Membrane Lipid Composition Regulates Tubulin Interaction with Mitochondrial Voltage-dependent Anion Channel

Elucidating molecular mechanisms by which lipids regulate protein function within biological membranes is critical for understanding the many cellular processes. Recently, we have found that dimeric αβ-tubulin, a subunit of microtubules, regulates mitochondrial respiration by blocking the voltage-dependent anion channel (VDAC) of mitochondrial outer membrane. Here, we show that the mechanism of VDAC blockage by tubulin involves tubulin interaction with the membrane as a critical step. The on-rate of the blockage varies up to 100-fold depending on the particular lipid composition used for bilayer formation in reconstitution experiments and increases with the increasing content of dioleoylphosphatidylethanolamine (DOPE) in dioleoylphosphatidylcholine (DOPC) bilayers. At physiologically low salt concentrations, the on-rate is decreased by the charged lipid. The off-rate of VDAC blockage by tubulin does not depend on the lipid composition. Using confocal fluorescence microscopy, we compared tubulin binding to the membranes of giant unilamellar vesicles (GUVs) made from DOPC and DOPC/DOPE mixtures. We found that detectable binding of the fluorescently labeled dimeric tubulin to GUV membranes requires the presence of DOPE. We propose that prior to the characteristic blockage of VDAC, tubulin first binds to the membrane in a lipid-dependent manner. We thus reveal a new potent regulatory role of the mitochondrial lipids in control of the mitochondrial outer membrane permeability and hence mitochondrial respiration through tuning VDAC sensitivity to blockage by tubulin. More generally, our findings give an example of the lipid-controlled protein-protein interaction where the choice of lipid species is able to change the equilibrium binding constant by orders of magnitude.     

Voltage-Dependent Anion Channel Proteins in Synaptosomes of the Torpedo Electric Organ: Immunolocalization, Purification, and Characterization

In this study, we purified and characterized the voltage-dependent anion channel (VDAC) from the Torpedo electric organ. Using immunogold labeling, VDAC was colocalized with the voltage-gated Ca²⁺ channel in the synaptic plasma membrane. By immunoblot analysis, five protein bands in synaptosomes isolated from the Torpedo electric organ cross reacted with two monoclonal anti-VDAC antibody. No more than about 7 to 10% mitochondrial contains could be detected in any synaptosomal membrane preparation tested. This was estimated by comparing the specific activity in mitochondria and synaptosomes of succinate–cytochrome-c oxidoreductase and antimycin-insensitive NADH–cytochrome-c oxidoreductase activities; mitochondrial inner and outer membrane marker enzymes, respectively. [¹⁴C]DCCD (dicyclohexylcarbodiimide), which specifically label mitochondrial VDAC, labeled four 30–35 kDa protein bands that were found to interact with the anti-VDAC antibody. The distribution of the Torpedo VDAC protein bands was different among membranes isolated from various tissues. VDAC was purified from synaptosomes and a separation between two of the proteins was obtained. The two purified proteins were characterized by their single channel activity and partial amino acid sequences. Upon reconstitution into a planar lipid bilayer, the purified VDACs showed voltage-dependent channel activity with properties similar to those of purified mitochondrial VDAC. Amino acid sequence of four peptides, derived from VDAC band II, exhibited high homology to sequences present in human VDAC1 (98%), VDAC2 (91.8%), and VDAC3 (90%), while another peptide, derived from VDAC band III, showed lower homology to either VDAC1 (88.4%) or VDAC2 (79%). Two more peptides show high homology to the sequence present in mouse brain VDAC3 (100 and 78%). In addition, we demonstrate the translocation of ATP into synaptosomes, which is inhibited by DCCD and by the anion transport inhibitor DIDS. The possible function of VDAC in the synaptic plasma membrane is discussed.     

High-Level Expression, Refolding and Probing the Natural Fold of the Human Voltage-Dependent Anion Channel Isoforms I and II

The voltage-dependent anion channel (VDAC) is the major protein found in the outer membrane of mitochondria. The channel is responsible for the exchange of ATP/ADP and the translocation of ions and other small metabolites over the membrane. In order to obtain large amounts of pure and suitably folded human VDAC for functional and structural studies, the genes of the human isoforms I and II (HVDAC1 and HVDAC2) were cloned in Escherichia coli. High-level expression led to inclusion body formation. Both proteins could be refolded in vitro by adding denatured protein to a solution of zwitterionic or nonionic detergents. A highly efficient and fast protocol for refolding was developed that yielded more than 50 mg of pure human VDACs per liter of cell culture. The native and functional state of the refolded porins was probed by Fourier transform infrared spectroscopy to determine the secondary structure composition and by electrophysiological measurements, demonstrating the pore-forming activity of HVDAC1. Furthermore, binding of HVDAC1 to immobilized ATP was demonstrated. Limited proteolysis of HVDAC1 protein embedded in detergent micelles in combination with matrix-assisted laser desorption ionization mass spectrometric analysis was applied to identify micelle-exposed regions of the protein and to develop an improved topology model. Our analysis strongly suggests a 16-stranded, antiparallel beta-barrel with one large and seven short loops and turns. Initial crystallization trials of the protein yielded crystals diffracting to 8 Angstrom resolution.     

Mitochondria-associated Endoplasmic Reticulum Membrane (MAM) Regulates Steroidogenic Activity via Steroidogenic Acute Regulatory Protein (StAR)-Voltage-dependent Anion Channel 2 (VDAC2) Interaction

Steroid hormones are essential for carbohydrate metabolism, stress management, and reproduction and are synthesized from cholesterol in mitochondria of adrenal glands and gonads/ovaries. In acute stress or hormonal stimulation, steroidogenic acute regulatory protein (StAR) transports substrate cholesterol into the mitochondria for steroidogenesis by an unknown mechanism. Here, we report for the first time that StAR interacts with voltage-dependent anion channel 2 (VDAC2) at the mitochondria-associated endoplasmic reticulum membrane (MAM) prior to its translocation to the mitochondrial matrix. In the MAM, StAR interacts with mitochondrial proteins Tom22 and VDAC2. However, Tom22 knockdown by siRNA had no effect on pregnenolone synthesis. In the absence of VDAC2, StAR was expressed but not processed into the mitochondria as a mature 30-kDa protein. VDAC2 interacted with StAR via its C-terminal 20 amino acids and N-terminal amino acids 221–229, regulating the mitochondrial processing of StAR into the mature protein. In the absence of VDAC2, StAR could not enter the mitochondria or interact with MAM-associated proteins, and therefore steroidogenesis was inhibited. Furthermore, the N terminus was not essential for StAR activity, and the N-terminal deletion mutant continued to interact with VDAC2. The endoplasmic reticulum-targeting prolactin signal sequence did not affect StAR association with the MAM and thus its mitochondrial targeting. Therefore, VDAC2 controls StAR processing and activity, and MAM is thus a central location for initiating mitochondrial steroidogenesis.     

High Resolution Structure and Double Electron-Electron Resonance of the Zebrafish Voltage-dependent Anion Channel 2 Reveal an Oligomeric Population

In recent years, there has been a vast increase in structural and functional understanding of VDAC1, but VDAC2 and -3 have been understudied despite having many unique phenotypes. One reason for the paucity of structural and biochemical characterization of the VDAC2 and -3 isoforms stems from the inability of obtaining purified, functional protein. Here we demonstrate the expression, isolation, and basic characterization of zebrafish VDAC2 (zfVDAC2). Further, we resolved the structure of zfVDAC2 at 2.8 Å resolution, revealing a crystallographic dimer. The dimer orientation was confirmed in solution by double electron-electron resonance spectroscopy and by cross-linking experiments disclosing a dimer population of ∼20% in lauryldimethine amine oxide detergent micelles, whereas in lipidic bicelles a higher population of dimeric and higher order oligomers species were observed. The present study allows for a more accurate structural comparison between VDAC2 and its better-studied counterpart VDAC1.     

The BH4 Domain of Anti-apoptotic Bcl-XL,but Not That of the Related Bcl-2, Limits the Voltage-dependent Anion Channel 1 (VDAC1)-mediated Transfer of Pro-apoptotic Ca2+ Signals to Mitochondria

Excessive Ca²⁺ fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca²⁺-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP₃Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca²⁺-dependent apoptosis by binding and inhibiting IP₃Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP₃Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca²⁺ uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1^−/− cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca²⁺ uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca²⁺ release in the cytosol or to prevent apoptosis, as done instead by an IP₃R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca²⁺ uptake into the mitochondria.     

Linearity, saturation and blocking in a large multiionic channel: divalent cation modulation of the OmpF porin conductance

Measurement of unitary conductance is a fundamental step in the characterization of a protein ion channel permeabilizing a membrane. We study here the effect of salts of divalent cations on the OmpF channel conductance with a particular emphasis in dissecting the role of the electrolyte itself, the role of the counterion accumulation induced by the protein channel charges and other effects not found in salts of monovalent cations. We show that current saturation and blocking are not exclusive properties of narrow (single-file) ion channels but may be observed in large, multiionic channels like bacterial porins. Single-channel conductance measurements performed over a wide range of salt concentrations (up to 3 M) combined with continuum electrodiffusion calculations demonstrate that current saturation cannot be simply ascribed to ion interaction with protein channel residues.     

Microcompartmentation of energy metabolism at the outer mitochondrial membrane: Role in diabetes mellitus and other diseases

Complexes made up of the kinases, hexokinase and glycerol kinase, together with the outer mitochondrial membrane voltage-dependent anion channel (VDAC) protein, porin, and the inner mitochondrial membrane protein, the adenine nucleotide translocator, are involved in tumorigenesis, diabetes mellitus, and central nervous system function. Identification of these two mitochondrial membrane proteins, along with an 18 kD protein, as components of the peripheral benzodiazepine receptor, provides independent confirmation of the interaction of porin and the adenine nucleotide translocator to form functional contact sites between the inner and outer mitochondrial membranes. We suggest that these are dynamic structures, with channel conductances altered by the presence of ATP, and that ligand-mediated conformational changes in the porin-adenine nucleotide translocator complexes may be a general mechanism in signal transduction.     

Characterization of a Chloride-Selective Channel from Rough Endoplasmic Reticulum Membranes of Rat Hepatocytes: Evidence for a Block by Phosphate

We have characterized the conduction and blocking properties of a chloride channel from rough endoplasmic reticulum membranes of rat hepatocytes after incorporation into a planar lipid bilayer. Our experiments revealed the existence of a channel with a mean conductance of 164 ± 5 pS in symmetrical 200 mm KCl solutions. We determined that the channel was ten times more permeable for Cl⁻ than for K⁺, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channel was voltage dependent, with an open probability value ranging from 0.9 at −20 mV to 0.4 at +60 mV. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to zero current level. Our results showed a decrease of the channel mean open time combined with an increase of the channel mean closed time at positive potentials. An analysis of the dwell time distributions for the open and closed intervals led to the conclusion that the observed fluctuation pattern was compatible with a kinetic scheme containing a single open state and a minimum of three closed states. The permeability sequence for test halides determined from reversal potentials was Br⁻ > Cl⁻ > I⁻≈ F⁻. The voltage dependence of the open probability was modified by the presence of halides in trans with a sequence reflecting the permeability sequence, suggesting that permeant anions such as Br⁻ and Cl⁻ have access to an internal site capable of controlling channel gating. Adding NPPB to the cis chamber inhibited the channel activity by increasing fast flickering and generating long silent periods, whereas channel activity was not affected by 50 μm DNDS in trans. The channel was reversibly inhibited by adding phosphate to the trans chamber. The inhibitory effect of phosphate was voltage-dependent and could be reversed by addition of Cl⁻. Our results suggest that channel block involves the interaction of HPO²⁻ ₄ with a site located at 70% of the membrane span. Received: 10 January 1997/Revised: 29 May 1997     

Modulation of the Calmodulin-induced Inhibition of Sarcoplasmic Reticulum Calcium Release Channel (Ryanodine Receptor) by Sulfhydryl Oxidation in Single Channel Current Recordings and [3H]Ryanodine Binding

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl–sulfonic acid (4-CMPS) and 4,4′-dithiodipyridine (4,4′-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [³H]ryanodine binding to HSR vesicles. 0.1 μm CaM reduced the open probability (P _o ) of the calcium release channel at maximally activating calcium concentrations (50–100 μm) from 0.502 ± 0.02 to 0.137 ± 0.022 (n= 28), with no effect on unitary conductance. 4-CMPS (10–40 μm) and 4,4′-DTDP (0.1–0.3 mm) induced a concentration dependent increase in P _o (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4′-DTDP to higher concentrations in single channel recordings and [³H]ryanodine binding. 40 μm 4-CMPS induced a near maximal (P _o > 0.9) and 0.3 mm 4,4′-DTDP a submaximal (P _o = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4′-DTDP affected Ca-[¹²⁵I]calmodulin binding to HSR. 1 mm MgCl₂ reduced P _o from 0.53 to 0.075 and 20–40 μm 4-CMPS induced a near maximal channel activation (P _o > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4′-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel. Received: 22 July 1999/Revised: 15 November 1999     

Translocation of Bax and Bid to Mitochondria, Endoplasmic Reticulum and Nuclear Envelope: Possible Control Points in Apoptosis

The cross-talk between endoplasmic reticulum (ER) and mitochondria was investigated during apoptosis in a breast cancer cell line (MCF-7) in culture. The effect of camptothecin, an inducer of apoptosis and a specific inhibitor of topoisomerase I, was investigated by morphological, immunocytochemical and histochemical techniques for electron microscopy. Our ultrastructural morphological data demonstrate alterations in ER configuration and communication with neighbouring mitochondria early after stimulation by camptothecin. Immunoelectron studies have demonstrated that Bax and Bid translocate from cytoplasm to mitochondria where they initiate mitochondrial dysfunction and cytochrome c release. Bax and Bid were also localized in ER and nuclear envelope. Since ER and mitochondria function as intracellular Ca2+ storage, we hypothesize that Bax and Bid are involved in the emptying of ER Ca2+ pool, triggers secondary changes in mitochondrial Ca2+ levels that contribute to cytochrome c release and cell death.     

A Mutation in Transmembrane Domain 7 (TM7) of Excitatory Amino Acid Transporters Disrupts the Substrate-dependent Gating of the Intrinsic Anion Conductance and Drives the Channel into a Constitutively Open State

In the mammalian central nervous system, excitatory amino acid transporters (EAATs) are responsible for the clearance of glutamate after synaptic release. This energetically demanding activity is crucial for precise neuronal communication and for maintaining extracellular glutamate concentrations below neurotoxic levels. In addition to their ability to recapture glutamate from the extracellular space, EAATs exhibit a sodium- and glutamate-gated anion conductance. Here we show that substitution of a conserved positively charged residue (Arg-388, hEAAT1) in transmembrane domain 7 with a negatively charged amino acid eliminates the ability of glutamate to further activate the anion conductance. When expressed in oocytes, R388D or R388E mutants show large anion currents that display no further increase in amplitude after application of saturating concentrations of Na⁺ and glutamate. They also show a substantially reduced transport activity. The mutant transporters appear to exist preferentially in a sodium- and glutamate-independent constitutive open channel state that rarely transitions to complete the transport cycle. In addition, the accessibility of cytoplasmic residues to membrane-permeant modifying reagents supports the idea that this substrate-independent open state correlates with an intermediate outward facing conformation of the transporter. Our data provide additional insights into the mechanism by which substrates gate the anion conductance in EAATs and suggest that in EAAT1, Arg-388 is a critical element for the structural coupling between the substrate translocation and the gating mechanisms of the EAAT-associated anion channel.     

Glutathione Adducts on Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase Cys-674 Regulate Endothelial Cell Calcium Stores and Angiogenic Function as Well as Promote Ischemic Blood Flow Recovery

The sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is key to Ca²⁺ homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca²⁺ uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. To determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared with wild type (WT). Ca²⁺ studies showed decreased nitric oxide (·NO)-induced ⁴⁵Ca²⁺ uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca²⁺ stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca²⁺ influx. Adenoviral overexpression of calreticulin, an ER Ca²⁺ binding protein, increased ionomycin-releasable stores, VEGF-induced Ca²⁺ influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive Cys-674 thiol on SERCA 2 is required for normal endothelial cell Ca²⁺ homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca²⁺ stores.     

Polycystic Kidney Disease Channel and Synaptotagmin Homologues Play Roles in Schizosaccharomyces pombe Cell Wall Synthesis/Repair and Membrane Protein Trafficking

Eukaryotic cells can sense a wide variety of environmental stresses, including changes in temperature, pH, osmolarity and nutrient availability. They respond to these changes through a variety of signal-transduction mechanisms, including activation of Ca²⁺-dependent signaling pathways. This research has discovered important implications in the function(s) of polycystic kidney disease (PKD) channels and the mechanisms through which they act in the control of cell growth and cell polarity in Schizosaccharomyces pombe by ion channel-mediated Ca²⁺ signaling. Pkd2 was expressed maximally during the exponential growth phase. At the cell surface pkd2 was localized at the cell tip during the G₂ phase of the cell cycle, although following cell wall damage, the cell surface-expressed protein relocalized to the whole plasma membrane. Pkd2 depletion affected Golgi trafficking, resulting in a buildup of vesicles at the cell poles, and strongly affected plasma membrane protein delivery. Surface-localized pkd2 was present in the plasma membrane for a very short time and was rapidly internalized. Internalization was dependent on Ca²⁺, enhanced by amphipaths and inhibited by gadolinium. The pkd2 protein was in a complex with a yeast synaptotagmin homologue and myosin V. Depletion of pkd2 severely affected the localization of glucan synthase. A role for pkd2 in a cell polarity and cell wall synthesis signaling complex with a synaptotagmin homologue, myosin V and glucan synthase is proposed.     

植物离子通道特征、功能、调节与分子生物学

本文对植物离子通道的特征、生理功能、影响通道启闭的因素和通道分子生物学研究的新进展作了较为系统的综述     

The Endoplasmic Reticulum Chaperone BiP/GRP78 Is Important in the Structure and Function of the Human Cytomegalovirus Assembly Compartment

We previously demonstrated that the endoplasmic reticulum (ER) chaperone BiP functions in human cytomegalovirus (HCMV) assembly and egress. Here, we show that BiP localizes in two cytoplasmic structures in infected cells. Antibodies to the extreme C terminus, which includes BiP''s KDEL ER localization sequence, detect BiP in regions of condensed ER near the periphery of the cell. Antibodies to the full length, N terminus, or larger portion of the C terminus detect BiP in the assembly compartment. This inability of C-terminal antibodies to detect BiP in the assembly compartment suggests that BiP''s KDEL sequence is occluded in the assembly compartment. Depletion of BiP causes the condensed ER and assembly compartments to dissociate, indicating that BiP is important for their integrity. BiP and pp28 are in association in the assembly compartment, since antibodies that detect BiP in the assembly compartment coimmunoprecipitate pp28 and vice versa. In addition, BiP and pp28 copurify with other assembly compartment components on sucrose gradients. BiP also coimmunoprecipitates TRS1. Previous data show that cells infected with a TRS1-deficient virus have cytoplasmic and assembly compartment defects like those seen when BiP is depleted. We show that a fraction of TRS1 purifies with the assembly compartment. These findings suggest that BiP and TRS1 share a function in assembly compartment maintenance. In summary, BiP is diverted from the ER to associate with pp28 and TRS1, contributing to the integrity and function of the assembly compartment.Human cytomegalovirus (HCMV), the largest of the human herpesviruses, is capable of encoding over 200 proteins, which are expressed in temporal fashion as immediate-early, early, delayed-early, and late genes. Despite the extensive coding capacity of HCMV, its replication cycle is slow. During this protracted period, the virus must maintain optimal replication conditions in the host cell. However, the increasing strain of the infection induces cellular stress responses with consequences that may be deleterious to the progress of the infection. We and others have previously shown that HCMV has multiple mechanisms to deal with the deleterious aspects of cellular stress responses while maintaining beneficial ones (2, 8-10, 14, 17, 18, 22-24, 26, 27, 50, 51).An example of these mechanisms is the viral control of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Due to the number of HCMV proteins that are glycosylated, or receive other ER-dependent posttranslational modifications, the load of proteins in the ER can exceed its capacity, resulting in ER stress and the activation of the UPR (18, 47, 51). However, we and others have shown that HCMV controls and modulates the UPR, maintaining aspects that may benefit the viral infection while inhibiting aspects that would be detrimental (18, 51).The UPR is normally controlled by transmembrane sensors which initiate the complex UPR signaling cascade when activated by ER stress (reviewed in references 20, 35, 38, and 52). The ER molecular chaperone BiP (immunoglobulin heavy chain-binding protein), also called glucose-regulated protein 78 (GRP78), is believed to bind these sensors and keep them inactive during unstressed conditions. However, when unfolded or misfolded proteins accumulate in the ER, BiP leaves these sensors to perform its chaperone function, thus allowing the sensors to activate UPR signaling. We have previously shown that during HCMV infection, BiP is vastly overproduced (8), suggesting that BiP may have other functions in the viral infection. Indeed, it has been shown that BiP binds to the viral proteins US2 and US11; this interaction is necessary for the virus-mediated degradation of major histocompatibility complex class I and II (15, 47). Further, we have shown that depletion of BiP, using either the BiP-specific subtilase cytotoxin SubAB (32) or short hairpin RNAs, caused infectious virion formation in the cytoplasm to cease and nucleocapsids to accumulate just outside the outer nuclear membrane (8). This result suggested that BiP has a significant role in virion formation and cytoplasmic egress.Although the exact mechanism of virion formation in the cytoplasm is not well understood, studies have identified a perinuclear structure, referred to as the cytoplasmic assembly compartment, that is involved in the process. Several viral proteins, for example, tegument proteins (pp28, pp65) (36) and viral glycoproteins (gB, gH, gL, gO, gp65) (36, 46), have been identified as part of this structure. Defining the exact origin of this compartment has been complicated by the observation of specific organellar markers in and around the compartment, while other markers of the same organelles are not detected. For example, immunofluorescence examination suggests that the early endosomal marker early endosome antigen 1 (EEA1) has been observed in the center of the assembly compartment (12, 13); however, Rab4 and Rab5, other early endosomal markers, were not detected (16). Such observations suggest that the virus directs specific viral and cellular proteins to the assembly compartment as needed for assembly compartment function.In the present study, we further examine the role of BiP during an HCMV infection, including its localization and interactions with other proteins. We show here that in infected cells, BiP localizes in two distinct structures, regions of condensed ER near the periphery of the cell and the assembly compartment. The data suggest that BiP diversion from the ER to the assembly compartment is due to occlusion of its ER localization signal. Depletion of BiP causes both condensed ER and assembly compartments to disperse, indicating that BiP is important for their formation or maintenance. BiP and pp28 appear to associate in the assembly compartment, since BiP from the assembly compartment coimmunoprecipitates pp28 and vice versa. In addition, both BiP and pp28 copurify with the assembly compartment on sucrose gradients. BiP also coimmunoprecipitates TRS1. Previous studies (1, 4) have shown that cells infected with HCMV with a mutation in the TRS1 gene show cytoplasmic and assembly compartment defects like those seen when BiP is depleted (reference 8 and the studies presented below). We show that a fraction of TRS1 purifies with the assembly compartment, indicating a shared assembly compartment function with BiP. In summary, our data suggest that BiP is diverted from the ER to associate with pp28 and TRS1, contributing to the integrity and function of the assembly compartment.     

Inactivation of the Peptide-Sensitive Channel from the Yeast Mitochondrial Outer Membrane: Properties,Sensitivity to Trypsin and Modulation by a Basic Peptide

The yeast Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane closes with slow kinetics at potentials of either polarity. The properties of this inactivation closely resemble those of the Voltage-Dependent Anion Channel (VDAC) slow kinetics closures. Addition of trypsin to one compartment suppresses the inactivation observed when this compartment is made positive, but does not affect the inactivation observed at potentials of reverse polarity. Both sides of the channel are sensitive. The reduced form of the Mast Cell Degranulating peptide (rMCD) increases the rate of inactivation, but only when the polarity of the compartment to which it is added is positive. The effect is not reversed by washing the peptide out, but is suppressed by trypsin. The peptide can bind to both sides of the membrane. The effect of rMCD on PSC closely resembles that of the ``modulator' on VDAC. The similarities between PSC and VDAC suggest that the former might be a cationic porin of the mitochondrial outer membrane possessing a structure closely related to that of VDAC. Received: 2 February 1996/Revised: 18 October 1996     

KCa3.1通道参与调控星形胶质细胞糖氧剥夺诱导的内质网应激

目的:研究KCa3.1在糖氧剥夺诱导的原代星形胶质细胞内质网应激(ERS)中的调控作用。方法:通过构建原代星形胶质细胞糖氧剥夺(OGD)模型,应用cck-8法、免疫荧光技术、western blotting等分子生物学技术研究KCa3.1在OGD引起的原代星形胶质细胞内质网应激中的作用。结果:OGD 4 h处理后星形胶质细胞内KCa3.1的表达明显上调。OGD处理后星形胶质细胞的细胞活力显著性降低,且具有时间依赖性。给予KCa3.1通道抑制剂TRAM-34可提高OGD 4 h处理后星形胶质细胞的细胞活力,并具有剂量依赖性。OGD处理0.5 h、1 h、3 h、4 h、6 h后,原代星形胶质细胞内ERS信号通路被激活,GRP78、p-eIF-2α的表达显著性上调。给予KCa3.1通道抑制剂TRAM-34后,OGD引起的星形胶质细胞内GRP78、p-eIF-2α的上调幅度显著性降低。结论:KCa3.1通道参与了星形胶质细胞内OGD引起的内质网应激通路的激活。     

The Association of Receptor of Activated Protein Kinase C 1(RACK1) with Infectious Bursal Disease Virus Viral Protein VP5 and Voltage-dependent Anion Channel 2 (VDAC2) Inhibits Apoptosis and Enhances Viral Replication

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Our previous report indicates that IBDV VP5 induces apoptosis via interaction with voltage-dependent anion channel 2 (VDAC2). However, the underlying molecular mechanism is still unclear. We report here that receptor of activated protein kinase C 1 (RACK1) interacts with both VDAC2 and VP5 and that they could form a complex. We found that overexpression of RACK1 inhibited IBDV-induced apoptosis in DF-1 cells and that knockdown of RACK1 by small interfering RNA induced apoptosis associated with activation of caspases 9 and 3 and suppressed IBDV growth. These results indicate that RACK1 plays an antiapoptotic role during IBDV infection via interaction with VDAC2 and VP5, suggesting that VP5 sequesters RACK1 and VDAC2 in the apoptosis-inducing process.