21 research outputs found
Nuclear Transport of Yeast Proteasomes
Proteasomes are key proteases in regulating protein homeostasis. Their holo-enzymes are composed of 40 different subunits which are arranged in a proteolytic core (CP) flanked by one to two regulatory particles (RP). Proteasomal proteolysis is essential for the degradation of proteins which control time-sensitive processes like cell cycle progression and stress response. In dividing yeast and human cells, proteasomes are primarily nuclear suggesting that proteasomal proteolysis is mainly required in the nucleus during cell proliferation. In yeast, which have a closed mitosis, proteasomes are imported into the nucleus as immature precursors via the classical import pathway. During quiescence, the reversible absence of proliferation induced by nutrient depletion or growth factor deprivation, proteasomes move from the nucleus into the cytoplasm. In the cytoplasm of quiescent yeast, proteasomes are dissociated into CP and RP and stored in membrane-less cytoplasmic foci, named proteasome storage granules (PSGs). With the resumption of growth, PSGs clear and mature proteasomes are transported into the nucleus by Blm10, a conserved 240 kDa protein and proteasome-intrinsic import receptor. How proteasomes are exported from the nucleus into the cytoplasm is unknown
Nuclear Transport of Yeast Proteasomes
Proteasomes are conserved protease complexes enriched in the nuclei of dividing yeast cells, a major site for protein degradation. If yeast cells do not proliferate and transit to quiescence, metabolic changes result in the dissociation of proteasomes into proteolytic core and regulatory complexes and their sequestration into motile cytosolic proteasome storage granuli. These granuli rapidly clear with the resumption of growth, releasing the stored proteasomes, which relocalize back to the nucleus to promote cell cycle progression. Here, I report on three models of how proteasomes are transported from the cytoplasm into the nucleus of yeast cells. The first model applies for dividing yeast and is based on the canonical pathway using classical nuclear localization sequences of proteasomal subcomplexes and the classical import receptor importin/karyopherin αβ. The second model applies for quiescent yeast cells, which resume growth and use Blm10, a HEAT-like repeat protein structurally related to karyopherin β, for nuclear import of proteasome core particles. In the third model, the fully-assembled proteasome is imported into the nucleus. Our still marginal knowledge about proteasome dynamics will inspire the discussion on how protein degradation by proteasomes may be regulated in different cellular compartments of dividing and quiescent eukaryotic cells
Nuclear Import and Biogenesis of 26S Proteasomes in Yeast Saccharomyces cerevisiae
Titelblatt und Inhaltsverzeichnis
Zusammenfassung
Einleitung
Ergebnisse und Diskussion
Literatur- und Publikationsverzeichnis
Eidesstattliche Erklärung und Danksagung
Zusammenfassung
Abstract26S Proteasomen ĂĽbernehmen die spezifische Degradation kurzlebiger Proteine im
Zyto- und Nukleoplasma, einen Prozess, der nahezu in alle Zellprozesse
eingreift. Das 20S Proteasom bildet den proteolytisch aktiven Kernkomplex, an
den sich zwei regulatorische 19S Komplexe anlagern. 20S Proteasomen bestehen
aus alpha- und beta-Untereinheiten, die sich in vier Ringen mit
alpha(1-7)beta(1-7)beta(1-7)alpha(1-7) Konfiguration anordnen. Ein reifes 20S
Proteasom entsteht aus zwei Vorläuferkomplexen, die sich aus einem alpha(1-7)
Ring und Vorläufern der beta-Untereinheiten zusammensetzen. Während der
Zusammenlagerung zweier Vorläuferkomplexe erfolgt in einer autokatalytischen
Reaktion die Prozessierung derr beta-Untereinheiten unter Freisetzung der
aktiven Zentren im Innenraum der Protease. Ein kleines Protein, genannt Ump1,
wird mit den Vorläuferkomplexen assoziiert vorgefunden und unterstützt den
Reifungsprozess, wobei es im Innenraum eingeschlossen und mit der
Vervollständigung der Maturierung als erstes Substrat des 20S Proteasoms
verdaut wird. In Hefe, einem Modellorganismus der eukaryontischen Zelle, haben
wir 26S Proteasomen vorwiegend im Kern und an der KernhĂĽllendoppelmembran
vorgefunden, so dass wir vermuten, dass proteasomale Proteolyse vorrangig in
diesem Zellkompartiment benötigt wird. Der Import von 26S Proteasomen in den
Zellkern findet über Vorläuferkomplexe des 20S Proteasoms und Subkomplexen des
regulatorischen 19S Komplexes statt, so dass nukleäre 26S Proteasomen im Kern
assembliert werden. Der Rezeptor fĂĽr klassische Kernlokalisationssequenzen,
Karyopherin / Importin alpha/beta, ist fĂĽr den Import proteasomaler
Komponenten verantwortlich. Klassische Kernlokalisationsequenzen proteasomaler
Untereinheiten wurden identifiziert. Eine dieser Kernlokalisationssequenzen
ist fĂĽr den Import des Basiskomplexes des regulatorischen 19S Komplexes
essentiell und somit für die Funktionalität des nukleären 26S Proteasoms
unentbehrlich. Ein weiteres hochmolekulares nukleäres Protein, genannt Blm3
(kürzlich umbenannt als Blm10), wurde mit einer späten Zwischenstufe der 20S
Proteasomenreifung assoziiert vorgefunden und dĂĽrfte eine regulatorische
Funktion bei der Maturierung des 20S Proteasoms im Kern einnehmen. Der
Einfluss von Blm3 und verwandter Proteine auf die Assemblierung des 26S
Proteasoms wird derzeit untersucht.26S proteasomes are protease complexes in the nucleo- and cytoplasm. They are
responsible for selective degradation of short-lived proteins which regulate
nearly all cellular processes. The proteolytically active core complex, the
20S proteasome, is faced by two regulatory 19S complexes. 20S proteasomes
consist of alpha and beta subunits which are stacked in four rings with
alpha(1-7) beta(1-7) beta(1-7) alpha(1-7) configuration. The mature 20S
proteasome is formed by dimerisation of two precursor complexes, which consist
of an alpha(1-7) ring and beta subunit precursors. During precursor complex
dimerisation the beta subunit proproteins are processed by an autocatalytic
reaction which yields the active site residues inside the proteolytic chamber.
A small protein, named Ump1, is associated with precursor complexes and
accompanies the maturation process. Burried inside the proteolytic chamber
Ump1 becomes the first substrate of the matured 20S proteasome. In yeast, an
eukaryotic model organism, the majority of proteasomes localizes to the
nucleus and around the nuclear membrane suggesting that proteasomal
proteolysis is mainly required in this subcellular compartment. Nuclear import
of 26S proteasomes occurs via precursor complexes of the 20S proteasome and
subcomplexes of the regulatory 19S complex. Thus, nuclear 26S proteasomes are
most likely assembled in the nucleus. The proteasomal subcomplexes are
recognized by the classical nuclear localization receptor, karyopherin /
importin alpha / beta. Classical nuclear localization signals are present in
proteasomal subunits. A subunit of the 19S base subcomplex harbours an
essential nuclear localization sequence which is crucial for nuclear 26S
proteasome function. Furthermore, the nuclear high molecular mass protein Blm3
(recently renamed Blm10) was found to be associated with late intermediates of
20S proteasome precursor complexes suggesting that Blm3 regulates late steps
in nuclear 20S proteasome maturation. The impact of Blm3 and related proteins
on 26S proteasome assembly is currently under investigation
Nuclear Transport of Yeast Proteasomes
Proteasomes are conserved protease complexes enriched in the nuclei of dividing yeast cells, a major site for protein degradation. If yeast cells do not proliferate and transit to quiescence, metabolic changes result in the dissociation of proteasomes into proteolytic core and regulatory complexes and their sequestration into motile cytosolic proteasome storage granuli. These granuli rapidly clear with the resumption of growth, releasing the stored proteasomes, which relocalize back to the nucleus to promote cell cycle progression. Here, I report on three models of how proteasomes are transported from the cytoplasm into the nucleus of yeast cells. The first model applies for dividing yeast and is based on the canonical pathway using classical nuclear localization sequences of proteasomal subcomplexes and the classical import receptor importin/karyopherin αβ. The second model applies for quiescent yeast cells, which resume growth and use Blm10, a HEAT-like repeat protein structurally related to karyopherin β, for nuclear import of proteasome core particles. In the third model, the fully-assembled proteasome is imported into the nucleus. Our still marginal knowledge about proteasome dynamics will inspire the discussion on how protein degradation by proteasomes may be regulated in different cellular compartments of dividing and quiescent eukaryotic cells
Targeting mechanism of proteasomes to nuclear / ER membranes and into the nucleus of yeast
Intracellular Dynamics of the Ubiquitin-Proteasome-System [v1; ref status: indexed, http://f1000r.es/5o5]
The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum (ER) membranes. In prolonged quiescence, proteasome granules drop off the NE / ER membranes and migrate as stable organelles throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus. Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm. Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells which comprise the majority of our body’s cells
Intracellular Dynamics of the Ubiquitin-Proteasome-System [version 2; referees: 3 approved]
The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum ( ER) membranes. In prolonged quiescence, proteasome granules drop off the nuclear envelopeNE / ER membranes and migrate as droplet-like entitiesstable organelles  throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus. Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm. Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells, which comprise the majority of our body’s cells
Structure and Function of p97 and Pex1/6 Type II AAA+ Complexes
Protein complexes of the Type II AAA+ (ATPases associated with diverse cellular activities) family are typically hexamers of 80–150 kDa protomers that harbor two AAA+ ATPase domains. They form double ring assemblies flanked by associated domains, which can be N-terminal, intercalated or C-terminal to the ATPase domains. Most prominent members of this family include NSF (N-ethyl-maleimide sensitive factor), p97/VCP (valosin-containing protein), the Pex1/Pex6 complex and Hsp104 in eukaryotes and ClpB in bacteria. Tremendous efforts have been undertaken to understand the conformational dynamics of protein remodeling type II AAA+ complexes. A uniform mode of action has not been derived from these works. This review focuses on p97/VCP and the Pex1/6 complex, which both structurally remodel ubiquitinated substrate proteins. P97/VCP plays a role in many processes, including ER- associated protein degradation, and the Pex1/Pex6 complex dislocates and recycles the transport receptor Pex5 from the peroxisomal membrane during peroxisomal protein import. We give an introduction into existing knowledge about the biochemical and cellular activities of the complexes before discussing structural information. We particularly emphasize recent electron microscopy structures of the two AAA+ complexes and summarize their structural differences
Nuclear Import of Yeast Proteasomes
Proteasomes are highly conserved protease complexes responsible for the degradation of aberrant and short-lived proteins. In highly proliferating yeast and mammalian cells, proteasomes are predominantly nuclear. During quiescence and cell cycle arrest, proteasomes accumulate in granules in close proximity to the nuclear envelope/ER. With prolonged quiescence in yeast, these proteasome granules pinch off as membraneless organelles, and migrate as stable entities through the cytoplasm. Upon exit from quiescence, the proteasome granules clear and the proteasomes are rapidly transported into the nucleus, a process reflecting the dynamic nature of these multisubunit complexes. Due to the scarcity of studies on the nuclear transport of mammalian proteasomes, we summarised the current knowledge on the nuclear import of yeast proteasomes. This pathway uses canonical nuclear localisation signals within proteasomal subunits and Srp1/Kap95, and the canonical import receptor, named importin/karyopherin αβ. Blm10, a conserved 240 kDa protein, which is structurally related to Kap95, provides an alternative import pathway. Two models exist upon which either inactive precursor complexes or active holo-enzymes serve as the import cargo. Here, we reconcile both models and suggest that the import of inactive precursor complexes predominates in dividing cells, while the import of mature enzymes mainly occurs upon exit from quiescence