Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) generates adenosine 3',5'-monophosphate (cAMP)-regulated chloride channels, indicating that CFTR is either a chloride channel or a chloride channel regulator. To distinguish between these possibilities, basic amino acids in the putative transmembrane domains were mutated. The sequence of anion selectivity of cAMP-regulated channels in cells containing either endogenous or recombinant CFTR was bromide greater than chloride greater than iodide greater than fluoride. Mutation of the lysines at positions 95 or 335 to acidic amino acids converted the selectivity sequence to iodide greater than bromide greater than chloride greater than fluoride. These data indicate that CFTR is a cAMP-regulated chloride channel and that lysines 95 and 335 determine anion selectivity.
Cystic fibrosis tnsmembrane conductance regulator (CFTR) generates cAMP-regulated channels; mutations in CFFR cause defective Cl-channel function in cystic fibrosis epithelia. We used the patch-chmp technique to determine the single channel properties of Cl-channels in cells expressing recombinant CFIR. In cell-attached patches, an increase in cellular cAMP reversibly activated low conductance Cl-channels. cAMP-dependent regulation is due to phosphorylation, because the catalytic subunit of cAMP-dependent protein kinase plus ATP reversibly activated the channel in excised, cell-free patches of membrane. In symmetrical a-solutions, the channel had a channel conductance of 10.4±0.2 (a = 7) pS and a linear current-voltage relation. The channel was more permeable to Cla than to I-and showed no appreciable time-dependent voltage effects. These biophysical properties are consistent with macroscopic studies of a-channels in single cells expressing CFTR and in the apical membrane of secretory epithelia. Identification of the single channel characteristics of CFIR-generated channels allows further studies oftheir regulation and the mechanism of ion permeation. (J. Cli,. Invest.
Ischemic tolerance is an endogenous neuroprotective mechanism in brain and other organs, whereby prior exposure to brief ischemia produces resilience to subsequent normally injurious ischemia. Although many molecular mechanisms mediate delayed (genemediated) ischemic tolerance, the mechanisms underlying rapid (protein synthesis-independent) ischemic tolerance are relatively unknown. Here we describe a novel mechanism for the induction of rapid ischemic tolerance mediated by the ubiquitin-proteasome system. Rapid ischemic tolerance is blocked by multiple proteasome inhibitors [carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), MG115 (carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal), and clasto-lactacystin--lactone].A proteomics strategy was used to identify ubiquitinated proteins after preconditioning ischemia. We focused our studies on two actin-binding proteins of the postsynaptic density that were ubiquitinated after rapid preconditioning: myristoylated, alanine-rich C-kinase substrate (MARCKS) and fascin. Immunoblots confirm the degradation of MARCKS and fascin after preconditioning ischemia. The loss of actin-binding proteins promoted actin reorganization in the postsynaptic density and transient retraction of dendritic spines. This rapid and reversible synaptic remodeling reduced NMDA-mediated electrophysiological responses and renders the cells refractory to NMDA receptor-mediated toxicity. The dendritic spine retraction and NMDA neuroprotection after preconditioning ischemia are blocked by actin stabilization with jasplakinolide, as well as proteasome inhibition with MG132. Together these data suggest that rapid tolerance results from changes to the postsynaptic density mediated by the ubiquitin-proteasome system, rendering neurons resistant to excitotoxicity.
The homologous proteins Oxa1, YidC, and Alb3 mediate the insertion of membrane proteins in mitochondria, bacteria, and chloroplast thylakoids, respectively. Depletion of YidC in Escherichia coli affects the integration of every membrane protein studied, and Alb3 has been shown previously to be required for the insertion of a signal recognition particle (SRP)-dependent protein, Lhcb1, in thylakoids. In this study we have analyzed the "global" role of Alb3 in the insertion of thylakoid membrane proteins. We show that insertion of two chlorophyll-binding proteins, Lhcb4.1 and Lhcb5, is almost totally blocked by preincubation of thylakoids with anti-Alb3 antibodies, indicating a requirement for Alb3 in the insertion pathway. Insertion of the related PsbS protein, on the other hand, is unaffected by Alb3 antibodies, and insertion of a group of SRP-independent, signal peptide-bearing proteins, PsbX, PsbW, and PsbY, is likewise completely unaffected. Proteinase K is furthermore able to completely degrade Alb3, but this treatment does not affect the insertion of these proteins. Among the thylakoid proteins studied here, Alb3 requirement correlates strictly with a requirement for stromal factors and nucleoside triphosphates. However, the majority of proteins tested do not require Alb3 or any other known form of translocation apparatus.The post-translational insertion of proteins into their target membranes has attracted a great deal of experimental attention in recent years in an effort to determine how hydrophobic regions are transferred from an aqueous environment into the membrane bilayer, and how the correct topology is achieved during this process. In bacteria, a complex "assisted" pathway (reviewed in Refs. 1 and 2) has been characterized in which newly synthesized membrane proteins interact with signal recognition particle (SRP), 1 FtsY and membrane-bound components of the secretory (Sec) apparatus (3-8). SRP appears to be involved in membrane protein biogenesis by virtue of its tendency to interact with particularly hydrophobic protein segments (6, 9). A broadly similar assisted pathway operates in plant thylakoids for the targeting of the major light-harvesting chlorophyll-binding (LHC) protein, Lhcb1, after import of this protein from the cytosol. Insertion of Lhcb1 into thylakoids requires nucleoside triphosphates (NTPs), stromal SRP, FtsY, and a thylakoid translocase minimally composed of Albino3 (Alb3) (10 -13). Post-translational formation of a SRP/Lhcb1 targeting complex requires a hydrophobic domain along with a novel SRP-binding element in Lhcb1, termed the L18 domain, which is found only in members of the LHC protein family (14, 15). These data along with studies on chloroplast-synthesized D1 (16) suggest that SRP is again used primarily to direct membrane proteins to the thylakoid membrane.For many years it was believed that other membrane proteins, in both bacteria and chloroplasts, were targeted by unassisted or "spontaneous" insertion pathways, in which the protein inserted directly into the bilayer...
The assembly of the chloroplast thylakoid membrane requires the import of numerous proteins from the cytosol and their targeting into or across the thylakoid membrane. It is now clear that multiple pathways are involved in the thylakoid-targeting stages, depending on the type of protein substrate. Two very different pathways are used by thylakoid lumen proteins; one is the Sec pathway which has been well-characterised in bacteria, and which involves the threading of the substrate through a narrow channel. In contrast, the more recently characterised twin-arginine translocation (Tat) system is able to translocate fully folded proteins across this membrane. Recent advances on bacterial Tat systems shed further light on the structure and function of this system. Membrane proteins, on the other hand, use two further pathways. One is the signal recognition particle-dependent pathway, involving a complex interplay between many different factors, whereas other proteins insert without the assistance of any known apparatus. This article reviews advances in the study of these pathways and considers the rationale behind the surprising complexity.Key words: chloroplast, membrane, protein transport, tat, thylakoid Received and accepted for publication 5 February 2001The chloroplast is well known as the site of photosynthesis in plants but it also carries out many other critical functions and consequently contains a vast range of protein typesprobably well over 1000. Some of these proteins, especially those involved in photosynthetic functions, are also present at very high concentrations, and the total protein concentration in the organelle is in the order of 300 mg/mL. A small proportion of these proteins are synthesised within the organelle, but the vast majority are encoded by nuclear genes and synthesised in the cytosol, with the net result that chloroplast biogenesis requires the import of a colossal amount of protein in a relatively short time. To complicate matters, this organelle is particularly complex in structural terms, comprising three separate membranes, which enclose three distinct 245 soluble phases. The chloroplast is bounded by a doublemembrane envelope, which encloses an intermembrane space, about which rather little is known. The major soluble phase is the stroma, which is the site of carbon fixation, amino acid synthesis and many other pathways, and the dominant membrane is the extensive interconnecting thylakoid network, where light is captured and ATP synthesised. Finally, the thylakoid membrane encloses a further soluble phase, the thylakoid lumen, which houses a number of extrinsic photosynthetic proteins as well as many others. Imported proteins are targeted into all six chloroplast subcompartments and the underlying mechanisms have been the focus of considerable attention over the past 25 years. Perhaps surprisingly, most proteins appear to be imported across the envelope membranes by a basically similar mechanism. This topic has been covered in a recent review (1) and will not be dealt with in det...
The ubiquitin-proteasome system is the major non-lysosymal system for degrading proteins in the cell; the work leading to its discovery was awarded the Nobel Prize in Chemistry in 2004. In addition to small ubiquitin-like modifiers (e.g. Sumo and Nedd8), ubiquitin is involved in the complex regulation of the levels and function of many proteins and signaling pathways involved in determining cell fate. The cell death regulatory proteins, such as Bcl-2 family proteins and caspases are targeted for degradation by the ubiquitin proteasome system (UPS). In addition to mediating the degradation of proteins, the UPS regulates function and translocation of proteins, many of which play a role in the determination of cell fate. For example the UPS can regulate the activity of transcription factors, such as P53, NF-kappaB and HIF-1 alpha, which control the expression of protein mediators of cell death. Aberrant UPS function has been reported in multiple neuropathologies including Parkinson's diseases and ischemia. With the number of ubiquitin conjugating and de-conjugating enzymes reaching close to the levels of protein kinases and phosphatases, it is clear that ubiquitination is an important biological regulatory step for proteins.
At 60-801% confluence keratinocyte cultures were assigned as controls or for gene transfer by infection with one or the other of two recombinant retrovirus vectors: (i) an amphotropic helper-free murine leukemia virus vector, MFG-lacZ, containing the (-galactosidase gene, lacZ (R.C.M., unpublished), or (ii) a vector, a-SGC-hGH, containing the gene sequence from hGH (L. Cohen and R.C.M., unpublished). Subconfluent keratinocyte cultures were incubated on 3 consecutive days with the retrovirus in Polybrene (8 pg/ml) and 10% fetal bovine serum containing Dulbecco's modified Eagle's medium (DMEM; GIBCO).During transfection the modified Waymouth medium was removed and replaced daily by an equal amount of DMEM containing the retrovirus at a concentration of 1-2 x 107 plaque-forming units/ml. After 6 hr the virus-containing medium was removed and replaced by Waymouth medium. This procedure was repeated on 3 consecutive days. At confluence, keratinocytes were released from the flasks with 0.01% trypsin and resuspended in normal unbuffered saline (0.9%O) containing 100 international units ofpenicillin and 100 pg of streptomycin per ml, at a concentration of 3 x 106 cells per ml. Keratinocytes resuspended in saline were found to be >90% viable, as determined by trypan blue staining.The procedure for making 170 reproducible standardized f-thickness skin wounds on the backs of 13 anesthetized pigs for these experiments was as follows. Under aseptic conditions using a 2.25-cm2 square template the skin was excised with a scalpel to the level ofthe panniculus carnosus muscle with careful hemostasis. Wound margins were tattooed with India ink, allowing for photoplanimetric evaluation of the surface area on sequential standardized photographs. The chambers, serving as an in vivo cell culture device, were applied to each wound (1, 4). The chamber (P. A. Medical, Columbia, TN) consists of a flexible transparent vinyl top bonded to an adhesive base. The base has a central opening fitting the wound margins. Chambers were filled with 1.2 ml of isotonic saline containing 100 pg of streptomycin and 100 international units penicillin per ml (1, 4). Twenty-two wounds were treated with lacZ transfected Abbreviations: hGH, human growth hormone; H&E, hematoxylin/ eosin; X-Gal, 5-bromo-4-chloro-3-indolyl -D-galactoside. t~o whom reprint requests should be addressed. 9307The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
We have previously shown that the cell death-promoting protein Bcl-2-interacting mediator of cell death (Bim) is ubiquitinated and degraded following a neuroprotection-conferring episode of brief ischemia (preconditioning). Here, we identify the E3 ligase that ubiquitinates Bim in this model, using a proteomics approach. Using phosphorylated GST-Bim as bait, we precipitated and identified by mass spectrometry tripartite motif protein 2 (TRIM2), a RING (really interesting new gene) domain-containing protein. The reaction between TRIM2 and Bim was confirmed using co-immunoprecipitation followed by immunoblotting. We show that TRIM2 binds to Bim when it is phosphorylated by p42/p44 MAPK but does not interact with a nonphosphorylatable Bim mutant (3ABim). 12-O-tetradecanoylphorbol-13-acetate activation of p42/p44 MAPK drives Bim ubiquitination in mouse embryonic fibroblast cells and is associated with an increased interaction between TRIM2 and Bim. One hour following preconditioning ischemia, the binding of Bim to TRIM2 increased, consistent with the time window of enhanced Bim degradation. Blocking p42/p44 MAPK activation following preconditioning ischemia with U0126 or using the nonphosphorylatable 3ABim reduced the binding between Bim and TRIM2. Immunodepletion of TRIM2 from cell lysates prepared from preconditioned cells reduced Bim ubiquitination. Finally, suppression of TRIM2 expression, using lentivirus transduction of shRNAmir, stabilized Bim protein levels and blocked neuroprotection observed in rapid ischemic tolerance. Taken together, these data support a role for TRIM2 in mediating the p42/p44 MAPK-dependent ubiquitination of Bim in rapid ischemic tolerance.Cerebral ischemia, the deprivation of oxygen and glucose to the brain, can result in neuronal death. However, prior exposure to a brief nonharmful dose of ischemia (preconditioning) activates an endogenous neuroprotective program, rendering the brain protected against subsequent ischemic injury (ischemic tolerance). Rapid (short term) ischemic tolerance in brain and cultured neurons occurs 1 h following a preconditioning stimulus, resulting in profound neuroprotection (1, 2). Although the exact molecular mechanisms of rapid tolerance are not fully resolved, they appear to be mediated by rapid biochemical events, including activation of adenosine receptors, ATP-activated potassium channels, multiple protein kinases, and the ubiquitin-proteasome system (1-7).The ubiquitin-proteasome system rapidly degrades short lived proteins in the cell. Ubiquitin is added to a target protein following a sequential series of reactions, whereby ubiquitin is bound first to an E1 ligase in a reaction requiring ATP. Ubiquitin is transferred to an E2 protein and then transferred via the E3 ligase to the target protein lysine residue (8). The HECT 3 (homologous to the E6-AP carboxyl terminus) class of E3 ligase binds to ubiquitin, prior to conjugating the ubiquitin to the target, whereas the RING (really interesting new gene)-containing E3 ligases appear to mediate the tran...
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