Translocation of chicken heart apocytochrome c and its mutants (C17S, H18D) across mitochondrial membrane

Cytochrome c is a component of mitochondrial respiratory chain, located at the outer side of mitochondrial inner membrane. Its precursor, apocytochrome c, is encoded by a nuclear gene, synthesized on cytoplasmic ribosomes, and posttranslationally imported into mitochondria, but apocytochrome c is unique in the translocation compared with most mitochondrial proteins. It does not carry a cleavable amino terminal targeting sequence; no proteinous receptor on the mitochondrial outer membrane is identified for its import and its translocation does not compete with other preproteins for translocation machinery in the outer membrane. Besides, neither ATP nor membrane potential is required for its translocation across mitochonctria.     

蛋白质跨线粒体膜运送的研究进展

线粒体拥有约1000种蛋白质,其中98％以上系由细胞核编码,在细胞质核糖体上以前体形式合成,之后再运至线粒体,经跨膜运送并分选定位于各部分。现对定位于外膜、基质和内膜的蛋白质的运送途径的研究进展作一扼要介绍。脱血红素细胞色素c是细胞色素c的前体,它不含导肽,对其转运的研究概况也作了评述。     

Region of heat-stable enterotoxin II of Escherichia coli involved in translocation across the outer membrane

Heat-stable enterotoxin II of Escherichia coli (STII) is synthesized as a precursor form consisting of pre- and mature regions. The pre-region is cleaved off from the mature region during translocation across the inner membrane, and the mature region emerges in the periplasm. The mature region, composed of 48 amino acid residues, is processed in the periplasm by DsbA to form an intramolecular disulfide bond between Cys-10 and Cys-48 and between Cys-21 and Cys-36. STII formed with these disulfide bonds is efficiently secreted out of the cell through the secretory system, including TolC. However, it remains unknown which regions of STII are involved in interaction with TolC. In this study, we mutated the STII gene and examined the secretion of these STIIs into the culture supernatant. A deletion of the part covering from amino acid residue 37 to the carboxy terminal end did not markedly reduce the efficiency of secretion of STII into the culture supernatant. On the other hand, the efficiency of secretion of the peptide covering from the amino terminal end to position 18 to the culture supernatant was significantly low. These observations indicated that the central region of STII from amino acid residue 19 to that at position 36 is involved in the secretion of STII into the milieu. The experiment using a dsbA-deficient strain of E. coli showed that the disulfide bond between Cys-21 and Cys-36 by DsbA is necessary for STII to adapt to the structure that can cross the outer membrane.     

Folding of apocytochrome c induced by the interaction with negatively charged lipid micelles proceeds via a collapsed intermediate state

Unfolded apocytochrome c acquires an alpha-helical conformation upon interaction with lipid. Folding kinetic results below and above the lipid's CMC, together with energy transfer measurements of lipid bound states, and salt-induced compact states in solution, show that the folding transition of apocytochrome c from the unfolded state in solution to a lipid-inserted helical conformation proceeds via a collapsed intermediate state (I(C)). This initial compact state is driven by a hydrophobic collapse of the polypeptide chain in the absence of the heme group and may represent a heme-free analogue of an early compact intermediate detected on the folding pathway of cytochrome c in solution. Insertion into the lipid phase occurs via an unfolding step of I(C) through a more extended state associated with the membrane surface (I(S)). While I(C) appears to be as compact as salt-induced compact states in solution with substantial alpha-helix content, the final lipid-inserted state (Hmic) is as compact as the unfolded state in solution at pH 5 and has an alpha-helix content which resembles that of native cytochrome c.     

Modes of proton translocation across the cell membrane of respiring cyanobacteria

Acidification of weakly buffered suspensions of the cyanobacteria Anacystis nidulans, Nostoc sp. strain MAC, Dermocarpa sp. and Anabaena variabilis was observed after the application of oxygen pulses to anaerobic cells. The acidification was caused by proton extrusion from the oxygen pulsed cells since it was eliminated by the uncoupler (H⁺ ionophore) carbonyl cyanide m-chlorophenylhydrazone. Results with the inhibitors dicyclohexylcarbodiimide or 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, orthovanadate and cyanide indicated the association of various fractions of the observed proton extrusion with different activities of the cell membrane, viz. a H⁺-translocating reversible F₀F₁-ATPase, a unidirectional H⁺-translocating ATP hydrolase, and a respiratory electron transport system, respectively. Further parameters investigated were the pH dependence and the H⁺/O stoichiometry of the H⁺ extrusion from oxygen pulsed cyanobacteria. H⁺/O ratios at neutral pH were between 4 (Anacystis nidulans) and 0.3 (Dermocarpa) with uninhibited, actively phosphorylating cells and between 2 (Anacystis nidulans) and 0.4 (Dermocarpa) with ATPase-inhibited (ATP-depleted) cells, respectively. It is significant that with all four cyanobacteria tested a major fraction of the observed H⁺ ejection remained unaffected by ATPase inhibitors even at concentration which completely abolished all oxidative phosphorylation. Vanadate had a major effect on the H⁺ extrusion from Anabaena only. From this it is concluded that in the cyanobacterial species investigated part of the H⁺ extrusion from oxygen pulsed cells is directly linked to some H⁺-translocating respiratory electron transport chain present in the cell membrane.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD N, N-dicyclohexylcarbodiimide - DCMU N-(3,4-dichlorophenyl-)N,N-dimethylurea - NBD 7-chloro-4-nitrobenzoxa-1,3-diazole - TPP⁺ tetraphenylphosphonium - Mes 2-(N-morpholino)ethanesulfonic acid - Pipes piperazine-N,N-bis-(2-ethanesulfonic acid) - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Taps tris (hydroxymethyl)-methyl-aminopropanesulfonic acid - Ches 2-(N-cyclohexylamino)-ethanesulfonic acid - Caps 3-cyclohexylamino)-1-propanesulfonic acid; according to most textbooks (e.g. Nicholls 1982) the terms proton electrochemical potential ( ) and protonmotive force (pmf, p), both of which equivalently describe the energetic state of energy-transducing membranes, were used synonymously and expressed in mV units throughout this article (however, cf. Lowe and Jones 1984) Dedicated to Prof. G. Drews on the occasion of his 60th birthday     

Peptide 68-88 of apocytochrome c plays a crucial role in its insertion into membrane and binding to mitochondria

Apocytochrome c (Apocyt. C) is the precursor of cytochrome c. It is synthesized in the cytosol and posttranslationally imported into mitochondria. In order to determine the crucial sequence in apocyt. Ctranslocation, deleted mutant and chemically synthesized peptides with different length were used. Obtained results showed that sequence 68-88 of apocyt. C plays a critical role in its insertion into membrane and binding to mitochondria.     

Peptide 68-88 of apocytochrome c plays a crucial role in its insertion into membrane and binding to mitochondria

Apocytochromec(Apocyt.c)istheheme-freeprecursorofcytochromec.Itissynthesizedinthecytosolandpost-translationallyimportedintomitochondriabyfollowingaquiteuniquepath-waycomparedtoothermitochondrialprecursorproteins[1].ItdoesnotpossessacleavableN-terminalpresequence,andneitheramembranepotentialnorATPisrequiredforitsimport[2].Further,noproteinaceouscomponentresponsibleforitsimporthaseverbeenidentified[3,4].Intheintermembranespaceapocyt.cisconvertedintoholocytochromecbycytochromechemelyase(CCHL)…     

Substrate-specific function of the translocon-associated protein complex during translocation across the ER membrane

Although the transport of model proteins across the mammalian ER can be reconstituted with purified Sec61p complex, TRAM, and signal recognition particle receptor, some substrates, such as the prion protein (PrP), are inefficiently or improperly translocated using only these components. Here, we purify a factor needed for proper translocation of PrP and identify it as the translocon-associated protein (TRAP) complex. Surprisingly, TRAP also stimulates vectorial transport of many, but not all, other substrates in a manner influenced by their signal sequences. Comparative analyses of several natural signal sequences suggest that a dependence on TRAP for translocation is not due to any single physical parameter, such as hydrophobicity of the signal sequence. Instead, a functional property of the signal, efficiency of its post-targeting role in initiating substrate translocation, correlates inversely with TRAP dependence. Thus, maximal translocation independent of TRAP can only be achieved with a signal sequence, such as the one from prolactin, whose strong interaction with the translocon mediates translocon gating shortly after targeting. These results identify the TRAP complex as a functional component of the translocon and demonstrate that it acts in a substrate-specific manner to facilitate the initiation of protein translocation.     

High-resolution crystal structure of a truncated ColE7 translocation domain: implications for colicin transport across membranes

ColE7 is a nuclease-type colicin released from Escherichia coli to kill sensitive bacterial cells by degrading the nucleic acid molecules in their cytoplasm. ColE7 is classified as one of the group A colicins, since the N-terminal translocation domain (T-domain) of the nuclease-type colicins interact with specific membrane-bound or periplasmic Tol proteins during protein import. Here, we show that if the N-terminal tail of ColE7 is deleted, ColE7 (residues 63-576) loses its bactericidal activity against E.coli. Moreover, TolB protein interacts directly with the T-domain of ColE7 (residues 1-316), but not with the N-terminal deleted T-domain (residues 60-316), as detected by co-immunoprecipitation experiments, confirming that the N-terminal tail is required for ColE7 interactions with TolB. The crystal structure of the N-terminal tail deleted ColE7 T-domain was determined by the multi-wavelength anomalous dispersion method at a resolution of 1.7 angstroms. The structure of the ColE7 T-domain superimposes well with the T-domain of ColE3 and TR-domain of ColB, a group A Tol-dependent colicin and a group B TonB-dependent colicin, respectively. The structural resemblance of group A and B colicins implies that the two groups of colicins may share a mechanistic connection during cellular import.     

pH-dependent conformational changes of ferricytochrome c induced by electrode surface microstructure

pH-dependent processes of bovine heart ferricytochrome c have been investigated by electronic absorption and circular dichroism (CD) spectra at functionalized single-wall carbon nanotubes (SWNTs) modified glass carbon electrode (SWNTs/GCE) using a long optical path thin layer cell. These methods enabled the pH-dependent conformational changes arising from the heme structure change to be monitored. The spectra obtained at functionalized SWNTs/GCE reflect electrode surface microstructure-dependent changes for pH-induced protein conformation, pK_a of alkaline transition and structural microenvironment of the ferricytochrome c heme. pH-dependent conformational distribution curves of ferricytochrome c obtained by analysis of in situ CD spectra using singular value decomposition least square (SVDLS) method show that the functionalized SWNTs can retain native conformational stability of ferricytochrome c during alkaline transition.     

CH-ILKBP regulates cell survival by facilitating the membrane translocation of protein kinase B/Akt

Cell survival depends on proper propagation of protective signals through intracellular signaling intermediates. We report here that calponin homology domain-containing integrin-linked kinase (ILK)-binding protein (CH-ILKBP), a widely expressed adaptor protein localized at plasma membrane-actin junctions, is essential for transmission of survival signals. Cells that are depleted of CH-ILKBP undergo extensive apoptosis despite the presence of cell-extracellular matrix contacts and soluble growth factors. The activating phosphorylation of protein kinase B (PKB/Akt), a key regulator of apoptosis, is impaired in the absence of CH-ILKBP. Importantly, loss of CH-ILKBP prevents the membrane translocation of PKB/Akt. Furthermore, forced membrane targeting of PKB/Akt bypasses the requirement of CH-ILKBP for the activating phosphorylation of PKB/Akt, suggesting that CH-ILKBP is required for the membrane translocation but not the subsequent phosphorylation of PKB/Akt. Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2. However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation. Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.     

Amino acid sequence motifs and mechanistic features of the membrane translocation of alpha-synuclein

Many lines of evidence suggest that alpha-synuclein can be secreted from cells and can penetrate into them, although the detailed mechanism is not known. In this study, we investigated the amino acid sequence motifs required for the membrane translocation of alpha-synuclein, and the mechanistic features of the phenomenon. We first showed that not only alpha-synuclein but also beta- and gamma-synucleins penetrated into live cells, indicating that the conserved N-terminal region might be responsible for the membrane translocation. Using a series of deletion mutants, we demonstrated that the 11-amino acid imperfect repeats found in synuclein family members play a critical role in the membrane translocation of these proteins. We further demonstrated that fusion peptides containing the 11-amino acid imperfect repeats of alpha-synuclein can transverse the plasma membrane, and that the membrane translocation efficiency is optimal when the peptide contains two repeat motifs. alpha-Synuclein appeared to be imported rapidly and efficiently into cells, with detectable protein in the cytoplasm within 5 min after exogenous treatment. Interestingly, the import of alpha-synuclein at 4 degrees C was comparable with the import observed at 37 degrees C. Furthermore, membrane translocation of alpha-synuclein was not significantly affected by treatment with inhibitors of endocytosis. These results suggest that the internalization of alpha-synuclein is temperature-insensitive and occurs very rapidly via a mechanism distinct from normal endocytosis.     

Nuclear translocation of EGF receptor regulated by Epstein-Barr virus encoded latent membrane protein 1

Epstein-Barrvirus(EBV),oneoftheDNAon-cogenicviruses,iscloselyassociatedwiththegenesisofBurkitt抯lymphoma,undifferentiatednasopharyn-gealcarcinoma(NPC),Hodgkin抯disease,gastriccancerand,lungcancer,etc.[1].EBVencodedlatentmembraneprotein1(LMP1)isconsideredtobethemajoroncogenicproteinofEBVencodedproteins.Biologicallyspeaking,LMP1isanintegralmembraneproteincontaining386aminoacids.Thethreedo-mains(CTAR1,CTAR2,CTAR3)intheC-terminusofLMP1havebeenshowntoinitiatethesignalingproc-ess.The…     

Role of the C-terminal pro-domain of aqualysin I in the maturation and translocation of the precursor across the cytoplasmic membrane in Escherichia coli

Aqualysin I, which is a subtilisin-type, extracellular protease secreted by Thermus aquaticus YT-1, is synthesized as a unique precursor bearing pro-domains at both N- and C-terminus of the mature protease domain as well as an N-terminal signal peptide. To investigate the function of the C-terminal pro-domain in maturation and export pathway of the precursor in E. coli cells, aqualysin I variants were constructed in which deletion mutants of the C-terminal pro-domain lacking its own signal peptide were inserted into pIN-III-ompA3. When E. coli harboring wild type and mutant plasmids were induced by 0.2 mM IPTG, active aqualysin I was produced by heat treatment at 65 °C. Aqualysin I precursors with deletions of more than 5 amino acid residues at the C-terminal end of pro-domain were much more rapidly processed than that of wild type, indicating that the C-terminal pro-domain functions as a inhibitor for processing of aqualysin I precursor. With the wild type, most of aqualysin I was present in membrane fraction (probably the outer membrane), whereas for the truncated mutants, it remained in the cytoplasm, indicating that for deletion mutants, their precursors expressed in cells were not translocated across the cytoplasmic membrane, despite the existence of an N-terminal signal peptide.     

Enhancement of protein translocation across the membrane by specific mutations in the hydrophobic region of the signal peptide.

The hydrophobic region of the signal peptide of the OmpA protein of the Escherichia coli outer membrane was extensively altered in its hydrophobicity and predicted secondary structure by site-specific mutagenesis. The mutated signal peptides were fused to nuclease A from Staphylococcus aureus, and the function of the signal peptide was examined by measuring the rate of processing of the signal peptide. Six of the 12 mutated signal peptides in the nuclease hybrid were processed faster than the wild-type. In particular, the processing of the mutated signal peptide in which the alanine residue at position 9 was substituted with a valine residue was enhanced almost twofold over the processing of the wild-type signal peptide. In addition, the production of nuclease A fused with this mutated signal peptide also increased twofold. However, these effects were not observed when the mutated signal peptide was fused to TEM beta-lactamase. Analysis of the present mutations suggests that both overall hydrophobicity and distinct structural requirements in the hydrophobic region have important roles in signal peptide function.     

Changes in levels of pancreatic endoplasmic reticulum proteins that function in translocation and maturation of secretory proteins in response to cholecystokinin

Two pathways operate to target newly-synthesised proteins to the endoplasmic reticulum. In one, the signal recognition particle attaches to the signal sequences of nascent chains on ribosomes and slows or stops translation until contact is made with the docking protein at the membrane. The second operates via molecular chaperons. The pathways converge at the level of a 43 kDa signal binding protein integrated into the membrane, where translocation through a proteinaceous pore is initiated. In the lumen, proteins fold and disulphide formation is catalysed by the enzyme protein disulphide isomerase. The heavy chain binding protein may attach to unassembled or unfolded proteins and prevent their exit from the ER to the Golgi. Cholecystokinin (CCK) treatment increases the biosynthesis and secretion of pancreatic proteins, increases the levels of PDI and the 43 kDa binding protein, and reduces levels of BiP. These proteins may be possible targets for genetic manipulation to improve processing of heterologous proteins from cultured mammalian cells.