scholarly journals Ubiquitin–Proteasome-dependent Degradation of a Mitofusin, a Critical Regulator of Mitochondrial Fusion

2008 ◽  
Vol 19 (6) ◽  
pp. 2457-2464 ◽  
Author(s):  
Mickael M.J. Cohen ◽  
Guillaume P. Leboucher ◽  
Nurit Livnat-Levanon ◽  
Michael H. Glickman ◽  
Allan M. Weissman
Keyword(s):  
Mitochondrial Fusion ◽  
26S Proteasome ◽  
Mitochondrial Outer Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Subsequent Degradation ◽  
Multistep Process ◽  
Ubiquitin Proteasome ◽  
Steady State Levels ◽  
F Box Protein

The mitochondrion is a dynamic membranous network whose morphology is conditioned by the equilibrium between ongoing fusion and fission of mitochondrial membranes. In the budding yeast, Saccharomyces cerevisiae, the transmembrane GTPase Fzo1p controls fusion of mitochondrial outer membranes. Deletion or overexpression of Fzo1p have both been shown to alter the mitochondrial fusion process indicating that maintenance of steady-state levels of Fzo1p are required for efficient mitochondrial fusion. Cellular levels of Fzo1p are regulated through degradation of Fzo1p by the F-box protein Mdm30p. How Mdm30p promotes degradation of Fzo1p is currently unknown. We have now determined that during vegetative growth Mdm30p mediates ubiquitylation of Fzo1p and that degradation of Fzo1p is an ubiquitin-proteasome–dependent process. In vivo, Mdm30p associates through its F-box motif with other core components of Skp1-Cullin-F-box (SCF) ubiquitin ligases. We show that the resulting SCFMdm30p ligase promotes ubiquitylation of Fzo1p at mitochondria and its subsequent degradation by the 26S proteasome. These results provide the first demonstration that a cytosolic ubiquitin ligase targets a critical regulatory molecule at the mitochondrial outer membrane. This study provides a framework for developing an understanding of the function of Mdm30p-mediated Fzo1p degradation in the multistep process of mitochondrial fusion.

The modified mitochondrial outer membrane carrier MTCH2 links mitochondrial fusion to lipogenesis

2021 ◽  
Vol 220 (11) ◽  
Author(s):  
Katherine Labbé ◽  
Shona Mookerjee ◽  
Maxence Le Vasseur ◽  
Eddy Gibbs ◽  
Chad Lerner ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fusion ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Carrier ◽  
Cellular Survival ◽  
Mitochondrial Elongation ◽  
Specific Lipid

Mitochondrial function is integrated with cellular status through the regulation of opposing mitochondrial fusion and division events. Here we uncover a link between mitochondrial dynamics and lipid metabolism by examining the cellular role of mitochondrial carrier homologue 2 (MTCH2). MTCH2 is a modified outer mitochondrial membrane carrier protein implicated in intrinsic cell death and in the in vivo regulation of fatty acid metabolism. Our data indicate that MTCH2 is a selective effector of starvation-induced mitochondrial hyperfusion, a cytoprotective response to nutrient deprivation. We find that MTCH2 stimulates mitochondrial fusion in a manner dependent on the bioactive lipogenesis intermediate lysophosphatidic acid. We propose that MTCH2 monitors flux through the lipogenesis pathway and transmits this information to the mitochondrial fusion machinery to promote mitochondrial elongation, enhanced energy production, and cellular survival under homeostatic and starvation conditions. These findings will help resolve the roles of MTCH2 and mitochondria in tissue-specific lipid metabolism in animals.

scholarly journals HSP27 Is a Ubiquitin-Binding Protein Involved in I-κBα Proteasomal Degradation

2003 ◽  
Vol 23 (16) ◽  
pp. 5790-5802 ◽  
Author(s):  
Arnaud Parcellier ◽  
Elise Schmitt ◽  
Sandeep Gurbuxani ◽  
Daphné Seigneurin-Berny ◽  
Alena Pance ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Cell Types ◽  
26S Proteasome ◽  
Necrosis Factor Alpha ◽  
Polyubiquitin Chains ◽  
Ubiquitinated Proteins ◽  
Activation Of Transcription ◽  
Ubiquitin Proteasome

ABSTRACT HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-α). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-κB by degrading its main inhibitor, I-κBα. HSP27 overexpression increases NF-κB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, ΤNF-α, and interleukin 1β. HSP27 does not affect I-κBα phosphorylation but enhances the degradation of phosphorylated I-κBα by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-κBα. A protein complex that includes HSP27, phosphorylated I-κBα, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-κBα. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-κB activity.

scholarly journals Regulation of mitochondrial fusion by the F-box protein Mdm30 involves proteasome-independent turnover of Fzo1

2006 ◽  
Vol 173 (5) ◽  
pp. 645-650 ◽  
Author(s):  
Mafalda Escobar-Henriques ◽  
Benedikt Westermann ◽  
Thomas Langer
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Critical Role ◽  
Mitochondrial Fusion ◽  
Yeast Cells ◽  
Mitochondrial Outer Membrane ◽  
Central Component ◽  
Mitochondrial Morphology ◽  
Proteolytic Pathway ◽  
F Box Protein

Mitochondrial morphology depends on balanced fusion and fission events. A central component of the mitochondrial fusion apparatus is the conserved GTPase Fzo1 in the outer membrane of mitochondria. Mdm30, an F-box protein required for mitochondrial fusion in vegetatively growing cells, affects the cellular Fzo1 concentration in an unknown manner. We demonstrate that mitochondrial fusion requires a tight control of Fzo1 levels, which is ensured by Fzo1 turnover. Mdm30 binds to Fzo1 and, dependent on its F-box, mediates proteolysis of Fzo1. Unexpectedly, degradation occurs along a novel proteolytic pathway not involving ubiquitylation, Skp1–Cdc53–F-box (SCF) E3 ubiquitin ligase complexes, or 26S proteasomes, indicating a novel function of an F-box protein. This contrasts to the ubiquitin- and proteasome-dependent turnover of Fzo1 in α-factor–arrested yeast cells. Our results therefore reveal not only a critical role of Fzo1 degradation for mitochondrial fusion in vegetatively growing cells but also the existence of two distinct proteolytic pathways for the turnover of mitochondrial outer membrane proteins.

scholarly journals The Saccharomyces cerevisiae ubiquitin–proteasome system

1999 ◽  
Vol 354 (1389) ◽  
pp. 1513-1522 ◽  
Author(s):  
M. Hochstrasser ◽  
P. R. Johnson ◽  
C. S. Arendt ◽  
A. Y. Amerik ◽  
S. Swaminathan ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Site Formation ◽  
Protein Protein Interaction ◽  
Proteasome Subunits ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Diploid Cells ◽  
Ubiquitin Conjugating Enzymes

Our studies of the yeast ubiquitin-proteasome pathway have uncovered a number of general principles that govern substrate selectivity and proteolysis in this complex system. Much of the work has focused on the destruction of a yeast transcription factor, MATα2. The α2 protein is polyubiquitinated and rapidly degraded in α–haploid cells. One pathway of proteolytic targeting, which depends on two distinct endoplasmic reticulum–localized ubiquitin–conjugating enzymes, recognizes the hydrophobic face of an amphipathic helix in α2. Interestingly, degradation of α2 is blocked in a /α–diploid cells by heterodimer formation between the α2 and a 1 homeodomain proteins. The data suggest that degradation signals may overlap protein–protein interaction surfaces, allowing a straightforward steric mechanism for regulated degradation. Analysis of α2 degradation led to the identification of both 20S and 26S proteasome subunits, and several key features of proteasome assembly and active–site formation were subsequently uncovered. Finally, it has become clear that protein (poly)ubiquitination is highly dynamic in vivo , and our studies of yeast de–ubiquitinating enzymes illustrate how such enzymes can facilitate the proteolysis of diverse substrates.

scholarly journals The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

1999 ◽  
Vol 19 (4) ◽  
pp. 2527-2534 ◽  
Author(s):  
Guillaume Hautbergue ◽  
Valérie Goguel
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Cyclin Dependent Kinase ◽  
Transcription Control ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Steady State Levels ◽  
Proteasome Pathway ◽  
Carboxy Terminal

ABSTRACT The yeast CTDK-I complex has been implicated in phosphorylation of the carboxy-terminal domain of the RNA polymerase II and in transcription control. It is composed of three polypeptides: Ctk1p and Ctk2p, a cyclin-dependent kinase and a C-type cyclin subunit, respectively; and Ctk3p, a third subunit of unknown function. Cyclins are regulatory proteins whose expression is tightly controlled at the protein level. In this study, we examined the regulation of Ctk2p expression in vivo. Surprisingly, unlike what has been described for cell cycle cyclins, steady-state levels of Ctk2p are composed of two relatively abundant forms, one of them phosphorylated. We show that this phosphorylated form is extremely unstable (half-life, 5 min) and that rapid proteolysis of Ctk2p exhibits growth-related regulation. Furthermore, our data establish that similar to the case for other naturally short-lived proteins, Ctk2p degradation is mediated by the ubiquitin-proteasome pathway. This is the first demonstration that a C-type cyclin is phosphorylated and targeted to the proteasome. Strikingly, neither phosphorylation nor destruction of Ctk2p requires its associated kinase Ctk1p, a feature fundamentally different from that which has been observed for cell cycle cyclins.

scholarly journals Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation

2012 ◽  
Vol 448 (1) ◽  
pp. 55-65 ◽  
Author(s):  
Jonas Boehringer ◽  
Christiane Riedinger ◽  
Konstantinos Paraskevopoulos ◽  
Eachan O. D. Johnson ◽  
Edward D. Lowe ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Functional Characterization ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Structural Motif ◽  
Protein Protein Interactions ◽  
Ubiquitin Proteasome

The ubiquitin–proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein–protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.

scholarly journals The Control Operated by the Cell Cycle Machinery on MEF2 Stability Contributes to the Downregulation of CDKN1A and Entry into S Phase

2015 ◽  
Vol 35 (9) ◽  
pp. 1633-1647 ◽  
Author(s):  
Eros Di Giorgio ◽  
Enrico Gagliostro ◽  
Andrea Clocchiatti ◽  
Claudio Brancolini
Keyword(s):  
Cell Cycle ◽  
Ubiquitin Proteasome System ◽  
S Phase ◽  
Open Chromatin ◽  
Cyclin Dependent Kinase ◽  
Murine Fibroblasts ◽  
Subsequent Degradation ◽  
Ubiquitin Proteasome ◽  
F Box Protein ◽  
Cdkn1a Gene

MEF2s are pleiotropic transcription factors (TFs) which supervise multiple cellular activities. During the cell cycle, MEF2s are activated at the G0/G1transition to orchestrate the expression of the immediate early genes in response to growth factor stimulation. Here we show that, in human and murine fibroblasts, MEF2 activities are downregulated during late G1. MEF2C and MEF2D interact with the E3 ligase F-box protein SKP2, which mediates their subsequent degradation through the ubiquitin-proteasome system. The cyclin-dependent kinase 4 (CDK4)/cyclin D1 complex phosphorylates MEF2D on serine residues 98 and 110, and phosphorylation of these residues is an important determinant for SKP2 binding. Unscheduled MEF2 transcription during the cell cycle reduces cell proliferation, whereas its containment sustains DNA replication. The CDK inhibitor p21/CDKN1A gene is a MEF2 target gene required to exert this antiproliferative influence. MEF2C and MEF2D bind a region within the first intron ofCDKN1A, presenting epigenetic markers of open chromatin. Importantly, H3K27 acetylation within this regulative region depends on the presence of MEF2D. We propose that following the initial engagement in the G0/G1transition, MEF2C and MEF2D must be polyubiquitylated and degraded during G1progression to diminish the transcription of theCDKN1Agene, thus favoring entry into S phase.

scholarly journals Mitochondrial inner-membrane protease Yme1 degrades outer-membrane proteins Tom22 and Om45

2017 ◽  
Vol 217 (1) ◽  
pp. 139-149 ◽  
Author(s):  
Xi Wu ◽  
Lanlan Li ◽  
Hui Jiang
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Ubiquitin Proteasome System ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Mitochondrial Proteome ◽  
Mitochondrial Inner Membrane ◽  
Ubiquitin Proteasome

Mitochondria are double-membraned organelles playing essential metabolic and signaling functions. The mitochondrial proteome is under surveillance by two proteolysis systems: the ubiquitin–proteasome system degrades mitochondrial outer-membrane (MOM) proteins, and the AAA proteases maintain the proteostasis of intramitochondrial compartments. We previously identified a Doa1–Cdc48-Ufd1-Npl4 complex that retrogradely translocates ubiquitinated MOM proteins to the cytoplasm for degradation. In this study, we report the unexpected identification of MOM proteins whose degradation requires the Yme1-Mgr1-Mgr3 i-AAA protease complex in mitochondrial inner membrane. Through immunoprecipitation and in vivo site-specific photo–cross-linking experiments, we show that both Yme1 adapters Mgr1 and Mgr3 recognize the intermembrane space (IMS) domains of the MOM substrates and facilitate their recruitment to Yme1 for proteolysis. We also provide evidence that the cytoplasmic domain of substrate can be dislocated into IMS by the ATPase activity of Yme1. Our findings indicate a proteolysis pathway monitoring MOM proteins from the IMS side and suggest that the MOM proteome is surveilled by mitochondrial and cytoplasmic quality control machineries in parallel.

scholarly journals The molecular chaperone Ydj1 is required for the p34CDC28-dependent phosphorylation of the cyclin Cln3 that signals its degradation.

1996 ◽  
Vol 16 (7) ◽  
pp. 3679-3684 ◽  
Author(s):  
J A Yaglom ◽  
A L Goldberg ◽  
D Finley ◽  
M Y Sherman
Keyword(s):  
Molecular Chaperone ◽  
Terminal Segment ◽  
Direct Role ◽  
Yeast Saccharomyces Cerevisiae ◽  
Free System ◽  
Close Proximity ◽  
Ubiquitin Proteasome ◽  
A Cell ◽  
Histone Kinase

The G1 cyclin Cln3 of the yeast Saccharomyces cerevisiae is rapidly degraded by the ubiquitin-proteasome pathway. This process is triggered by p34CDC28-dependent phosphorylation of Cln3. Here we demonstrate that the molecular chaperone Ydj1, a DnaJ homolog, is required for this phosphorylation. In a ydj1 mutant at the nonpermissive temperature, both phosphorylation and degradation of Cln3 were deficient. No change was seen upon inactivation of Sis1, another DnaJ homolog. The phosphorylation defect in the ydj1 mutant was specific to Cln3, because no reduction in the phosphorylation of Cln2 or histone H1, which also requires p34CDC28, was observed. Ydj1 was required for Cln3 phosphorylation and degradation rather than for the proper folding of this cyclin, since Cln3 produced in the ydj1 mutant was fully active in the stimulation of p34CDC28 histone kinase activity. Moreover, Ydj1 directly associates with Cln3 in close proximity to the segment that is phosphorylated and signals degradation. Thus, binding of Ydj1 to this domain of Cln3 seems to be essential for the phosphorylation and breakdown of this cyclin. In a cell-free system, purified Ydj1 stimulated the p34CDC28-dependent phosphorylation of the C-terminal segment of Cln3 and did not affect phosphorylation of Cln2 (as was found in vivo). The reconstitution of this process with pure components provides evidence of a direct role for the chaperone in the phosphorylation of Cln3.

scholarly journals Critical Role for a Central Part of Mdm2 in the Ubiquitylation of p53

2003 ◽  
Vol 23 (14) ◽  
pp. 4929-4938 ◽  
Author(s):  
Erik Meulmeester ◽  
Ruth Frenk ◽  
Robert Stad ◽  
Petra de Graaf ◽  
Jean-Christophe Marine ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
P53 Protein ◽  
Critical Role ◽  
Ring Finger ◽  
26S Proteasome ◽  
Functional Difference ◽  
Subsequent Degradation ◽  
The Stability ◽  
Internal Domain

ABSTRACT The stability of the p53 protein is regulated by Mdm2. By acting as an E3 ubiquitin ligase, Mdm2 directs the ubiquitylation of p53 and its subsequent degradation by the 26S proteasome. In contrast, the Mdmx protein, although structurally similar to Mdm2, cannot ubiquitylate or degrade p53 in vivo. To ascertain which domains determine this functional difference between Mdm2 and Mdmx and consequently are essential for p53 ubiquitylation and degradation, we generated Mdm2-Mdmx chimeric constructs. Here we show that, in addition to a fully functional Mdm2 RING finger, an internal domain of Mdm2 (residues 202 to 302) is essential for p53 ubiquitylation. Strikingly, the function of this domain can be fulfilled in trans, indicating that the RING domain and this internal region perform distinct activities in the ubiquitylation of p53.

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