Albert Neutzner

The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division

We identify a mitochondrial E3 ubiquitin ligase, MARCH5, as a critical regulator of mitochondrial fission. MARCH5 RING mutants and MARCH5 RNA interference induce an abnormal elongation and interconnection of mitochondria indicative of an inhibition of mitochondrial division. The aberrant mitochondrial phenotypes in MARCH5 RING mutant–expressing cells are reversed by ectopic expression of Drp1, but not another mitochondrial fission protein Fis1. Moreover, as indicated by abnormal clustering and mitochondrial accumulation of Drp1, as well as decreased cellular mobility of YFP-Drp1 in cells expressing MARCH5 RING mutants, MARCH5 activity regulates the subcellular trafficking of Drp1, likely by impacting the correct assembly at scission sites or the disassembly step of fission complexes. Loss of this activity may account for the observed mitochondrial division defects. Finally, MARCH5 RING mutants and endogenous Drp1, but not wild-type MARCH5 or Fis1, co-assemble into abnormally enlarged clusters in a Drp1 GTPase-dependent manner, suggesting molecular interactions among these proteins. Collectively, our data suggest a model in which mitochondrial division is regulated by a MARCH5 ubiquitin-dependent switch.

Loss of MARCH5 mitochondrial E3 ubiquitin ligase induces cellular senescence through dynamin-related protein 1 and mitofusin 1

Mitochondria constantly divide and combine through fission and fusion activities. MARCH5, a mitochondrial E3 ubiquitin ligase, has been identified as a molecule that binds mitochondrial fission 1 protein (hFis1), dynamin-related protein 1 (Drp1) and mitofusin 2 (Mfn2), key proteins in the control of mitochondrial fission and fusion. However, how these interactions control mitochondrial dynamics, and cellular function has remained obscure. Here, we show that shRNA-mediated MARCH5 knockdown promoted the accumulation of highly interconnected and elongated mitochondria. Cells transfected with MARCH5 shRNA or a MARCH5 RING domain mutant displayed cellular enlargement and flattening accompanied by increased senescence-associated β-galactosidase (SA-β-Gal) activity, indicating that these cells had undergone cellular senescence. Notably, a significant increase in Mfn1 level, but not Mfn2, Drp1 or hFis1 levels, was observed in MARCH5-depleted cells, indicating that Mfn1 is a major ubiquitylation substrate. Introduction of Mfn1T109A, a GTPase-deficient mutant form of Mfn1, into MARCH5-RNAi cells not only disrupted mitochondrial elongation, but also abolished the increase in SA-β-Gal activity. Moreover, the aberrant mitochondrial phenotypes in MARCH5-RNAi cells were reversed by ectopic expression of Drp1, but not by hFis1, and reversion of the mitochondria morphology in MARCH5-depleted cells was accompanied by a reduction in SA-β-Gal activity. Collectively, our data indicate that the lack of MARCH5 results in mitochondrial elongation, which promotes cellular senescence by blocking Drp1 activity and/or promoting accumulation of Mfn1 at the mitochondria.

Neurodegeneration as a consequence of failed mitochondrial maintenance

Maintaining the functional integrity of mitochondria is pivotal for cellular survival. It appears that neuronal homeostasis depends on high-fidelity mitochondria, in particular. Consequently, mitochondrial dysfunction is a fundamental problem associated with a significant number of neurological diseases, including Parkinson’s disease (PD), Huntington’s disease (HD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS) and various peripheral neuropathies, as well as the normal aging process. To ensure optimal mitochondrial function, diverse, evolutionarily conserved mitochondrial quality control mechanisms are in place, including the scavenging of toxic reactive oxygen species (ROS) and degradation of damaged mitochondrial proteins, but also turnover of whole organelles. In this review we will discuss various mitochondria-associated conditions, focusing on the role of protein turnover in mitochondrial maintenance with special emphasis on neurodegenerative disorders.

Instability of the Mitofusin Fzo1 Regulates Mitochondrial Morphology during the Mating Response of the Yeast Saccharomyces cerevisiae

Mitochondria form a highly dynamic network that is shaped by continuous fission and fusion of these organelles. In the yeast Saccharomyces cerevisiae two machineries are involved in this process, one of which includes the mitochondrial fusion promoting GTPase Fzo1. Although a role for the F-box protein Mdm30 in regulating the stability of Fzo1 has been proposed, the molecular basis for the regulation of the fission to fusion ratio of mitochondria remains unknown. To discern the mechanism of the regulation of mitochondrial morphology, we arrested cells at different stages of the cell cycle and examined mitochondrial morphology as well as the stability of mitochondrial fission and fusion proteins. In response to a G1 arrest evoked by the mating pheromone α factor the mitochondrial network fragmented into small pieces, which was accompanied by dramatic down-regulation of Fzo1. Mating pheromone also triggered the degradation of Fzo1 produced under the control of a constitutive promoter, and Fzo1 was stabilized upon proteasome inhibition, indicating a role for the proteasome system in the degradation of Fzo1. However, deletion of MDM30 did not stabilize Fzo1 after mating pheromone treatment, showing a different mechanism from the previously reported process of steady state Fzo1 regulation. We show an example for a regulated change of the mitochondrial fission to fusion ratio during the life cycle of budding yeast. Proteasomal degradation of Fzo1 in response to the mating pheromone is proposed to mediate the remodeling of the mitochondrial network during the process of mating.

Role of the ubiquitin conjugation system in the maintenance of mitochondrial homeostasis

Mitochondria are essential for a variety of cellular functions, including ATP production, lipid biosynthesis, and calcium homeostasis. Moreover, a number of major cell signaling pathways, including apoptosis, require mitochondria. Consistent with a major role of mitochondria in the control of cell function, defects of these organelles are thought to induce a variety of pathologies ranging from diabetic neuropathies to Parkinsons and Alzheimers diseases. Here we discuss the role of ubiquitin (Ub) conjugation in the maintenance of mitochondrial function. We found that at least four putative RING finger E3 Ub ligases, the main determinants of substrate specificity in the ubiquitination system, localize to mitochondria. Our data also demonstrate that mitochondrial membrane dynamics is under the regulatory control of the Ub‐conjugation system. These and data recently published by others indicate that the Ub‐conjugation system and proteasomal degradation of mitochondrial proteins might be vital for the maintenance of mitochondrial homeostasis and lead to cell demise when dysfunctional.

Novel Structure of the N Terminus in Yeast Fis1 Correlates with a Specialized Function in Mitochondrial Fission

Mitochondrial fission is facilitated by a multiprotein complex assembled at the division site. The required components of the fission machinery in Saccharomyces cerevisiae include Dnm1, Fis1, and Mdv1. In the present study, we determined the protein structure of yeast Fis1 using NMR spectroscopy. Although the six α-helices, as well as their folding, in the yeast Fis1 structure are similar to those of the tetratricopeptide repeat (TPR) domains of the human Fis1 structure, the two structures differ in their N termini. The N-terminal tail of human Fis1 is flexible and unstructured, whereas a major segment of the longer N terminus of yeast Fis1 is fixed to the concave face formed by the six α-helices in the TPR domains. To investigate the role of the fixed N terminus, exogenous Fis1 was expressed in yeast lacking the endogenous protein. Expression of yeast Fis1 protein rescued mitochondrial fission in Δfis1 yeast only when the N-terminal TPR binding segment was left intact. The presence of this segment is also correlated to the recruitment of Mdv1 to mitochondria. The conformation of the N-terminal segment embedded in the TPR pocket indicates an intra-molecular regulation of Fis1 bioactivity. Although the TPR-like helix bundle of Fis1 mediates the interaction with Dnm1 and Mdv1, the N terminus of Fis1 is a prerequisite to recruit Mdv1 to facilitate mitochondrial fission.

Role of Membrane Association and Atg14-Dependent Phosphorylation in Beclin-1-Mediated Autophagy

ABSTRACT During autophagy, a double membrane envelops cellular material for trafficking to the lysosome. Human beclin-1 and its yeast homologue, Atg6/Vps30, are scaffold proteins bound in a lipid kinase complex with multiple cellular functions, including autophagy. Several different Atg6 complexes exist, with an autophagy-specific form containing Atg14. However, the roles of Atg14 and beclin-1 in the activation of this complex remain unclear. We here addressed the mechanism of beclin-1 complex activation and reveal two critical steps in this pathway. First, we identified a unique domain in beclin-1, conserved in the yeast homologue Atg6, which is involved in membrane association and, unexpectedly, controls autophagosome size and number in yeast. Second, we demonstrated that human Atg14 is critical in controlling an autophagy-dependent phosphorylation of beclin-1. We map these novel phosphorylation sites to serines 90 and 93 and demonstrate that phosphorylation at these sites is necessary for maximal autophagy. These results help clarify the mechanism of beclin-1 and Atg14 during autophagy.

IBRDC2, an IBR-type E3 ubiquitin ligase, is a regulatory factor for Bax and apoptosis activation

Bax, a pro‐apoptotic protein from the Bcl‐2 family, is central to apoptosis regulation. To suppress spontaneous apoptosis, Bax must be under stringent control that may include regulation of Bax conformation and expression levels. We report that IBRDC2, an IBR‐type RING‐finger E3 ubiquitin ligase, regulates the levels of Bax and protects cells from unprompted Bax activation and cell death. Downregulation of IBRDC2 induces increased cellular levels and accumulation of the active form of Bax. The ubiquitination‐dependent regulation of Bax stability is suppressed by IBRDC2 downregulation and stimulated by IBRDC2 overexpression in both healthy and apoptotic cells. Although mostly cytosolic in healthy cells, upon induction of apoptosis, IBRDC2 accumulates in mitochondrial domains enriched with Bax. Mitochondrial accumulation of IBRDC2 occurs in parallel with Bax activation and also depends on the expression levels of Bcl‐xL. Furthermore, IBRDC2 physically interacts with activated Bax. By applying Bax mutants in HCT116 Bax−/− cells, combined with the use of active Bax‐specific antibodies, we have established that both mitochondrial localization and apoptotic activation of Bax are required for IBRDC2 translocation to the mitochondria.

A Systematic Search for Endoplasmic Reticulum (ER) Membrane-associated RING Finger Proteins Identifies Nixin/ZNRF4 as a Regulator of Calnexin Stability and ER Homeostasis

To identify novel regulators of endoplasmic reticulum (ER)-linked protein degradation and ER function, we determined the entire inventory of membrane-spanning RING finger E3 ubiquitin ligases localized to the ER. We identified 24 ER membrane-anchored ubiquitin ligases and found Nixin/ZNRF4 to be central for the regulation of calnexin turnover. Ectopic expression of wild type Nixin induced a dramatic down-regulation of the ER-localized chaperone calnexin that was prevented by inactivation of the Nixin RING domain. Importantly, Nixin physically interacts with calnexin in a glycosylation-independent manner, induces calnexin ubiquitination, and p97-dependent degradation, indicating an ER-associated degradation-like mechanism of calnexin turnover.

Enzymes of ubiquitination and deubiquitination

Ubiquitination, the covalent attachment of the small protein modifier ubiquitin to a substrate protein is involved in virtually all cellular processes by mediating the regulated degradation of proteins. Aside from proteasomal degradation, ubiquitination plays important roles in transcriptional regulation, protein trafficking, including endocytosis and lysosomal targeting, and activation of kinases involved in signalling processes. A three-tiered enzymatic cascade consisting of E1 or ubiquitin-activating enzyme, E2 or ubiquitin-conjugating enzyme, and E3, or ubiquitin ligases, is necessary to achieve the many forms of ubiquitination known to date. In this chapter, we summarize the current knowledge on the enzymatic machinery necessary for ubiquitin activation and ligation, as well as its removal, and provide some insight into the complexity of regulatory processes governed by ubiquitination.