scholarly journals The AAA ATPase Rix7 powers progression of ribosome biogenesis by stripping Nsa1 from pre-60S particles

2008 ◽  
Vol 181 (6) ◽  
pp. 935-944 ◽  
Author(s):  
Dieter Kressler ◽  
Daniela Roser ◽  
Brigitte Pertschy ◽  
Ed Hurt
Keyword(s):  
Ribosome Biogenesis ◽  
Aaa Atpase ◽  
Energy Consuming ◽  
Ribosome Formation

Ribosome biogenesis takes place successively in the nucleolar, nucleoplasmic, and cytoplasmic compartments. Numerous nonribosomal factors transiently associate with the nascent ribosomes, but the mechanisms driving ribosome formation are mostly unknown. Here, we show that an energy-consuming enzyme, the AAA-type (ATPases associated with various cellular activities) ATPase Rix7, restructures a novel pre-60S particle at the transition from the nucleolus to nucleoplasm. Rix7 interacts genetically with Nsa1 and is targeted to the Nsa1-defined preribosomal particle. In vivo, Nsa1 cannot dissociate from pre-60S particles in rix7 mutants, causing nucleolar Nsa1 to escape to the cytoplasm, where it remains associated with aberrant 60S subunits. Altogether, our data suggest that Rix7 is required for the release of Nsa1 from a discrete preribosomal particle, thereby triggering the progression of 60S ribosome biogenesis.

scholarly journals Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation

2012 ◽  
Vol 199 (5) ◽  
pp. 771-782 ◽  
Author(s):  
Lisa Kappel ◽  
Mathias Loibl ◽  
Gertrude Zisser ◽  
Isabella Klein ◽  
Gernot Fruhmann ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Ribosome Biogenesis ◽  
Atp Hydrolysis ◽  
Large Subunit ◽  
Exact Role ◽  
Aaa Atpase ◽  
Energy Consuming

Formation of eukaryotic ribosomes is driven by energy-consuming enzymes. The AAA-ATPase Drg1 is essential for the release of several shuttling proteins from cytoplasmic pre-60S particles and the loading of late joining proteins. However, its exact role in ribosome biogenesis has been unknown. Here we show that the shuttling protein Rlp24 recruited Drg1 to pre-60S particles and stimulated its ATPase activity. ATP hydrolysis in the second AAA domain of Drg1 was required to release shuttling proteins. In vitro, Drg1 specifically and exclusively extracted Rlp24 from purified pre-60S particles. Rlp24 release required ATP and was promoted by the interaction of Drg1 with the nucleoporin Nup116. Subsequent ATP hydrolysis in the first AAA domain dissociated Drg1 from Rlp24, liberating both proteins for consecutive cycles of activity. Our results show that release of Rlp24 by Drg1 defines a key event in large subunit formation that is a prerequisite for progression of cytoplasmic pre-60S maturation.

Phenotype–genotype relationships in peroxisome biogenesis disorders of PEX1-defective complementation group 1 are defined by Pex1p–Pex6p interaction

2001 ◽  
Vol 357 (2) ◽  
pp. 417-426 ◽  
Author(s):  
Shigehiko TAMURA ◽  
Naomi MATSUMOTO ◽  
Atsushi IMAMURA ◽  
Nobuyuki SHIMOZAWA ◽  
Yasuyuki SUZUKI ◽  
...  
Keyword(s):  
Zellweger Syndrome ◽  
Complementation Group ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Peroxisome Biogenesis ◽  
Causative Gene ◽  
Aaa Atpase ◽  
Peroxisome Biogenesis Disorders ◽  
The Stability

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD) and infantile Refsum disease (IRD), are fatal autosomal recessive diseases caused by impaired peroxisome biogenesis, of which 12 genotypes have been reported. ZS patients manifest the severest clinical and biochemical abnormalities, whereas those with NALD and IRD show less severity and the mildest features respectively. We have reported previously that temperature-sensitive peroxisome assembly is responsible for the mildness of the clinical features of IRD. PEX1 is the causative gene for PBDs of complementation group E (CG-E, CG1 in the U.S.A. and Europe), the PBDs of highest incidence, encoding the peroxin Pex1p of the AAA ATPase family. It has been also reported that Pex1p and Pex6p interact with each other. In the present study we investigated phenotype–genotype relationships of CG1 PBDs. Pex1p from IRD such as Pex1p with the most frequently identified mutation at G843D was largely degraded in vivo at 37°C, whereas a normal level of Pex1p was detectable at the permissive temperature. In contrast, PEX1 proteins derived from ZS patients, including proteins with a mutation at L664P or the deletion of residues 634–690, were stably present at both temperatures. Pex1p-G843D interacted with Pex6p at approx. 50% of the level of normal Pex1p, whereas Pex1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p. Taking these results together, we consider it most likely that the stability of Pex1p reflects temperature-sensitive peroxisome assembly in IRD fibroblasts. Failure in Pex1p–Pex6p interaction gives rise to more severe abnormalities, such as those manifested by patients with ZS.

scholarly journals Cytoplasmic Recycling of 60S Preribosomal Factors Depends on the AAA Protein Drg1

2007 ◽  
Vol 27 (19) ◽  
pp. 6581-6592 ◽  
Author(s):  
Brigitte Pertschy ◽  
Cosmin Saveanu ◽  
Gertrude Zisser ◽  
Alice Lebreton ◽  
Martin Tengg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
Dominant Negative ◽  
Cytoplasmic Localization ◽  
Atpase Domain ◽  
Aaa Atpase ◽  
Cytoplasmic Accumulation ◽  
Functional Inactivation ◽  
Aaa Protein

ABSTRACT Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.

Cellular Samurai: katanin and the severing of microtubules

2000 ◽  
Vol 113 (16) ◽  
pp. 2821-2827 ◽  
Author(s):  
L. Quarmby
Keyword(s):  
Epithelial Cells ◽  
Essential Role ◽  
Aaa Atpase ◽  
C Elegans ◽  
Microtubule Severing ◽  
Biochemical Studies ◽  
New Evidence

Recent biochemical studies of the AAA ATPase, katanin, provide a foundation for understanding how microtubules might be severed along their length. These in vitro studies are complemented by a series of recent reports of direct in vivo observation of microtubule breakage, which indicate that the in vitro phenomenon of catalysed microtubule severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells such as neurons and epithelial cells. Although it has been difficult to establish the role of katanin in mitosis, new genetic evidence indicates that a katanin-like protein, MEI-1, plays an essential role in meiosis in C. elegans. Finally, new proteins involved in the severing of axonemal microtubules have been discovered in the deflagellation system of Chlamydomonas.

scholarly journals Formation of a Complex between Nucleolin and Replication Protein a after Cell Stress Prevents Initiation of DNA Replication

2000 ◽  
Vol 149 (4) ◽  
pp. 799-810 ◽  
Author(s):  
Yaron Daniely ◽  
James A. Borowiec
Keyword(s):  
Dna Replication ◽  
Ribosome Biogenesis ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
Cell Stress ◽  
Replication Protein ◽  
Binding Studies

We used a biochemical screen to identify nucleolin, a key factor in ribosome biogenesis, as a high-affinity binding partner for the heterotrimeric human replication protein A (hRPA). Binding studies in vitro demonstrated that the two proteins physically interact, with nucleolin using an unusual contact with the small hRPA subunit. Nucleolin significantly inhibited both simian virus 40 (SV-40) origin unwinding and SV-40 DNA replication in vitro, likely by nucleolin preventing hRPA from productive interaction with the SV-40 initiation complex. In vivo, use of epifluorescence and confocal microscopy showed that heat shock caused a dramatic redistribution of nucleolin from the nucleolus to the nucleoplasm. Nucleolin relocalization was concomitant with a tenfold increase in nucleolin–hRPA complex formation. The relocalized nucleolin significantly overlapped with the position of hRPA, but only poorly with sites of ongoing DNA synthesis. We suggest that the induced nucleolin–hRPA interaction signifies a novel mechanism that represses chromosomal replication after cell stress.

scholarly journals Cooperative subunit dynamics modulate p97 function

2018 ◽  
Vol 116 (1) ◽  
pp. 158-167 ◽  
Author(s):  
Rui Huang ◽  
Zev A. Ripstein ◽  
John L. Rubinstein ◽  
Lewis E. Kay
Keyword(s):  
Bound State ◽  
Nmr Studies ◽  
Biologically Relevant ◽  
Aaa Atpase ◽  
Cellular Processes ◽  
Allosteric Communication ◽  
Functional Dynamics ◽  
Nmr Study ◽  
Wide Range

p97 is an essential hexameric AAA+ ATPase involved in a wide range of cellular processes. Mutations in the enzyme are implicated in the etiology of an autosomal dominant neurological disease in which patients are heterozygous with respect to p97 alleles, containing one copy each of WT and disease-causing mutant genes, so that, in vivo, p97 molecules can be heterogeneous in subunit composition. Studies of p97 have, however, focused on homohexameric constructs, where protomers are either entirely WT or contain a disease-causing mutation, showing that for WT p97, the N-terminal domain (NTD) of each subunit can exist in either a down (ADP) or up (ATP) conformation. NMR studies establish that, in the ADP-bound state, the up/down NTD equilibrium shifts progressively toward the up conformation as a function of disease mutant severity. To understand NTD functional dynamics in biologically relevant p97 heterohexamers comprising both WT and disease-causing mutant subunits, we performed a methyl-transverse relaxation optimized spectroscopy (TROSY) NMR study on a series of constructs in which only one of the protomer types is NMR-labeled. Our results show positive cooperativity of NTD up/down equilibria between neighboring protomers, allowing us to define interprotomer pathways that mediate the allosteric communication between subunits. Notably, the perturbed up/down NTD equilibrium in mutant subunits is partially restored by neighboring WT protomers, as is the two-pronged binding of the UBXD1 adaptor that is affected in disease. This work highlights the plasticity of p97 and how subtle perturbations to its free-energy landscape lead to significant changes in NTD conformation and adaptor binding.

scholarly journals Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes

Plants ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 892
Author(s):  
Alexandre Augusto Pereira Firmino ◽  
Michal Gorka ◽  
Alexander Graf ◽  
Aleksandra Skirycz ◽  
Federico Martinez-Seidel ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Gradient Centrifugation ◽  
Leaf Tissue ◽  
Proteome Analysis ◽  
Sucrose Density Gradient ◽  
Cross Linking ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytoplasmic Ribosome ◽  
Plant Chloroplast

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

scholarly journals From Snapshots to Flipbook—Resolving the Dynamics of Ribosome Biogenesis with Chemical Probes

2020 ◽  
Vol 21 (8) ◽  
pp. 2998 ◽  
Author(s):  
Lisa Kofler ◽  
Michael Prattes ◽  
Helmut Bergler
Keyword(s):  
Ribosome Biogenesis ◽  
Chemical Probes ◽  
Rdna Transcription ◽  
Dynamic Nature ◽  
Tremendous Progress ◽  
Cell Cycle Perturbation ◽  
The Individual ◽  
Ribosome Formation ◽  
Molecular Compounds ◽  
Initial Assembly

The synthesis of ribosomes is one of the central and most resource demanding processes in each living cell. As ribosome biogenesis is tightly linked with the regulation of the cell cycle, perturbation of ribosome formation can trigger severe diseases, including cancer. Eukaryotic ribosome biogenesis starts in the nucleolus with pre-rRNA transcription and the initial assembly steps, continues in the nucleoplasm and is finished in the cytoplasm. From start to end, this process is highly dynamic and finished within few minutes. Despite the tremendous progress made during the last decade, the coordination of the individual maturation steps is hard to unravel by a conventional methodology. In recent years small molecular compounds were identified that specifically block either rDNA transcription or distinct steps within the maturation pathway. As these inhibitors diffuse into the cell rapidly and block their target proteins within seconds, they represent excellent tools to investigate ribosome biogenesis. Here we review how the inhibitors affect ribosome biogenesis and discuss how these effects can be interpreted by taking the complex self-regulatory mechanisms of the pathway into account. With this we want to highlight the potential of low molecular weight inhibitors to approach the dynamic nature of the ribosome biogenesis pathway.

scholarly journals Post-transcriptional regulation of ribosome formation in the nucleus of regenerating rat liver

1983 ◽  
Vol 210 (1) ◽  
pp. 183-192 ◽  
Author(s):  
K P Dudov ◽  
M D Dabeva
Keyword(s):  
Rat Liver ◽  
Ribosome Biogenesis ◽  
Rrna Processing ◽  
Regenerating Liver ◽  
Regenerating Rat Liver ◽  
Rrna Species ◽  
Rrna Maturation ◽  
The Individual ◽  
Post Transcriptional Regulation

Kinetic experiments on RNA labelling in vivo with [14C]orotate were performed with normal and 12h-regenerating rat liver. The specific radioactivities of nucleolar, nucleoplasmic and cytoplasmic rRNA species were analysed by computer according to the models of rRNA processing and nucleo-cytoplasmic migration given previously [Dudov, Dabeva, Hadjiolov & Todorov, Biochem. J. (1978) 171, 375-383]. The rates of formation and the half-lives of the individual pre-rRNA and rRNA species were determined in both normal and regenerating liver. The results show clearly that the formation of ribosomes in regenerating rat liver is post-transcriptionally activated: (a) the half-lives of all the nucleolar pre-rRNA and rRNA species are decreased by 30% on average; (b) the pre-rRNA processing is directed through the shortest maturation pathway: 45 S leads to 32 S + 18 S leads to 28 S; (c) the nucleo-cytoplasmic transfer of ribosomes is accelerated. As a consequence, the time for formation and appearance of ribosomes in the cytoplasm is shortened 1.5-fold for the large and 2-fold for the small subparticle. A new scheme for endonuclease cleavage of 45 S pre-rRNA is proposed, which explains the alterations in pre-rRNA processing in regenerating liver. Its validity for pre-rRNA processing in other eukaryotes is discussed. It is concluded that: (i) the control sites in the intranucleolar formation of 28 S and 18 S rRNA are the immediate precursor of 28 S rRNA, 32 S pre-rRNA, and the primary pre-rRNA, 45 S pre-rRNA, respectively; (ii) the limiting step in the post-transcriptional stages of ribosome biogenesis is the pre-rRNA maturation.

scholarly journals Essential Assembly Factor Rpf2 Forms Novel Interactions within the 5S RNP in Trypanosoma brucei

mSphere ◽  
2017 ◽  
Vol 2 (5) ◽  
Author(s):  
Anyango D. Kamina ◽  
Daniel Jaremko ◽  
Linda Christen ◽  
Noreen Williams
Keyword(s):  
Trypanosoma Brucei ◽  
Ribosome Biogenesis ◽  
Sleeping Sickness ◽  
5S Rrna ◽  
Ribonucleoprotein Particle ◽  
Content Type ◽  
Assembly Factor ◽  
Specific Proteins ◽  
First Time ◽  
Ribosome Formation

ABSTRACT Trypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the regulatory checkpoints during ribosome biogenesis. We have previously characterized the 5S RNP in T. brucei and showed that trypanosome-specific proteins P34 and P37 are part of this complex. In this study, we characterize for the first time the interactions of the homolog of the assembly factor Rpf2 with members of the 5S RNP in another organism besides fungi. Our studies show that Rpf2 is essential in T. brucei and that it forms unique interactions within the 5S RNP, particularly with P34 and P37. These studies have identified parasite-specific interactions that can potentially function as new therapeutic targets against sleeping sickness. Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in Trypanosoma brucei, the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the T. brucei homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. T. brucei Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the T. brucei 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T. brucei. IMPORTANCE Trypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the regulatory checkpoints during ribosome biogenesis. We have previously characterized the 5S RNP in T. brucei and showed that trypanosome-specific proteins P34 and P37 are part of this complex. In this study, we characterize for the first time the interactions of the homolog of the assembly factor Rpf2 with members of the 5S RNP in another organism besides fungi. Our studies show that Rpf2 is essential in T. brucei and that it forms unique interactions within the 5S RNP, particularly with P34 and P37. These studies have identified parasite-specific interactions that can potentially function as new therapeutic targets against sleeping sickness.

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