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 übernehmen 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 Kernhüllendoppelmembran 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 für klassische Kernlokalisationssequenzen, Karyopherin / Importin alpha/beta, ist für den Import proteasomaler Komponenten verantwortlich. Klassische Kernlokalisationsequenzen proteasomaler Untereinheiten wurden identifiziert. Eine dieser Kernlokalisationssequenzen ist für 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 dürfte 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

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