Outer membrane protein 25-a mitochondrial anchor and inhibitor of stress-activated protein kinase-3

Stress-activated protein kinase-3 (SAPK3) is unique amongst the mitogen-activated protein kinase (MAPK) family with its C-terminal 5 amino acids directing interaction with the PDZ domain-containing substrates alpha1-Syntrophin and SAP90/PSD95. Here, we identify three additional PDZ domain-containing binding partners, Lin-7C, Scribble, and outer membrane protein 25 (OMP25). This latter protein is localised together with SAPK3 at the mitochondria but it is not a SAPK3 substrate. Instead, OMP25 inhibits SAPK3 activity towards PDZ domain-containing substrates such as alpha1-Syntrophin and substrates without PDZ domains such as the mitochondrial protein Sab. This is a new mechanism for the regulation of SAPK3 and suggests that its intracellular activity should not be solely assessed by its phosphorylation status.     

Identification and characterization of a synaptojanin 2 splice isoform predominantly expressed in nerve terminals

We have previously identified synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of synaptojanin 2 that shares several properties with synaptojanin 1. This isoform, synaptojanin 2B, undergoes further alternative splicing to generate synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners synaptojanin 1, bind synaptojanin 2B2, whereas only amphiphysin binds synaptojanin 2B1. Sequence similar to the endophilin-binding site in synaptojanin 1 is present only in synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of synaptojanin 2, but not of synaptojanin 1. These results suggest that synaptojanin 2B has a partially overlapping function with synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.     

VDAC electronics: 5. Mechanism and computational model of hexokinase-dependent generation of the outer membrane potential in brain mitochondria

Glycolysis plays a key role in brain energy metabolism. The initial and rate-limiting step of brain glycolysis is catalyzed mainly by hexokinase I (HKI), the majority of which is bound to the mitochondrial outer membrane (MOM), mostly through the mitochondrial inter-membrane contact sites formed by the voltage-dependent anion channel (VDAC, outer membrane) and the adenine nucleotide translocator (ANT, inner membrane). Earlier, we proposed a mechanism for the generation of the mitochondrial outer membrane potential (OMP) as a result of partial application of the inner membrane potential (IMP) to MOM through the electrogenic ANT-VDAC-HK inter-membrane contact sites. According to this previous mechanism, the Gibbs free energy of the hexokinase reaction might modulate the generated OMP (Lemeshko, Biophys. J., 2002). In the present work, a new computational model was developed to perform thermodynamic estimations of the proposed mechanism of IMP-HKI-mediated generation of OMP. The calculated OMP was high enough to electrically regulate MOM permeability for negatively charged metabolites through free, unbound VDACs in MOM. On the other hand, the positive-inside polarity of OMP generated by the IMP-HKI-mediated mechanism is expected to protect mitochondria against elevated concentrations of cytosolic Ca²⁺. This computational analysis suggests that metabolically-dependent generation of OMP in the brain mitochondria, controlled by many factors that modulate VDAC1-HKI interaction, VDAC's voltage-gating properties and permeability, might represent one of the physiological mechanisms of regulation of the brain energy metabolism and of neuronal death resistance, and might also be involved in various neurodegenerative disorders, such as Alzheimer's disease.     

VDAC as a voltage-dependent mitochondrial gatekeeper under physiological conditions

Mitochondria, composed of two membranes, play a key role in energy production in eukaryotic cells. The main function of the inner membrane is oxidative phosphorylation, while the mitochondrial outer membrane (MOM) seems to control the energy flux and exchange of various charged metabolites between mitochondria and the cytosol. Metabolites cross MOM via the various isoforms of voltage-dependent anion channel (VDAC). In turn, VDACs interact with some enzymes, other proteins and molecules, including drugs. This work aimed to analyze various literature experimental data related to targeting mitochondrial VDACs and VDAC-kinase complexes on the basis of the hypothesis of generation of the outer membrane potential (OMP) and OMP-dependent reprogramming of cell energy metabolism. Our previous model of the VDAC-hexokinase-linked generation of OMP was further complemented in this study with an additional regulation of the MOM permeability by the OMP-dependent docking of cytosolic proteins like tubulin to VDACs. Computational analysis of the model suggests that OMP changes might be involved in the mechanisms of apoptosis promotion through the so-called transient hyperpolarization of mitochondria. The high concordance of the performed computational estimations with many published experimental data allows concluding that OMP generation under physiological conditions is highly probable and VDAC might function as an OMP-dependent gatekeeper of mitochondria, controlling cell life and death. The proposed model of OMP generation allows understanding in more detail the mechanisms of cancer death resistance and anticancer action of various drugs and treatments influencing VDAC voltage-gating properties, VDAC content, mitochondrial hexokinase activity and VDAC-kinase interactions in MOM.     

EphrinB-EphB signalling regulates clathrin-mediated endocytosis through tyrosine phosphorylation of synaptojanin 1

Recent studies show that Eph receptors act mainly through the regulation of actin reorganization. Here, we show a novel mode of action for EphB receptors. We identify synaptojanin 1 - a phosphatidylinositol 5'-phosphatase that is involved in clathrin-mediated endocytosis - as a physiological substrate for EphB2. EphB2 causes tyrosine phosphorylation in the proline-rich domain of synaptojanin 1, and inhibits both the interaction with endophilin and the 5'-phosphatase activity of synaptojanin 1. Treatment with the EphB ligand, ephrinB2, elevates the cellular level of phosphatidylinositol 4,5-bisphosphate and promotes transferrin uptake. A kinase inactive mutant of EphB2 and a phosphorylation site mutant of synaptojanin 1 both neutralize the increase of transferrin uptake after ephrinB2 treatment. These mutants also inhibit AMPA glutamate receptor endocytosis in hippocampal neurons. Interestingly, incorporated transferrin does not reach endosomes, suggesting dual effects of EphB signalling on the early and late phases of clathrin-mediated endocytosis. Our results indicate that ephrinB-EphB signalling regulates clathrin-mediated endocytosis in various cellular contexts by influencing protein interactions and phosphoinositide turnover through tyrosine phosphorylation of synaptojanin 1.     

Synaptojanin 2 functions at an early step of clathrin-mediated endocytosis

Synaptojanin 2 is a ubiquitously expressed polyphosphoinositide phosphatase that displays a high degree of homology in its catalytic domains with synaptojanin 1 [1,2]. Neurons of synaptojanin 1-deficient mice display an increase in clathrin-coated vesicles and delayed reentry of recycling vesicles into the fusion-competent vesicle pool, but no defects in early steps of endocytosis [3,4]. Here we show that inhibition of synaptojanin 2 expression via small interfering (si) RNA causes a strong defect in clathrin-mediated receptor internalization in a lung carcinoma cell line. This inhibitory phenotype is rescued by overexpression of wild-type synaptojanin 2, but not of wild-type synaptojanin 1 or mutant synaptojanin 2 that is deficient in 5'-phosphatase activity. In addition, electron-microscopic analysis shows that synaptojanin 2 depletion causes a decrease in clathrin-coated pits and vesicles. These results suggest a role for synaptojanin 2 in clathrin-coated pit formation and imply that lipid hydrolysis is required at an early stage of clathrin-mediated endocytosis. Taken together, our results also indicate that synaptojanin 2 is functionally distinct from synaptojanin 1.     

Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae

The type 2 secretion system (T2SS) occurring in Gram-negative bacteria is composed of 12-15 different proteins which form large assemblies spanning two membranes and secreting several virulence factors in folded state across the outer membrane. The T2SS component EpsC of Vibrio cholerae plays an important role in this machinery. While anchored in the inner membrane, by far the largest part of EpsC is periplasmic, containing a so-called homology region (HR) domain and a PDZ domain. Here we report studies on the structure and function of both periplasmic domains of EpsC. The crystal structures of two variants of the PDZ domain of EpsC from V. cholerae were determined at better than 2 A resolution. Compared to the short variant, the longer variant contains an additional N-terminal helix, and reveals a significant difference in the position of helix alphaB with respect to the beta-sheet. Both our structures show that the PDZ domain of EpsC adopts a more open form than in previously reported structures of other PDZ domains. Most interestingly, in the crystals of the short EpsC-PDZ domain the peptide binding groove interacts with an alpha-helix from a neighboring subunit burying approximately 921 A2 solvent accessible surface. This makes it possible that the PDZ domain of this bacterial protein binds proteins in a manner which is altogether different from that seen in any other PDZ domain so far. We also determined that the HR domain of EpsC is primarily responsible for the interaction with the secretin EpsD, while the PDZ is not, or much less, so. This new finding, together with studies of others, leads to the suggestion that the PDZ domain of EpsC may interact with exoproteins to be secreted while the HR domain plays a key role in linking the inner-membrane sub-complex of the T2SS in V. cholerae to the outer membrane secretin.     

Apparent “mild depolarization of the inner mitochondrial membrane” as a result of a possible generation of the outer membrane potential

Recently reported kinase-linked mild depolarization of mitochondria, which prevents the generation of the reactive oxygen species (ROS) and disappears in various organs of the old mice, has been assumed to represent a crucial component of the mitochondrial anti-aging program. To measure mitochondrial inner membrane potential (IMP), the authors used fluorescent probe safranin O⁺. It is widely accepted that the accumulation of such cationic probes in the mitochondrial matrix depends exclusively on IMP, thus completely ignoring the possibility of the outer membrane potential (OMP) generation. However, computational analysis performed in the presented work suggests that the kinase-linked generation of the positive OMP might take place under the described conditions, because the measured potential includes the algebraic sum of both IMP and OMP. Alternatively to the suggested mild depolarization of mitochondria, the reported experimental data might reflect mainly a change of the positive OMP generated by the VDAC-kinase complexes. We also demonstrate that the reported in the literature mitochondrial hyperpolarization induced by erastin (known to prevent VDAC-tubulin interactions) and the depolarization caused by the mitochondrial VDAC knockdowns in the cancer cells might actually represent a decrease or increase, respectively, of the magnitude of the kinase-linked positive OMP. This is consistent with our hypothesis that VDAC voltage gating by the kinase-linked metabolically-dependent OMP plays a very important physiological role in regulating the cell energy metabolism under normal and pathological conditions, in the maintenance of the cell death resistance and even in the genetic aging program.     

Allosteric activation of a bacterial stress sensor

In Gram-negative bacteria, envelope stress signals such as unfolded outer membrane proteins (OMP) activate the periplasmic protease DegS. This protease then triggers a cellular pathway to alleviate the stress. Now Sohn et al. (2007) show conclusively that inhibition of DegS is relieved allosterically by binding of the C-terminal sequences in unfolded OMPs to the PDZ domain of DegS.     

PDZ domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator

The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-DeltaTRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-DeltaTRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from approximately 24 to approximately 13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-DeltaTRL did not differ. However, endocytic recycling of CFTR-DeltaTRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells.     

Metaxin 1 interacts with metaxin 2, a novel related protein associated with the mammalian mitochondrial outer membrane.

A recently described protein, metaxin 1, serves as a component of a preprotein import complex in the outer membrane of the mammalian mitochondrion. A yeast two-hybrid screen with metaxin 1 as bait has now identified a novel protein, which we have termed metaxin 2, as a metaxin 1-binding protein. Metaxin 2 shares 29% identity with metaxin 1 at the amino acid level, but metaxin 2, unlike metaxin 1, lacks a C-terminal mitochondrial outer membrane signal-anchor domain. Two C. elegans hypothetical proteins, CelZC97.1 and CelF39B2.i, share high sequence similarity with metaxin 2 and metaxin 1, respectively, and likely represent the C. elegans orthologs. Affinity-purified antibodies against metaxin 2 were prepared against the recombinant protein produced in E. coli and were used to analyze the subcellular distribution of metaxin 2. In subcellular fractions of mouse liver, a 29 kD immunoreactive protein, consistent in size with the predicted translation product of metaxin 2 cDNA, was found solely in mitochondria. Alkali extraction of mitochondria indicated that metaxin 2 is peripherally associated with mitochondrial membranes. Metaxin 2 in intact mitochondria was susceptible to digestion with proteinase K, indicating that metaxin 2 is located on the cytosolic face of the mitochondrial outer membrane. Finally, baculoviruses encoding a His6-tagged metaxin 2 and an untagged metaxin 1 lacking its C-terminal transmembrane domain were produced and used separately or in combination to infect Sf21 insect cells. Metaxin 1 bound to a Ni2+-chelate affinity column only in the presence of metaxin 2, indicating that metaxin 1 and metaxin 2 interact when overexpressed in insect cells. These results suggest that metaxin 2 is bound to the cytosolic face of the mitochondrial outer membrane by means of its interaction with membrane-bound metaxin 1, and that this complex may play a role in protein import into mammalian mitochondria.     

Structural and functional analysis of the ligand specificity of the HtrA2/Omi PDZ domain

The mitochondrial serine protease HtrA2/Omi helps to maintain mitochondrial function by handling misfolded proteins in the intermembrane space. In addition, HtrA2/Omi has been implicated as a proapoptotic factor upon release into the cytoplasm during the cell death cascade. The protein contains a C-terminal PDZ domain that packs against the protease active site and inhibits proteolytic activity. Engagement of the PDZ domain by peptide ligands has been shown to activate the protease and also has been proposed to mediate substrate recognition. We report a detailed structural and functional analysis of the human HtrA2/Omi PDZ domain using peptide libraries and affinity assays to define specificity, X-ray crystallography to view molecular details of PDZ-ligand interactions, and alanine-scanning mutagenesis to probe the peptide-binding groove. We show that the HtrA2/Omi PDZ domain recognizes both C-terminal and internal stretches of extended, hydrophobic polypeptides. High-affinity ligand recognition requires contacts with up to five hydrophobic side chains by distinct sites on the PDZ domain. However, no particular residue type is absolutely required at any position, and thus, the HtrA2/Omi PDZ domain appears to be a promiscuous module adapted to recognize unstructured, hydrophobic polypeptides. This type of specificity is consistent with the biological role of HtrA2/Omi in mitochondria, which requires the recognition of diverse, exposed stretches of hydrophobic sequences in misfolded proteins. The findings are less consistent with, but do not exclude, a role for the PDZ domain in targeting the protease to specific substrates during apoptosis.     

Regulation of SLC26A3 activity by NHERF4 PDZ-mediated interaction

SLC26A3 functions as a chloride/bicarbonate anion exchanger expressed in the secretory epithelial cells in the intestine, pancreas, and salivary glands. SLC26A3 has a C-terminal class I PDZ binding motif that assembles regulatory factors or other transporters by anchoring to various PDZ scaffold proteins. NHERF4 is an epithelial-enriched PDZ domain scaffold protein that has attracted attention because of its enriched tissue expression in the intestine and kidney. In this study, we identified SLC26A3 as a novel binding transporter of NHERF4. We investigated the functional role of NHERF4 in the regulation of SLC26A3 by using integrated biochemical and physiological approaches. A direct protein-protein interaction was identified between the PDZ-binding motif of SLC26A3 and the third PDZ domain of NHERF4. Interaction with NHERF4 decreased the level of SLC26A3 expression on the plasma membrane, which led to reduced SLC26A3 anion exchange activity. Notably, interaction with NHERF4 induced rapid internalisation of SLC26A3 from the plasma membrane. The SLC26A3-NHERF4 interaction was modulated by phosphorylation; serine 329 of NHERF4-PDZ3 played a critical role in modulating binding selectivity. Our findings suggest that NHERF4 is a novel modulator of luminal fluidity in the intestine by adjusting SLC26A3 expression and activity through a phosphorylation-dependent mechanism.     

Involvement of Porin N,N-dicyclohexylcarbodiimide-Reactive Domain in Hexokinase Binding to the Outer Mitochondrial Membrane

The proportion of hexokinase that is bound to the outer mitochondrial membrane is tissue specific and metabolically regulated. This study examined the role of the N,N-dicyclohexylcarbodiimide-binding domain of mitochondrial porin in binding to hexokinase I. Selective proteolytic cleavage of porin protein was performed and peptides were assayed for their, effect on hexokinase I binding to isolated mitochondria. Specificity of DCCD-reactive domain binding to hexokinase I was demonstrated by competition of the peptides for porin binding sites on hexokinase as well as by blockage hexokinase binding by N,N-dicyclohexylcarbodiimide. One of the peptides, designated as 5 kDa (the smallest of the porin peptides, which contains a DCCD-reactive site), totally blocked binding of the enzyme to the mitochondrial membrane, and significantly enhanced the release of the mitochondrially bound enzyme. These experiments demonstrate that there exists a direct and specific interaction between the DCCD-reactive domain of VDAC and hexokinase I. The peptides were further characterized with respect to their effects on certain functional properties of hexokinase I. None had any detectable effect on catalytic properties, including inhibition by glucose 6-phosphate. To evaluate further the outer mitochondrial membranes role in the hexokinase binding, insertion of VDAC was examined using isolated rat mitochondria. Pre-incubation of mitochondria with purified porin strongly increases hexokinase I binding to rat liver mitochondria. Collectively, the results imply that the high hexokinase-binding capability of porin-enriched mitochondria was due to a quantitative difference in binding sites.     

Crystal structure of yeast mitochondrial outer membrane translocon member Tom70p

A majority of the proteins targeted to the mitochondria are transported through the translocase of the outer membrane (TOM) complex. Tom70 is a major surface receptor for mitochondrial protein precursors in the TOM complex. To investigate how Tom70 receives the mitochondrial protein precursors, we have determined the crystal structure of yeast Tom70p to 3.0 A. Tom70p forms a homodimer in the crystal. Each subunit consists primarily of tetratricopeptide repeat (TPR) motifs, which are organized into a right-handed superhelix. The TPR motifs in the N-terminal domain of Tom70p form a peptide-binding groove for the C-terminal EEVD motif of Hsp70, whereas the C-terminal domain of Tom70p contains a large pocket that may be the binding site for mitochondrial precursors. The crystal structure of Tom70p provides insights into the mechanisms of precursor transport across the mitochondrion's outer membrane.     

An internal EELD domain facilitates mitochondrial targeting of Mcl-1 via a Tom70-dependent pathway

Mcl-1 functions at an apical step in many regulatory programs that control cell death. Although the mitochondrion is one major subcellular organelle where Mcl-1 functions, the molecular mechanism by which Mcl-1 is targeted to mitochondria remains unclear. Here, we demonstrate that Mcl-1 is loosely associated with the outer membrane of mitochondria. Furthermore, we demonstrate that Mcl-1 interacts with the mitochondrial import receptor Tom70, and such interaction requires an internal domain of Mcl-1 that contains an EELD motif. A Tom70 antibody that blocks Mcl-1-Tom70 interaction blocks mitochondrial import of Mcl-1 in vitro. Furthermore, Mcl-1 is significantly less targeted to mitochondria in Tom70 knockdown than in the control cells. Similar targeting preference is also observed for the DM mutant of Mcl-1 whose mutation at the EELD motif markedly attenuates its Tom70 binding activity. Together, our results indicate that the internal EELD domain facilitates mitochondrial targeting of Mcl-1 via a Tom70-dependent pathway.     

Molecular requirements for the regulation of the renal outer medullary K(+) channel ROMK1 by the serum- and glucocorticoid-inducible kinase SGK1

The serum- and glucocorticoid- inducible kinase SGK1 stimulates the renal outer medullary K(+) channel ROMK1 in the presence of the Na(+)/H(+) exchanger regulating factor NHERF2. SGK1/NHERF2 are effective through enhancement of ROMK1 abundance within the cell membrane. The present study aims to define the molecular requirements for the interaction of ROMK1 with SGK1/NHERF2. Pull down assays reveal that SGK1 interacts with NHERF2 through the second PDZ domain of NHERF2. According to chemiluminescence and electrophysiology, deletion of the second PDZ domain of NHERF2 or the putative PDZ binding motif on ROMK1 abrogates the stimulating effect of SGK1 on ROMK1 protein abundance in the plasma membrane and K(+) current.     

Acid stress activation of the σE stress response in Salmonella enterica serovar Typhimurium

The alternative sigma factor σ^E is activated by unfolded outer membrane proteins (OMPs) and plays an essential role in Salmonella pathogenesis. The canonical pathway of σ^E activation in response to envelope stress involves sequential proteolysis of the anti-sigma factor RseA by the PDZ proteases DegS and RseP. Here we show that σ^E in Salmonella enterica sv. Typhimurium can also be activated by acid stress. A σ^E-deficient mutant exhibits increased susceptibility to acid pH and reduced survival in an acidified phagosomal vacuole. Acid activation of σ^E-dependent gene expression is independent of the unfolded OMP signal or the DegS protease but requires processing of RseA by RseP. The RseP PDZ domain is indispensable for acid induction, suggesting that acid stress may disrupt an inhibitory interaction between RseA and the RseP PDZ domain to allow RseA proteolysis in the absence of antecedent action of DegS. These observations demonstrate a novel environmental stimulus and activation pathway for the σ^E regulon that appear to be critically important during Salmonella –host cell interactions.