MARCH-V is a novel mitofusin 2- and Drp1-binding protein able to change mitochondrial morphology

Mitofusins and Drp1 are key components in mitochondrial membrane fusion and division, but the molecular mechanism underlying the regulation of their activities remains to be clarified. Here, we identified human membrane-associated RING-CH (MARCH)-V as a novel transmembrane protein of the mitochondrial outer membrane. Immunoprecipitation studies demonstrated that MARCH-V interacts with mitofusin 2 (MFN2) and ubiquitinated forms of Drp1. Overexpression of MARCH-V promoted the formation of long tubular mitochondria in a manner that depends on MFN2 activity. By contrast, mutations in the RING finger caused fragmentation of mitochondria. We also show that MARCH-V promotes ubiquitination of Drp1. These results indicate that MARCH-V has a crucial role in the control of mitochondrial morphology by regulating MFN2 and Drp1 activities.     

Identification of a novel protein complex containing annexin A4, rabphilin and synaptotagmin

Rabphilin is a synaptic vesicle-associated protein proposed to play a role in regulating neurotransmitter release. Here we report the isolation and identification of a novel protein complex containing rabphilin, annexin A4 and synaptotagmin 1. We show that the rabphilin C2B domain interacts directly with the N-terminus of annexin A4 and mediates the co-complexing of these two proteins in PC12 cells. Analyzing the cellular localisation of these co-complexing proteins we find that annexin A4 is located on synaptic membranes and co-localises with rabphilin at the plasma membrane in PC12 cells. Given that rabphilin and synaptotagmin are synaptic vesicle proteins involved in neurotransmitter release, the identification of this complex suggests that annexin A4 may play a role in synaptic exocytosis.     

Identification of toposome, a novel multisubunit complex containing topoisomerase IIalpha

Topoisomerase IIalpha plays essential roles in chromosome segregation. However, it is not well understood how topoisomerase IIalpha exerts its function during mitosis. In this report, we find that topoisomerase IIalpha forms a multisubunit complex, named toposome, containing two ATPase/helicase proteins (RNA helicase A and RHII/Gu), one serine/threonine protein kinase (SRPK1), one HMG protein (SSRP1), and two pre-mRNA splicing factors (PRP8 and hnRNP C). Toposome separates entangled circular chromatin DNA about fourfold more efficiently than topoisomerase IIalpha. Interestingly, this decatenation reaction yields knotted circles, which are not seen in reactions provided with monomeric circular DNA. Our results also show that interaction among toposome-associated proteins is highest in G2/M phase but drastically diminishes in G1/S phase. These results suggest that toposome is a dynamic complex whose assembly or activation is subject to cell cycle regulation.     

Identification of a novel PP2C-type mitochondrial phosphatase

A novel phosphatase has been cloned and partially characterized. It has a mitochondrial leader sequence and its amino acid sequence places it in the PP2C family like two known mitochondrial phosphatases. Western blot analysis of subcellular fractions and confocal microscopy of 3T3L1 preadipocytes expressing the GFP-tagged protein confirm its mitochondrial localization. Western blot analysis indicates that the protein is expressed in several mouse tissues, with highest expression in brain, heart, liver, and kidney. The recombinant protein exhibits Mn(2+)-dependent phosphoserine phosphatase activity against the branched-chain alpha-keto acid dehydrogenase complex, suggesting the enzyme may play a role in regulation of branched chain amino acid catabolism. Whether there are other mitochondrial substrates for the enzyme is not known.     

Modulation of T cell activation by stomatin-like protein 2

T cell activation through the Ag receptor (TCR) requires sustained signaling from signalosomes within lipid raft microdomains in the plasma membrane. In a proteomic analysis of lipid rafts from human T cells, we identified stomatin-like protein (SLP)-2 as a candidate molecule involved in T cell activation through the Ag receptor. In this study, we show that SLP-2 expression in human primary lymphocytes is up-regulated following in vivo and ex vivo activation. In activated T cells, SLP-2 interacts with components of TCR signalosomes and with polymerized actin. More importantly, up-regulation of SLP-2 expression in human T cell lines and primary peripheral blood T cells increases effector responses, whereas down-regulation of SLP-2 expression correlates with loss of sustained TCR signaling and decreased T cell activation. Our data suggest that SLP-2 is an important player in T cell activation by ensuring sustained TCR signaling, which is required for full effector T cell differentiation, and point to SLP-2 as a potential target for immunomodulation.     

Identification and characterization of a novel Nogo-interacting mitochondrial protein (NIMP)

Nogo is a potent inhibitor of regeneration following spinal cord injury. To develop a better understanding of the mechanisms responsible for regenerative failure we used a yeast two-hybrid approach to try and identify proteins that interact with Nogo. We identified a novel mitochondrial protein designated Nogo-interacting mitochondrial protein (NIMP) in a screen of an adult human brain cDNA library. This interaction was confirmed by co-immunoprecipitation in both brain tissue (endogenous) and transfected HEK293T cells (overexpressed). In support of these studies we demonstrate that Nogo interacts with the UQCRC1 and UQCRC2 components of complex III, within the mitochondrial respiratory chain. The mitochondrial localization of NIMP was evidenced by confocal image analysis and western blot analysis of isolated mitochondria. NIMP is highly conserved and ubiquitously expressed in mitochondria-enriched tissues. Within the CNS, NIMP-like immunoreactivity is present in neurons and astrocytes. These data suggest that NIMP is a novel mitochondrial protein that interacts with Nogo. The interaction of Nogo with mitochondrial proteins may provide insight into the mechanisms for Nogo-induced inhibition of neurite growth.     

Identification of a novel protein complex containing annexin VI, Fyn, Pyk2, and the p120(GAP) C2 domain

p120(GAP) (RasGAP) has been proposed to function as both an inhibitor and effector of Ras. Previously we have shown that RasGAP contains a C2 domain which mediates both Ca(2+)-dependent membrane association and protein-protein interactions. Specifically, three proteins have been isolated in a complex with the C2 domain of RasGAP; these are the Ca(2+)-dependent lipid binding protein annexin VI (p70) and two previously unidentified proteins, p55 and p120. Here we provide evidence that p55 is the Src family kinase Fyn and p120 is the focal adhesion kinase family member Pyk2. In addition, in vitro binding assays indicate that Fyn, but not Pyk2 binds directly to annexin VI. Finally, co-immunoprecipitation studies in Rat-1 fibroblasts confirm that Fyn, Pyk2, annexin VI and RasGAP can form a protein complex in mammalian cells.     

Identification of a novel small Arabidopsis protein interacting with gamma-tubulin complex protein 3

In higher plants, microtubules (MTs) show dynamic structural changes during cell cycle and development progression. A precise control of MT nucleation at dispersed sites is one way used to regulate the cytoskeletal organization. Some gamma-tubulin complex proteins (GCPs) were previously identified in Arabidopsis thaliana (At). They are directly involved in the nucleation process. Nevertheless, no additional player which may anchor the nucleating complex or regulate the nucleation activity has been found in plant cells so far. Therefore, our aim was the identification of Arabidopsis proteins interacting with MT nucleating complexes and particularly with AtGCP3. Performing a yeast two-hybrid screen, we discovered a new protein which we called AtGCP3 Interacting Protein 1 (AtGIP1). The possible role of this protein during the nucleation process is discussed.     

Stress-induced phosphorylation and proteasomal degradation of mitofusin 2 facilitates mitochondrial fragmentation and apoptosis

Mitochondria play central roles in integrating pro- and antiapoptotic stimuli, and JNK is well known to have roles in activating apoptotic pathways. We establish a critical link between stress-induced JNK activation, mitofusin 2, which is an essential component of the mitochondrial outer membrane fusion apparatus, and the ubiquitin-proteasome system (UPS). JNK phosphorylation of mitofusin 2 in response to cellular stress leads to recruitment of the ubiquitin ligase (E3) Huwe1/Mule/ARF-BP1/HectH9/E3Histone/Lasu1 to mitofusin 2, with the BH3 domain of Huwe1 implicated in this interaction. This results in ubiquitin-mediated proteasomal degradation of mitofusin 2, leading to mitochondrial fragmentation and enhanced apoptotic cell death. The stability of a nonphosphorylatable mitofusin 2 mutant is unaffected by stress and protective against apoptosis. Conversely, a mitofusin 2 phosphomimic is more rapidly degraded without cellular stress. These findings demonstrate how proximal signaling events can influence both mitochondrial dynamics and apoptosis through phosphorylation-stimulated degradation of the mitochondrial fusion machinery.     

Identification of a novel small molecule targeting UQCRB of mitochondrial complex III and its anti-angiogenic activity

Our recent study has shown that ubiquinol-cytochrome c reductase binding protein (UQCRB), the 13.4-kDa subunit of mitochondrial complex III, plays a crucial role in hypoxia-induced angiogenesis via mitochondrial reactive oxygen species (ROS)-mediated signaling. Here we report a new synthetic small molecule targeting the mitochondrial oxygen sensor UQCRB that was identified by pharmacophore-based virtual screening and in vitro and in vivo competition binding analyses. 6-((1-Hydroxynaphthalen-4-ylamino)dioxysulfone)-2H-naphtho[1,8-bc]thiophen-2-one (HDNT) binds to the hydrophobic pocket of UQCRB and potently inhibits in vitro angiogenesis of human umbilical vein endothelial cells without cytotoxicity. Furthermore, the binding of HDNT to UQCRB suppressed mitochondrial ROS-mediated hypoxic signal transduction. These results demonstrated that HDNT is a novel synthetic small molecule targeting UQCRB and exhibits anti-angiogenic activity by modulating the oxygen-sensing function of UQCRB.     

Identification of CBS2 as a mitochondrial protein in Saccharomyces cerevisiae

Summary The nuclear genome encoded yeast protein CBS2 is required for translational activation of mitochondrial cytochrome b RNA. Genetic studies have shown that the target sequence of the CBS2 protein is the 5 untranslated leader sequence of cytochrome b RNA. Here we report on the intracellular localization of CBS2. CBS2 protein, expressed in Escherichia coli and prepared from inclusion bodies, was used as an antigen to raise a polyclonal rabbit antiserum. Affinity-purified CBS2 antibodies detect a 45 kDa protein in mitochondrial lysates of wild-type cells, which is absent in a strain in which the CBS2 gene has been deleted. The protein is overexpressed in mitochondrial extracts of a transformant carrying the CBS2 gene on a high copy number plasmid, but undetectable in the post-mitochondrial supernatant. Intramitochondrial localization of CBS2 was verified by in vitro import of CBS2 protein that had been synthesized in a reticulocyte lysate programmed with CBS2 mRNA transcribed in vitro. Mitochondrial import of CBS2 is not accompanied by any detectable proteolytic processing.     

A novel caspase-2 complex containing TRAF2 and RIP1

The enzymatic activity of caspases is implicated in the execution of apoptosis and inflammation. Here we demonstrate a novel nonenzymatic function for caspase-2 other than its reported proteolytic role in apoptosis. Caspase-2, unlike caspase-3, -6, -7, -9, -11, -12, and -14, is a potent inducer of NF-kappaB and p38 MAPK activation in a TRAF2-mediated way. Caspase-2 interacts with TRAF1, TRAF2, and RIP1. Furthermore, we demonstrate that endogenous caspase-2 is recruited into a large and inducible protein complex, together with TRAF2 and RIP1. Structure-function analysis shows that NF-kappaB activation occurs independent of enzymatic activity of the protease and that the caspase recruitment domain of caspase-2 is sufficient for the activation of NF-kappaB and p38 MAPK. These results demonstrate the inducible assembly of a novel protein complex consisting of caspase-2, TRAF2, and RIP1 that activates NF-kappaB and p38 MAPK through the caspase recruitment domain of caspase-2 independently of its proteolytic activity.     

Identification and characterization of a novel mitochondrial tricarboxylate carrier

Here we report the identification and functional characterization of a novel mitochondrial tricarboxylate carrier protein, designated BBG-TCC, in rat brain. The cDNA encodes the predicted protein of 342-amino acid residues with five putative membrane-spanning domains. The protein has apparent similarity with a mitochondrial tricarboxylate carrier TCC, but is distinct from the other mitochondria anion transporters. BBG-TCC shows a citrate transport activity. It is specifically expressed in the brain and localizes in the mitochondria of Bergmann glial cells. In contrast, the expression of TCC is rather ubiquitous and strong in neuronal cells in the brain. This new family of proteins may contribute to biosynthesis and bioenergetics in the brain.     

Mass spectrometrical verification of stomatin-like protein 2 (SLP-2) primary structure

Stomatin-like protein 2 (SLP-2) (syn.: EPB72-like 2 [NP_038470], HSPC108 [AAF29073]), a protein of unknown function, has been described in several tissues and cells but its primary structure is still not completely elucidated. Moreover, sequence conflicts appear in several databases. It was the aim of the study to further describe SLP-2 primary sequence and to solve existing sequence conflicts. For this purpose a protein extract was run on two-dimensional gel electrophoresis and SLP-2 was identified by MALDI-TOF/TOF. SLP-2 was digested with trypsin, chymotrypsin, Lys-C, and de novo sequencing studies as well as Nano-HPLC-ESI-MS/MS analysis were carried out. By the use of several proteases sequence coverage of 90% was obtained but the N-terminal 34 amino acids harbouring database conflict 1 were not covered. The presence of Leucine 129 (sequence conflict 2) and Alanine 202 (sequence conflict 3) was verified by three independent approaches. High sequence coverage resulting from multiple proteolytic cleavage, MALDI-TOF/TOF, Nano-HPLC-ESI-MS/MS and de novo sequencing completed unambiguous analysis of SLP-2 primary structure of approximately = 90% of sequence coverage. In addition, methodology used was able to solve so far pending sequence conflicts in databases and literature. SLP-2 is a high abundance protein in several tissues and cells and may play an important biological role and therefore characterization of its primary structure is of importance.     

Identification of a novel mitochondrial protein ("mitoNEET") cross-linked specifically by a thiazolidinedione photoprobe

Thiazolidinediones address underlying causes of type 2 diabetes, although their mechanism of action is not clearly understood. The compounds are thought to function as direct activators of the nuclear receptor PPARgamma (peroxisome proliferator-activated receptor-gamma), although pioglitazone, the weaker agonist of the two thiazolidinediones now in clinical use, seems to have more useful effects on circulating lipids. We have used tritiated pioglitazone and a photoaffinity cross-linker to identify a novel binding site in mitochondria. A saturable binding site for [3H]pioglitazone was solubilized from the membranes with CHAPS and migrated as a large complex by size exclusion chromatography. The binding correlated with a <17-kDa protein (m17), marked by a photoaffinity cross-linker, in both subcellular location and selectivity of competition by analogs. The protein was isolated and identified by mass spectrometry analysis and NH2-terminal sequencing. Three synthetic peptides with potential antigenic properties were synthesized from the predicted nontransmembrane sequence to generate antibodies in rabbits. Western blots show that this protein, which we have termed "mitoNEET," is located in the mitochondrial fraction of rodent brain, liver, and skeletal muscle, showing the identical subcellular location and migration on SDS-PAGE as the protein cross-linked specifically by the thiazolidinedione photoprobe. The protein exists in low levels in preadipocytes, and expression increases exponentially in differentiated adipocytes. The synthetic protein bound to solid phase associated with a complex of solubilized mitochondrial proteins, including the trifunctional beta-oxidation protein. It is possible that thiazolidinedione modification of the function of the mitochondrial target may contribute to lipid lowering and/or antidiabetic actions.     

Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, we carried out a two-hybrid screen with the regulatory B/PR55 subunit. We isolated a human homolog of C. elegans HCP6, a protein distantly related to the condensin subunit hCAP-D2, and we named this homolog hHCP-6. Both C. elegans HCP-6 and condensin are required for chromosome organization and segregation. HCP-6 binding partners are unknown, whereas condensin is composed of the structural maintenance of chromosomes proteins SMC2 and SMC4 and of three non-SMC subunits. Here we show that hHCP-6 becomes phosphorylated during mitosis and that its dephosphorylation by PP2A in vitro depends on B/PR55, suggesting that hHCP-6 is a B/PR55-specific substrate of PP2A. Unlike condensin, hHCP-6 is localized in the nucleus in interphase, but similar to condensin, hHCP-6 associates with chromosomes during mitosis. hHCP-6 is part of a complex that contains SMC2, SMC4, kleisin-beta, and the previously uncharacterized HEAT repeat protein FLJ20311. hHCP-6 is therefore part of a condensin-related complex that associates with chromosomes in mitosis and may be regulated by PP2A.