis ... 68
4.1.5 Ssb associates with ribosomal precursor complexes in the nucleus and cytosol and crosslinks to proteins of the pre-60S particle ... 71
4.1.6 Ssb contains a functional but non-essential nuclear export signal and partially localizes to the nucleus ... 74
4.1.7 Nuclear accumulation of Ssb complements all aspects of ribosome biogenesis but results in translational defects ... 76
4.2 Discussion and outlook (part B) ... 81
5 Results, discussion and outlook (part C) ... 86
The principle of antagonism ensures protein targeting specificity at the endoplasmic reticulum ... 86
5.1 Contributions ... 86
5.2 Objective ... 86
5.3 Results from own contribution ... 87
5.3.1 Mutation of the beta-NAC RRK motif abolishes ribosome binding in vitro ... 87
5.3.2 NAC prevents interaction of ribosomes and microsomes ... 88
5.3.3 NAC releases ribosomes from microsomes ... 88
5.4 Summary, discussion and outlook ... 89
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6 Materials and methods ... 94
6.1 Materials ... 94
6.1.1 Chemicals ... 94
6.1.2 Growth media and general buffer ... 94
6.1.3 Antibiotics, inductors and inhibitors ... 95
6.1.4 Enzymes and antibodies ... 95
6.1.5 Dyes, kits and markers ... 95
6.1.6 Primer ... 96
6.1.7 Plasmids ... 98
6.1.8 E. coli strains ... 99
6.1.9 S. cerevisiae strains ... 99
6.1.10 Peptides ... 99
6.2 Methods ... 99
6.2.1 Cloning and mutagenesis ... 99
- Polymerase chain reaction (PCR) ... 100
- Mutagenesis ... 100
- Fusion PCR ... 100
- Agarose gel electrophoresis ... 100
- DNA digestion, dephosphorylation and ligation ... 100
6.2.2 Transformation of bacteria and yeast cells ... 100
- Generation of chemically competent E. coli cells ... 100
- Transformation of E. coli cells ... 101
- Transformation of S. cerevisiae (fast / high efficiency protocol) ... 101
6.2.3 Growth analyses, cultivation and storage of cells ... 101
- Cultivation and storage of E. coli strains ... 101
- Cultivation and storage of S. cerevisiae strains ... 101
- Growth analyses (spot test) ... 101
6.2.4 SDS-PAGE and Western blot analysis ... 101
- Yeast lysate preparation ... 101
- TCA precipitation ... 102
- Bradford assay / BCA assay ... 102
- SDS-PAGE ... 102
- Bis-Tris-SDS-PAGE ... 102
- Tricine-PAGE ... 102
- Coomassie staining ... 102
- Silver staining ... 102
- Western blot analysis ... 102
6.2.5 Polysome profiling ... 103
6.2.6 Ribosome sedimentation assay ... 103
6.2.7 Preparation of cellular aggregates ... 103
6.2.8 Northern blot analysis ... 103
- Construction of RNA-probes ... 103
- Isolation of yeast RNA ... 104
- Northern blotting ... 104
6.2.9 Microscopy ... 104
6.2.10 TAP-pulldown ... 104
- General protocol ... 104
- Nuclear vs. cytosolic interactome ... 105
- ATP-dependency ... 105
- Crosslinking of TAP-elutions ... 105
Table of contents
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6.2.11 Isolation of yeast nuclei ... 105
6.2.12 Isolation of yeast ribosomes and generation of ribosomal peptides ... 106
- Purification of yeast ribosomes ... 106
- Isolation of r-proteins ... 106
- Generation of r-peptides ... 106
6.2.13 Expression and purification of proteins ... 106
- Purification of Ssb1 constructs ... 106
- Purification of DnaK ... 107
- Purification of NAC constructs ... 107
6.2.14 Protein-ribosome interaction study (add back) ... 107
- Binding of Ssb1 variants to ribosomes ... 107
- Binding of NAC variants to ribosomes ... 108
6.2.15 In vitro protein-peptide interaction studies ... 108
- Gel filtration approach ... 108
- Fluorescence anisotropy measurements ... 108
- Peptide release measurements ... 108
6.2.16 In silico protein-peptide interaction studies ... 108
- Modeling of the Ssb1 structure ... 108
- Ssb1 simulations ... 109
6.2.17 In vitro ribosome-translocon binding analyses ... 109
- Generation of salt-stripped rough microsomes ... 109
- Binding of ribosomes to microsomes ... 109
- Generation of native rough microsomes ... 109
- Detachment of ribosomes from native rough microsomes ... 109
7 Abbreviations ... 110
8 List of figures ... 112
9 Literature ... 114
9.1 Publications and manuscripts of this thesis ... 114
9.1.1 Publications ... 114
9.1.2 Manuscripts ... 114
9.2 Cited references ... 115
10 Danksagung (Acknowledgements) ... 130
Appendix ... IX I Identified protein hits ... IX I.I Ssb1-TAP plus RAC ... IX I.II Ssb1-TAP minus RAC ... X I.III Ssb1-TAP (nuclear fraction) ... X I.IV Ssb1-TAP (cytosolic fraction) ... X I II Identified crosslinks ... X I II.I Inter-links upon Nop7-TAP pulldown ... X I II.II Intra-links upon Nop7-TAP pulldown ... X III
Manuscript (I): Multivalent contacts of the Hsp70 chaperone Ssb contribute to its architecture on ribosomes and nascent polypeptide interaction
Manuscript (II): An important role of the yeast Hsp70 Ssb during nuclear maturation of ribosomal particles Paper: The principle of antagonism ensures protein targeting specificity at the endoplasmic reticulum
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" " Introduction
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1 Introduction
1.1 Cellular protein homeostasis
The primary sequence of all proteins largely determines their cellular function, however, polypeptides can adopt different folding states in response to changes of the environment, some of which may be even deleterious to the organism (POWERS & BALCH, 2013). Therefore, species of all kingdoms of life have evolved a protein homeostasis, or proteostasis network that helps to maintain the cellular protein composition and to adapt it during different environmental, developmental or age-related challenges (BALCH et al., 2008). These networks consist of chaperones and folding factors, degradation components, signaling pathways and specialized compartmentalized modules that manage protein folding in response to environmental stimuli and variation (POWERS & BALCH, 2013). Thus, the concentration of a protein, its three-dimensional conformation and its ability to interact with other proteins as well as its sub-cellular localization can be tightly controlled ranging from the adjustment of protein synthesis to its deposition or final degradation (Fig. 1).
Proteostasis is maintained by a complex network which comprises several pathways that control protein synthesis, folding and unfolding, trafficking, aggregation and disaggregation as well as terminal deposition or degradation of proteins (POWERS et al., 2009). One process that influences proteostasis amongst others is the protein quality control, which regulates the constant competition between protein folding and degradation.
Figure 1: Pathways of the cellular proteostasis network. Arrows indicate proteostasis pathways that are influenced by regulators. All components illustrated can be adjusted according to the respective cellular conditions.
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The protein quality control system includes components offering alternatives to protein degradation, like a large set of chaperones and folding enzymes that enhance the cellular protein folding efficiency (BALCH et al., 2008).
To maintaining the cellular proteome, the temporal adaptation of proteostasis is necessary as a result of organismal development, changing cellular or environmental conditions or age-related accumulation of misfolded proteins. Therefore, cells use stress sensors and inducible pathways like the cellular heat shock response (HSR) (WESTERHEIDE & MORIMOTO, 2005) or the unfolded protein response (UPR) e.g. of the endoplasmic reticulum (RON &WALTER, 2007) to quickly adapt to the loss of proteostatic control.
Due to the complexity of the proteostasis network defects in any one branch can provoke breakdown of the entire network and become manifest in numerous metabolic, oncologic, cardiovascular and neurodegenerative diseases (DOUGLAS &DILLIN, 2010). There are different reasons that may decrease the ability of the proteostasis network to cope with the cellular proteome like inherited misfolding-prone proteins, metabolic and environmental stress or aging that goes along with decreasing cellular proteostasis capacity and increasing protein damage. A misbalance of the complex proteostasis network may cause illnesses that include loss-of-function diseases like cystic fibrosis or gain-of-toxic-function diseases like Alzheimer's, Parkinson's or Huntington's disease (POWERS et al., 2009).
The following chapters will highlight the basic principles and mechanisms as well as the importance of protein folding and the involvement of molecular chaperones in this process. The main focus will be the role of ribosome-associated factors that directly control folding, modification or targeting of newly synthesized polypeptides and therefore represent an important branch of the cellular proteostasis network.
1.2 Ribosomes and protein synthesis
“The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential informations. It states that such information cannot be transferred back from protein to either protein or nucleic acid” (CRICK, 1970). With this statement Francis Crick laid the foundation of understanding the flow of the genetic information within all biological systems. The central dogma describes the process of transcription that transforms the genetic sequence information of the DNA into a messenger, the mRNA, which is further translated into a polypeptide composed of amino acids during the process of translation. This procedure of protein synthesis is provided by the ribosome, a large and highly complex cellular machine that can be found in all living cells.
1.2.1 Architecture and function of ribosomes
Discovery: Ribosomes were first observed in the mid-1950s (PALADE, 1955), since 1958 they are termed ribosomes, due to their composition of a “body” (greek: soma) containing ribonucleic acid (ROBERTS, 1958).
Since 2000 the structure of the prokaryotic 70S ribosome is known (BAN et al., 2000; SCHLUENZEN et al., 2000;
WIMBERLY et al., 2000) and since 2011 the more complex structure of the eukaryotic 80S ribosome is solved (Fig. 2) (BEN-SHEM et al., 2011; KLINGE et al., 2011; RABL et al., 2011), which led to a deeper understanding of processes related to the translation machine. Each ribosomal subunit has its characteristic structural landmarks:
the small subunit displays a body, platform, head and beak, while the large subunit has a more massive, rounded body, with central and lateral protuberances, the acidic stalks (GAMALINDA &WOOLFORD, 2015).
" " Introduction
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Figure 2: Structure and architecture of the eukaryotic 80S ribosome. Ribosomal proteins are colored and labeled according to the new nomenclature (BAN et al., 2014). The ribosomal rRNA is shown in grey. Proteins colored in red, orange and yellow belong to the large subunit, proteins colored in blue, cyan and teal belong to the small subunit. If a protein is partially obstructed from view, it may be labeled more than once, even though all ribosomal proteins appear in only a single copy. A) View from the E-site. B) View from the 40S small subunit side. C) View from the A-site. D) View from the 60S large subunit side (adapted from YUSOPOVA &YUSOPOV, 2014).
General features: Ribosomes are ribonucleoprotein particles as they are composed of ribosomal RNA (rRNA) and proteins (r-proteins). They represent central machineries of all living cells that convert the genetic information of the mRNA into proteins during the process of translation. Ribosomes are highly abundant enzymes that can make up ~ 30 % of the total cell mass, and ~ 80 % of the total RNA is rRNA (WARNER, 1999).
Furthermore, prokaryotic cells may possess up to 105 ribosomes which synthesize proteins with an elongation rate of ~15-20 amino acids per second. Eukaryotic cells, in contrast, may contain several millions of ribosomes depending on the cellular state but are characterized by a slower elongation rate of ~5-7 amino acids per second (WEGRZYN & DEUERLING, 2005).
All types of ribosomes are composed of a small and a large subunit that both contain r-proteins and rRNA. Their composition within the different kingdoms of life varies strongly resulting e.g. in a 2.3 MDa ribosome of
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bacteria in contrast to the 4.3 MDa ribosome of human cells (Fig. 3). The functional divergence underlying these structural differences are still not fully understood. Despite these variations the different ribosomal versions share one common core of 34 r-proteins and 3 rRNAs. 4400 bases of the ribosomal RNA are conserved, which harbor the major functional centers of the ribosome as a ribozyme, such as the decoding site, the peptidyltransferase center and the tRNA-binding site. Recent data even describe structural and functional variations of ribosomes within one species, which might change under different conditions of growth or stress (GUNDERSON et al., 1987; MCINTOSH & WARNER, 2007; GILBERT, 2011). In general, the prokaryotic 70S ribosome (3 rRNAs; 54 r-proteins) is composed of a small 30S (16S rRNA; 21 r-proteins) and a large 50S (5S, 23S rRNA; 33 r-proteins) subunit, whereas the eukaryotic 80S ribosome (4 rRNAs, 79 r-proteins) e.g. of Saccharomyces cerevisiae is composed of a small 40S (18S rRNA; 33 r-proteins) and a large 60S (5S, 5.8S, 25S rRNA; 46 r-proteins) subunit (Fig. 3).
Figure 3: Composition of bacterial and eukaryotic ribosomes and the common core. Bacterial and eukaryotic ribosomes share conserved rRNA (light blue) and r-proteins (light red) in addition to their own set of proteins, extensions and insertions in conserved proteins (dark red) and extension segments in ribosomal RNA (dark blue). Dashed lines indicate positions of flexible stalks; for simplicity these lines are not shown in the other structures. The 80S structure of higher eukaryotes has recently be determined (KHATTER et al., 2015). This figure shows a prediction, is highly similar to the yeast ribosome, based on genetic analysis and cryo-EM studies (adapted from MELNICOV et al., 2012).
Subunits and translation: Within the translating ribosome the two subunits fulfill distinct functions: The small subunit is responsible for decoding the mRNA sequence upon initiation of translation as it selects the correct aminoacyl-tRNA. Its major functional regions are the mRNA path, the decoding center and the tRNA binding sites. The A-site serves for binding the aminoacyl-tRNA, the P-site holds the tRNA that carries the nascent polypeptide and the E-site marks the exit of the dissociated empty tRNA. During translational elongation the tRNAs translocate from the A- to the P-site and finally dissociate from the ribosome in the E-site. The large subunit catalyzes peptide-bond formation within the peptidyltransferase center (PTC). Its further functional sites are the tRNA binding sites (A, P and E) as well as the peptide exit tunnel that extends through the body of the
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whole subunit. The PTC is located at the entrance to the polypeptide tunnel in a conserved region on the interface to the small subunit. Upon peptide-bond formation in the PTC, the nascent polypeptide chain is transferred from the peptidyl-tRNA in the P-site to the aminoacyl-tRNA in the A-site, thus extending the nascent chain by one amino acid (aa). Subsequently, the ribosome translocates on the mRNA towards the 3’ end to the next codon and a new aminoacyl-tRNA can bind via its anticodon. During translation the two ribosomal subunits rotate and swivel like a ratchet relative to one another to allow translocation of tRNA and mRNA along the subunit interface (FRANK &AGRAWAL, 2000; HORAN & NOLLER, 2007; ZHANG et al., 2009). The ribosome translates the mRNA sequence from its 5’ to 3’ end into a new polypeptide that is synthesized from its N-terminus to the C-terminus. Upon reaching a stop codon within the mRNA to which no tRNA matches, translation is terminated, the ribosomal subunits dissociate and the nascent polypeptide is released. Translation is assisted by a plethora of factors that provide energy and control the different steps of this complex process. They are termed initiation factors (IFs in prokaryotes, eIFs in eukaryotes), elongation factors (EFs or eEFs), release factors (RFs or eRFs) and recycling factors and differ in their composition and complexity between bacteria and eukaryotes (MELNICOV et al., 2012).
Eukaryotic ribosomes: The eukaryotic 80S ribosome acquired several architecture- and assembly-related features that cannot be found in the structure of the bacterial ribosome. These features include long dynamic rRNA helices on the solvent side of both ribosomal subunits, larger protein clusters assembled round the single-stranded rRNA and unusual interactions mediated by protein tails (MELNICOV et al., 2012; YUSOPOVA &
YUSOPOV, 2014). Remarkably, the interface between both subunits is highly conserved between prokaryotic and eukaryotic ribosomes, indicating that subunit association and the basic mechanism of tRNA recruitment involves conserved mechanisms (SPAHN et al., 2001). Furthermore, the peptidyltransferase active sites show also a high degree of structural conservation, although there are notable differences in the surrounding area (KLINGE
et al., 2011). Many eukaryotic ribosomal proteins, like eL4, uL14, eL22, eL29 or uS3, show unusual folds and contain long tails and loops extending from globular domains. Most of these elongated proteins are not buried within the 80S ribosome but are located at the surface where they can potentially interact with other proteins, likely to control ribosomal function (BEN-SHEM et al., 2011; KLINGE et al., 2011; RABL et al., 2011). Several of these elongated r-proteins interact with long rRNA segments, so-called expansion segments (ES) (MELNICOV
et al., 2012). Some of these segments are tightly associated with rRNAs or r-proteins whereas others comprise long helices that are attached to the ribosome only at their basis, e.g. ES27L (ARMACHE et al., 2010). These types of elongated rRNA segments can adopt different conformations, but the biological relevance of this feature is still not fully understood. In case of ES27L it was suggested that it docks non-ribosomal factors like chaperones or modifying enzymes to the nascent chain that emerges through the ribosomal tunnel exit (BECKMANN et al., 2001).
The ribosomal tunnel: The ribosomal tunnel - through which the nascent polypeptide extends while it is still bound to the peptidyltransferase center - connects the PTC with the cytosol. The tunnel with a length of approximately 80-100 Å and a diameter of about 10-20 Å (NISSEN et al., 2000) is large enough to protect a segment of the growing polypeptide of 30-35 aa in an extended conformation (HARDESTY &KRAMER, 2001) but limits protein folding to the formation of alpha-helices (BHUSHAN et al., 2010). Only in the last 20 Å of the
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tunnel also tertiary structures of the nascent chain might be allowed, as the tunnel widens up in this region (KOSOLAPOV &DEUTSCH, 2009).
In prokaryotes, the tunnel wall is mainly formed by the conserved parts of the 23S rRNA and contains loops of the proteins uL4, uL22 and uL23 (Fig. 4A/B) (BAN et al., 2000; HARMS et al., 2001). In eukaryotes, the area corresponding to the bacteria-specific uL23 overlaps with eL39 (Fig. 4) (BEN-SHEM et al., 2011; KLINGE
et al., 2011). Hydrated polar groups primarily line the tunnel wall that lacks extended hydrophobic patches, which allows passage of all kinds of nascent chains (NISSEN et al., 2000). Together with rRNA the highly conserved proteins uL4 and uL22 form a constriction of the exit tunnel, located ~30 Å from the PTC. This constriction is even narrower in eukaryotic ribosomes, which is suggested to protect the translating 80S ribosome from some macrolide antibiotics that hamper bacterial translation (TU et al., 2005).
Figure 4: The ribosomal tunnel and exit site of prokaryotic and eukaryotic ribosomes. A) Prokaryotic peptide tunnel exit indicated on the slice of the large subunit. Ribosomal proteins involved in the tunnel structure are colored. B) Eukaryotic 60S as in A) C) Structure of the peptide tunnel exit on the solvent side of the 50S subunit. Coloring as in A).
D) Eukaryotic 60S as in C) (adapted from YUSOPOVA &YUSOPOV, 2014).
Until recently the ribosomal tunnel was thought to be inert, however, several studies provide evidence that it plays a more active role in regulating translation and early protein folding. Thus, some proteins interact with the tunnel wall during their synthesis which might regulate translation of the downstream open reading frame and modulate ribosome activity e.g. by the induction of stalling (LOVETT &ROGERS, 1996; TENSON &EHRENBERG, 2002; MANKIN, 2006; HOOD et al., 2009). The tunnel carries an overall negative potential (LU et al., 2007)
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which leads to electrostatic interactions of nascent polypeptides containing elongated stretches of consecutive positively charged amino acids and a transient elongation arrest (LU & DEUTSCH, 2008). In addition to translational regulation, the ribosomal tunnel also provides a defined environment for first protein folding.
Recent data suggest a communication between the nascent chain within the tunnel and exit site proteins that might regulate the recruitment of downstream acting factors (WILSON &BECKMANN, 2011). Taken together, the tunnel represents a universally conserved functional domain of the ribosome, which appears to play a diverse role in protein biogenesis.
1.2.2 Modification, folding and targeting of nascent polypeptides
The tunnel exit site: During translation the growing nascent polypeptide passes through the exit tunnel and emerges at the solvent side, where it undergoes processing and early folding. The exit site is surrounded by rRNA, e.g. by the expansion segment ES27L, as well as by several ribosomal proteins. Four of these, uL22, uL23, uL24 and uL29, are conserved whereas eL19, eL31 and eL39 are not found in the prokaryotic ribosome (Fig. 4C/D) (YUSOPOVA & YUSOPOV, 2014). These variations are associated with the different N-termini processing of nascent chains in bacteria and eukaryotes: In prokaryotes, the nascent polypeptide contains a formyl group at the N-terminus which is due to the special formylation-modification of the aminoacylated initiator tRNA. As the eukaryotic initiator tRNA is not formylated, the positions corresponding to the bacterial bL17 and bL32 are occupied by the non-homologous protein eL31 (Fig. 4D) that serves as a global docking site for several non-ribosomal factors (MELNICOV et al., 2012).
The ribosome and especially the region of the tunnel exit serves as a platform for the spatially and temporally regulated association of targeting factors, enzymes or chaperones that act on the nascent polypeptide as it emerges from the tunnel exit (Fig. 5). Thus, the translation machinery provides opportunities to coordinate the synthesis of a polypeptide with its targeting or folding process (KRAMER et al., 2009). Amongst the early acting proteins are targeting factors like the signal recognition particle (SRP), and proteins that chemically modify the nascent chain like methionine aminopeptidases (MAPs), peptide deformylases (PDFs) or N-acetyl transferases (NATs). Chaperones like the bacterial Trigger Factor (TF) or the eukaryotic ribosome-associated factors NAC (nascent polypeptide-associated complex) and Ssb-RAC (ribosome-associated complex) guide initial protein folding. The ribosome quality control complex (RQC) and the Ccr4-Not complex are involved in mRNA surveillance as well as nascent chain ubiquitination and degradation (Fig. 5).
Nascent chain modifications: Many cellular proteins are subjected to chemical modifications some of which occur already during protein biosynthesis at the ribosomal tunnel exit. Among the factors that interact with nascent chains are enzymes that are involved in the N-terminal deformylation and methionine excision or in the enzymatic modification of the nascent chain by acetylation. Such modification is thought to influence the half-life of a protein as well as its ability to interact with other factors; thus, modification affects both the function and stability of a protein (KRAMER et al., 2009).
Methionine aminopeptidases that remove the N-terminal methionine of 30-50 % of nascent chains are ubiquitous and essential in all kingdoms (GIGLIONE et al., 2004). One homologue is found in bacteria and two in eukaryotes. MAPs can act co-translationally and bind directly to the ribosome (VETRO &CHANG, 2002) but need a minimal size of the nascent polypeptide of 40 aa for full functionality (BALL & KÄSBERG, 1973). At the ribosome MAPs bind via a positively charged loop to bL17 and uL23 which is in close proximity to the tunnel
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exit (SANDIKCI et al., 2013). In bacteria or mitochondria peptide deformylases (PDF) have to initially remove the formyl moiety from the N-terminal methionine before MAPs can act (PINE, 1969). PDFs associate with the ribosome via a C-terminal helix that binds to a groove between uL22 and bL32 (BINGEL-ERLENMEYER
et al., 2008). They have fast association and dissociation kinetics and compete with MAPs for ribosome binding (SANDIKCIet al., 2013). Simultaneous binding of MAP and PDF with the targeting factor SRP is possible in bacteria, indicating co-translational processing and targeting of nascent chains. In contrast, premature recruitment of the chaperone Trigger Factor or early polypeptide folding negatively affects the processing efficiency (SANDIKCI et al., 2013).
N-terminal acetylation is another modification that occurs co-translationally in approximately 80-90 % of mammalian cytosolic proteins, in 50 % of yeast proteins and only occasionally in prokaryotes. Several studies provide evidence for N-acetyl transferase activity, however the biological relevance of this modification is still unclear (POLEVODA &SHERMAN, 2003). Eukaryotes possess at least five different types of NATs, some of which acetylate the N-terminal methionine whereas others rely on the previous activity of MAPs. In yeast the N-acetyltransferase NatA could be crosslinked to nascent polypeptides, indicating its localization close to the ribosomal tunnel exit (GAUTSCHI et al., 2003) and pulldown experiments suggest NatA binding to the ribosomal proteins uL23 and uL29 (POLEVODA et al., 2008).
Figure 5: Co-translational processes on the nascent chain. Ribosome-associated factors interact with the nascent polypeptide and initiate transport to desired destinations, protein modification and/or folding by chaperones. Quality control factors prevent the accumulation of aberrant mRNAs and misfolded proteins. * deformylation and Trigger Factor (TF) are restricted to prokaryotes; # myristoylation, ubiquitination and the presence of the RQC and the Ccr4-Not complex are restricted to eukaryotes; + Ssb is specific to fungi.
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After methionine excision 1-4 % of eukaryotic proteins are subjected to co-translational myristoylation, which is the covalent attachment of a myristic acid moiety to an N-terminal glycine residue. It still remains elusive how myristoylation and translation are coupled (MEINNEL &GIGLIONE, 2008) and which function this modification provides. Proteins carrying a myristic acid modification are mostly targeted to lipid membranes where they are thought to play a role in the cellular communication network (GIGLIONE et al., 2015).
Nascent chain targeting by SRP: The signal recognition particle SRP is an abundant and universally conserved cytosolic ribonucleoprotein that co-translationally recognizes proteins containing a signal sequences (ss) or a transmembrane domain. These secretory or transmembrane proteins are targeted by SRP to the inner membrane of bacteria or to the endoplasmic reticulum (ER) in eukaryotes. Upon recognition of an N-terminal hydrophobic signal sequence, SRP binds to the ribosome nascent chain complex (RNC) and targets it to the membrane integrated SRP receptor SR (FtsY in bacteria). After transferring the RNC to the Sec61 translocation channel in the ER membrane (SecYEG in bacteria), the SRP-receptor complex dissociates. This cycle of protein targeting is regulated by guanosine triphosphatases (GTPases) present in SRP and its receptor, and GTP is hydrolyzed in a shared active site. The composition of SRP and its receptor varies strongly between kingdoms, although a combination of proteins and RNA is characteristic. In eukaryotes SRP consists of six proteins (SRP9, 14, 68, 72, 19 and 54) and the 7SL RNA with GTPase activity. The prokaryotic SRP is much less complex and composed of the 4.5S RNA and one protein with GTPase activity (Ffh = Fifty-four homologue) (GRUDNIK et al., 2009). The structure of eukaryotic SRP can be divided into an Alu- and a S-domain. The Alu-domain contains the SRP9/14 heterodimer and is involved in the elongation arrest of translation. The S-domain is composed of the remaining proteins and provides ss-binding and receptor interaction (HALIC et al., 2004). The overall composition of yeast SRP is similar to that of higher eukaryotes but with notable differences: it is characterized by the functional replacement of SRP9/14 by an SRP14 homodimer and the presence of a yeast-specific protein, SRP21, which is structurally related to SRP9. Furthermore, the yeast SRP RNA possesses a specific structure due to frequent insertions (STRUB et al., 1999), and the yeast homologue of SRP19 (Sec65) is a much larger protein (HANN &
WALTER, 1991).
SRP recognizes signal seqeunces that vary in their composition and length but share the common feature of a hydrophobic core region (VON HEIJNE, 1990). Remarkably, short nascent chains that are still enclosed within the ribosomal tunnel are sufficient for recruiting bacterial SRP, that possesses now a ~100-fold increased affinity for the translation machinery (BORNEMANN et al., 2008). This indicates a communication of tunnel wall proteins with those of the exit site. The transfer of this signal from the inside of the tunnel to the ribosomal surface occurs via a loop of the exit site protein uL23 that reaches into the tunnel (BORNEMANN et al., 2008). A similar mechanism could be observed in yeast, where SRP shows increased affinity for RNCs harboring nascent chains in the exit tunnel that contain a membrane anchor sequence (BERNDT et al., 2009). SRP contacts the ribosome via SRP54 and parts of its RNA and binds to the exit site proteins uL23 and uL29 (CROSS et al., 2009). Both, uL23 and either uL29 (HALIC et al., 2006; BECKER et al., 2009) or eL29 (VOORHEES et al., 2014) are also involved in the docking of RNCs to the translocon pore Sec61.
SRP binding to the ribosome induces arrest of translation elongation regulated via SRP9/14 and the RNA part of the Alu domain (THOMAS et al., 1997); a feature that is not found in bacteria. Upon translational arrest SRP delivers the RNC to the Sec61 translocon by interaction with its receptor in the ER membrane (HALIC
et al., 2006). The RNC engages the Sec61 translocon, which leads to resumption of translation, enabling the
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nascent chain to be directly transported into the ER lumen where it can fold into its final conformation. During translocation enzymes such as oligosaccharyl transferases (OST) and signal peptidases (SPase) can associate with the translocon and either N-glycosylate or cleave the signal peptide from the translocating nascent chain (NYATHI et al., 2013).
Although the exact interplay and spatial-temporal coordination of SRP and other ribosome-associated factors is not fully understood, studies in bacteria suggest the following order: SRP is the first factor interacting with the nascent chain (! 25 aa) followed by PDF and MAP (! 44 aa) which exclude each other from binding. Lastly TF binds nascent chains with a length of ~100 aa (SANDIKCI et al., 2013). In eukaryotes, where the composition of ribosome-associated factors is considerably more complex, further analyses are necessary to better understand the binding modes of all factors at the ribosome. A recent study analyzing binding of different factors to the universal adaptor site at the tunnel exit suggests that in the absence of NAC MAPs and SRP antagonize each other, proposing a role of NAC in regulating the access of MAP and SRP to the ribosome (NYATHI &POOL, 2015).
For the sake of completeness it should be mentioned that targeting in bacteria is more complex, as mainly membraneproteins are targeted co-translationally via SRP whereas periplasmic, outer membrane or secretory proteins that need to be translocated through the inner membrane into the periplasm are targeted via a late or post-translational pathway involving a set of Sec proteins (RAPOPORT, 2007).
De novo protein folding: Folding of the nascent polypeptide already starts within the ribosomal exit tunnel in which alpha-helical structures can form before and after the tunnel constriction (BHUSHAN et al., 2010). The formation of tertiary structures such as beta-hairpins has also been observed near the exit 80 Å away from the PTC where the tunnel opens gradually (KOSOLAPOV &DEUTSCH, 2009).
One mRNA transcript may be translated by several ribosomes simultaneously (so-called polysomes), where the ribosomes are arranged in a staggered or pseudo-helical organization around the mRNA with the tunnel exit sites facing outward (BRANDT et al., 2009). This arrangement maximizes the distances between the different nascent polypeptides emerging from neighboring ribosomes, preventing unfavorable interactions between them.
Different sets of ribosome-associated chaperones like TF in bacteria or Ssb-RAC in yeast interact early with the emerging nascent chain to prevent unspecific interactions or misfolding and aggregation (WEGRZYN &
DEUERLING, 2005). The principle of de novo protein folding is closer illuminated in a separate chapter (1.3.2) and the different systems of ribosome-associated chaperones are described in chapter 1.4.3.
1.2.3 Eukaryotic assembly of new ribosomal particles
Ribosome biogenesis is one of the most energy consuming and complex processes of a living cell (STRUNK &
KARBSTEIN, 2009). It is tightly controlled in a hierarchical manner and involves a plethora of accessory factors.
In eukaryotes, ribosome assembly starts in the nucleolus with the synthesis of the ribosomal RNA and the formation of a large 90S ribosomal precursor complex which already includes the majority of ribosomal proteins. Subsequently, nucleolar and nuclear precursors of the large and small ribosomal subunits undergo several processing and maturation steps until they are exported to the cytosol where last maturation occurs resulting finally in translation-competent 40S and 60S ribosomal subunits (Fig. 6; VENEMA &TOLLERVEY, 1999;
RAUE, 2005; WOOLFORD &BASERGA, 2013; NERURKAR et al., 2015).
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Figure 6: Ribosome assembly of Saccharomyces cerevisiae. Simplified overview of the ribosome biogenesis pathway which starts with the synthesis of the primary transcript of rRNA by RNA-Polymerase I (Pol I) in the nucleolus; upon rRNA cleavage, the 90S pre-ribosome is separated into a pre-40S and a pre-60S ribosomal subunit, which are further processed in the nucleoplasm, exported through the nuclear pore complexes and finally matured in the cytosol. Different sets of ribosome biogenesis factors bind to and dissociate from specific precursors. The sizes of the different ribosomal precursors and subunits as well as their rRNA content (in brackets) are indicated.
Synthesis and assembly of rRNA and r-proteins: The biogenesis of new ribosomal particles starts in the nucleolus with the synthesis of the rRNA by RNA-Polymerases I and III. The 18S, 5.8S and 25S rRNAs are organized in tandemly repeated operons transcribed as a 35S rRNA by RNA Pol-I, whereas Pol-III transcribes the 5S rRNA separately (Fig. 7). In yeast, the site of rRNA synthesis is the RDN1 locus containing a 1-2 Mb cluster of 150-200 rDNA tandem copies of a 9.1 kb repeat, which represents about 10 % of the yeast genome.
These polycistronic repeats comprise the sequences of the rRNAs as well as non-transcribed (NTS), external transcribed (ETS) and internal transcribed spacers (ITS) (Fig. 7; RAUE, 2005). The rRNA genes are localized in the nucleolus, a non-membrane bound substructure of the nucleus that is a “genetically determined element”, as its localization depends on the position of rDNA, rRNA, r-proteins and accessory factors (RASKA et al., 2006).
There are three prominent structural entities within the nucleolus: the fibrillar center (FC) surrounded by the dense fibrillar component (DFC) which in turn is surrounded by the granular component (GC). rRNA transcription is generally thought to occur in the FC at its boundary with the DFC and during subsequent processing and assembly the pre-rRNA moves from the DFC to the GC (RAUE, 2005). Upon transcription of the 35S primary transcript, internal and external transcribed spacers are stepwise removed which goes along with rRNA modifications by small nucleolar ribonucleoprotein particles (snoRNPs) (VENEMA &TOLLERVEY, 1999).
These particles consist of a guiding snoRNA that contains an antisense element to the surroundings of the base to be modified, and a snoProtein that introduces either 2’-O-ribose-methylation (box C/D snoRNPs) or pseudouridylation (box H/ACA snoRNPs). The precise effects of these base modifications that take place in functionally important domains are still under investigation. Pseudouridin adds another option of H-bonding and methylated RNA is protected from hydrolysis indicating that rRNA modification serves for stabilization, facilitated folding and enhanced r-protein interaction (BACHELLERIE et al., 2002). Cleavage of the 35S rRNA at A0, A1 and A2 (Fig. 7) results in the 20S rRNA embedded in the pre-40S small ribosomal subunit and in the 27S rRNA within the pre-60S precursor. In rapidly dividing cells the majority of pre-rRNA undergoes co-transcriptional cleavage at the A0, A1 and A2 sites before transcription is completed. The pre-40S ribosome is then exported through the nuclear pore complexes into the cytosol, where the 20S rRNA is finally matured to the 18S form. Processing of the pre-60S particles is more complex, involves several pre-stages and can proceed in two different ways: 85 % of the 27S rRNA maturation involves cleavage at the A3 site and processing at B1S,
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resulting via the 7S precursor in a small version of the 5.8SS rRNA and the 25S rRNA (Fig. 7). 15 % of the 27S rRNA precursor processing takes place at position B1L resulting in a 6 nt longer version of the 5.8SL rRNA as well as the normal 25S rRNA (VENEMA &TOLLERVEY, 1999). Mutants producing only one of the two 5.8S versions are viable (RAUE, 2005) and the physiological significance of the phenomenon of having two different versions of this rRNA remains unclear, although it suggests a mode of structurally and probably also functionally adapting the translation machinery.
Figure 7: Simplified scheme of synthesis and processing of eukaryotic rRNA. The 35S pre-rRNA is transcribed by RNA polymerase I (Pol-I) and contains sequences of the 18S, 5.8S and 25S rRNAs flanked and separated by external (ETS) and internal transcribed spacers (ITS). Non-transcribed spacers (NTS) are only present in the rDNA locus; the 5S rRNA is transcribed by RNA polymerase III (Pol-III). Spacer sequences are removed from pre-rRNA by the indicated series of endonucleolytic and exonucleolytic processing steps within pre-ribosomes and in parallel to base modifications. Assembly starts in the nucleolus, later steps occur in the nucleoplasm and, after nuclear pore passage, in the cytoplasm.
Genes encoding r-proteins are spread over the entire genome and show some special features in comparison to other yeast genes. First, most r-proteins are encoded by duplicated genes (59 of 79) resulting in an A and B version of the respective r-protein (WOOLFORD &BASERGA, 2013) and, although yeast is characterized by only few genes containing introns, many of the r-genes contain 1-2 of these (102 of 138) (PLANTA, 1997). Thus, adapted expression and incorporation of r-protein paralogues under different conditions may provide additional flexibility to the translational machinery (WOOLFORD &BASERGA, 2013). R-proteins are translated in the cytosol and are in most cases subsequently imported into the nucleolus where they assemble into various processing intermediates in an ordered hierarchical manner. However, no detailed assembly maps are available, in contrast to the less complex bacterial ribogenesis. It is still a matter of dispute how most of the r-proteins are imported into the nucleus and directed to the particular region of the nucleolus where they are assembled (RAUE, 2005).
In vivo analyses addressing the influence of individual r-proteins on the assembly of ribosomes revealed that the
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absence of any r-protein leads to a defect in a distinct pre-rRNA processing step (WOOLFORD & BASERGA, 2013). Importantly, recent investigations discovered that several r-proteins and factors known to be critical for ribosome biogenesis may have an additional role in other cellular processes such as cell cycle control, DNA replication or secretion (DU &STILLMAN, 2002).
Ribosomal precursor particles: The different assembly and processing steps of ribosomal precursors are guided by more than 250 trans-acting, or ribosome biogenesis factors (GERHARDY et al., 2014). Most r-proteins associate with nascent ribosomes co-transcriptionally in the nucleolus and become more stably associated as assembly proceeds (DE LA CRUZ et al., 2015). Biogenesis of the small subunit machinery (SSU) already starts during transcription of the 35S rRNA and appears to be initiated by association of the U3 snoRNP with the pre-rRNA transcript (Fig. 6; DRAGON et al., 2002). Next, the majority of the 40S r-proteins associates, resulting in a 90S pre-ribosome that still lacks most of the trans-acting factors and r-proteins required for formation of the large subunit (LSU). Only the initial stages of the LSU assembly occur co-transcriptionally whereas the majority of pre-rRNA processing and remodeling takes place post-transcriptionally. The 5S rRNA transcribed by RNA-Pol-III is incorporated early into the pre-60S ribosome as part of a 5S-ribonucleoprotein particle containing uL18 and uL5 (ZHANG et al., 2007). Assembly of the LSU processome probably starts after removal of the 5’-ETS and the two ribosomal precursors are then separated by cleavage at A2 (see Fig. 7), which produces the pre-40S and pre-60S ribosomal particles. The former goes through at least two subsequent stages before it is exported to the cytoplasm for final conversion into the mature 40S species, whereas maturation of the pre-60S particle is more complex and involves at least five precursor stages until cytoplasmic maturation (Fig. 6;
RAUE, 2005). Subdomains of both the pre-40S and pre-60S particles are assembled hierarchically: the body of the SSU is assembled first, followed by the head and the mRNA-binding channel. In the case of pre-60S particle maturation the solvent-exposed surface is assembled first, followed by the surrounding of the polypeptide exit tunnel, the intersubunit surface and finally the central protuberance. Thus, the active sites of each subunit are assembled late, likely to prevent inactive nascent subunits from prematurely entering translation (DE LA CRUZ
et al., 2015). In yeast, endonucleases involved in rRNA processing are known, however the exonucleases responsible for cleavage at A0, A1, D, A2, B1L and C2 remain to be identified. Furthermore, the exact function of several trans-acting factors is currently unknown. Most ribogenesis factors are present at several consecutive assembly intermediates, whereby early precursors display the highest complexity of bound factors. During remodeling of the ribosomal precursor complexes some assembly factors are released and only a small number of new factors joins the intermediate pre-stages. A distinct set of export and shuttling factors guides the transition of the pre-40S and pre-60S subunits through the nuclear pore complexes and late acting factors as well as some r-proteins dock to cytoplasmic precursors to facilitate final maturation (WOOLFORD &BASERGA, 2013).
Cytoplasmic quality control: During late nuclear steps of ribosome assembly and export to the cytoplasm pre-ribosomes undergo a structural proofreading for correct assembly. Pre-40S particles exported to the cytoplasm contain several biogenesis factors but still lack some functionally important r-proteins. These biogenesis factors shield the pre-40S subunit from premature association with translation initiation factors as well as with 60S ribosomal subunits (STRUNK et al., 2011). Proofreading of 60S-precursors includes binding of different shuttling factors like Nmd3, which protects the subunit interface (SENGUPTA et al., 2010), Arx1 which blocks the surrounding of the polypeptide tunnel exit (BRADATSCH et al., 2012) and Mex67/Mtr2 which shield
