Cell membranes are phospholipid bilayers with a large number of embedded transmembrane proteins. Some of these proteins, such as scramblases, have properties that facilitate lipid flip-flop from one membrane leaflet to another. Scramblases and similar transmembrane proteins could also affect the translocation of other amphiphilic molecules, including cell-penetrating or antimicrobial peptides. We studied the effect of transmembrane proteins on the translocation of amphiphilic peptides through the membrane. Using two very different models, we consistently demonstrate that transmembrane proteins with a hydrophilic patch enhance the translocation of amphiphilic peptides by stabilizing the peptide in the membrane. Moreover, there is an optimum amphiphilicity because the peptide could become overstabilized in the transmembrane state, in which the peptide-protein dissociation is hampered, limiting the peptide translocation. The presence of scramblases and other proteins with similar properties could be exploited for more efficient transport into cells. The described principles could also be utilized in the design of a drug-delivery system by the addition of a translocation-enhancing peptide that would integrate into the membrane.

• MeSH
• buněčná membrána MeSH
• fosfolipidy MeSH
• lipidové dvojvrstvy * MeSH
• membránové proteiny MeSH
• peptidy * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfolipidy MeSH
• lipidové dvojvrstvy * MeSH
• membránové proteiny MeSH
• peptidy * MeSH

Hundreds of mitochondrial proteins with N-terminal presequences are translocated across the outer and inner mitochondrial membranes via the TOM and TIM23 complexes, respectively. How translocation of proteins across two mitochondrial membranes is coordinated is largely unknown. Here, we show that the two domains of Tim50 in the intermembrane space, named core and PBD, both have essential roles in this process. Building upon the surprising observation that the two domains of Tim50 can complement each other in trans, we establish that the core domain contains the main presequence-binding site and serves as the main recruitment point to the TIM23 complex. On the other hand, the PBD plays, directly or indirectly, a critical role in cooperation of the TOM and TIM23 complexes and supports the receptor function of Tim50. Thus, the two domains of Tim50 both have essential but distinct roles and together coordinate translocation of proteins across two mitochondrial membranes.

• MeSH
• mitochondriální importní komplex MeSH
• mitochondriální membrány * metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   MeSH
• transportní proteiny mitochondriální membrány genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální importní komplex MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• transportní proteiny mitochondriální membrány MeSH

Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide (Allen et al., 2016). Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogendeuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP-induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.

• Klíčová slova
• E. coli, SecA, SecYEG, biochemistry, chemical biology, computational biology, electron paramagnetic resonance (EPR) spectroscopy, hydrogen deuterium exchange (HDX) mass spectrometry, molecular dynamics, protein translocation, systems biology,
• MeSH
• adenosintrifosfát chemie   metabolismus   MeSH
• adenosintrifosfatasy chemie   metabolismus   MeSH
• Escherichia coli metabolismus   MeSH
• membránové transportní proteiny chemie   metabolismus   MeSH
• molekulární modely MeSH
• proteinové prekurzory metabolismus   MeSH
• proteiny SecA chemie   metabolismus   MeSH
• proteiny z Escherichia coli chemie   metabolismus   MeSH
• sbalování proteinů * MeSH
• translokační kanály SEC chemie   metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• adenosintrifosfatasy MeSH
• membránové transportní proteiny MeSH
• proteinové prekurzory MeSH
• proteiny SecA MeSH
• proteiny z Escherichia coli MeSH
• translokační kanály SEC MeSH

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.

• Klíčová slova
• E. coli, SecA, SecYEG, fluorescence, kinetics, molecular biophysics, single molecule, stochastic, structural biology,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenosintrifosfatasy chemie   genetika   metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• fluorescenční mikroskopie metody   MeSH
• hydrolýza MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• mutace MeSH
• proteiny - lokalizační signály genetika   MeSH
• proteiny SecA MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• protonmotorická síla * MeSH
• translokační kanály SEC chemie   genetika   metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• adenosintrifosfatasy MeSH
• bakteriální proteiny MeSH
• proteiny - lokalizační signály MeSH
• proteiny SecA MeSH
• proteiny z Escherichia coli MeSH
• translokační kanály SEC MeSH

Potato and tomato plant uncoupling mitochondrial protein (PUMP) was reconstituted into liposomes, and K+ or H+ fluxes associated with fatty acid (FA)-induced ion movement were measured using fluorescent ion indicators potassium binding benzofuraneisophthalate and 6-methoxy-N-(3-sulfopropyl)-quinolinium. We suggest that PUMP, like its mammalian counterpart, the uncoupling protein of brown adipose tissue mitochondria (Garlid, K. D., Orosz, D. E., Modrianský, M., Vassanelli, S., and Jeek, P. (1996), J. Biol. Chem. 271, 2615-2702), allows for H+ translocation via a FA cycling mechanism. Reconstituted PUMP translocated anionic linoleic and heptylbenzoic acids, undecanesulfonate, and hexanesulfonate, but not phenylvaleric and abscisic acids or Cl-. Transport was inhibited by ATP and GDP. Internal acidification of protein-free liposomes by linoleic or heptylbenzoic acid indicated that H+ translocation occurs by FA flip-flopping across the lipid bilayer. However, addition of valinomycin after FA-initiated GDP-sensitive H+ efflux solely in proteoliposomes, indicating that influx of anionic FA via PUMP precedes a return of protonated FA carrying H+. Phenylvaleric acid, unable to flip-flop, was without effect. Kinetics of FA and undecanesulfonate uniport suggested the existence of an internal anion binding site. Exponential flux-voltage characteristics were also studied. We suggest that regulated uncoupling in plant mitochondria may be important during fruit ripening, senescence, and seed dormancy.

• MeSH
• biologický transport MeSH
• iontové kanály MeSH
• kinetika MeSH
• kyseliny sulfonové metabolismus   MeSH
• mastné kyseliny metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• mitochondriální proteiny MeSH
• proteolipidy metabolismus   MeSH
• protony MeSH
• Solanum lycopersicum metabolismus   MeSH
• Solanum tuberosum metabolismus   MeSH
• transportní proteiny metabolismus   MeSH
• uncoupling protein 1 MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• iontové kanály MeSH
• kyseliny sulfonové MeSH
• mastné kyseliny MeSH
• membránové proteiny MeSH
• mitochondriální proteiny MeSH
• proteolipidy MeSH
• proteoliposomes MeSH Prohlížeč
• protony MeSH
• transportní proteiny MeSH
• uncoupling protein 1 MeSH

The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.

• Klíčová slova
• Dynamic Allostery, Protein Translocation, SecA, SecYEG, Single‐Molecule FRET,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• adenosintrifosfatasy genetika   metabolismus   MeSH
• bakteriální proteiny * metabolismus   MeSH
• nukleotidy metabolismus   MeSH
• proteiny SecA metabolismus   MeSH
• proteiny z Escherichia coli * metabolismus   MeSH
• translokační kanály SEC chemie   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• adenosintrifosfatasy MeSH
• bakteriální proteiny * MeSH
• nukleotidy MeSH
• proteiny SecA MeSH
• proteiny z Escherichia coli * MeSH
• translokační kanály SEC MeSH

The metazoan Sec61 translocon transports polypeptides into and across the membrane of the endoplasmic reticulum via two major routes, a well-established co-translational pathway and a post-translational alternative. We have used two model substrates to explore the elements of a secretory protein precursor that preferentially direct it towards a co- or post-translational pathway for ER translocation. Having first determined the capacity of precursors to enter ER derived microsomes post-translationally, we then exploited semi-permeabilized mammalian cells specifically depleted of key membrane components using siRNA to address their contribution to the membrane translocation process. These studies suggest precursor chain length is a key factor in the post-translational translocation at the mammalian ER, and identify Sec62 and Sec63 as important components acting on this route. This role for Sec62 and Sec63 is independent of the signal sequence that delivers the precursor to the ER. However, the signal sequence can influence the subsequent membrane translocation process, conferring sensitivity to a small molecule inhibitor and dictating reliance on the molecular chaperone BiP. Our data support a model where secretory protein precursors that fail to engage the signal recognition particle, for example because they are short, are delivered to the ER membrane via a distinct route that is dependent upon both Sec62 and Sec63. Although this requirement for Sec62 and Sec63 is unaffected by the specific signal sequence that delivers a precursor to the ER, this region can influence subsequent events, including both Sec61 mediated transport and the importance of BiP for membrane translocation. Taken together, our data suggest that an ER signal sequence can regulate specific aspects of Sec61 mediated membrane translocation at a stage following Sec62/Sec63 dependent ER delivery.

• MeSH
• endoplazmatické retikulum metabolismus   MeSH
• intracelulární membrány metabolismus   MeSH
• malá interferující RNA MeSH
• posttranslační úpravy proteinů genetika   fyziologie   MeSH
• transport proteinů fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malá interferující RNA MeSH

Bordetella adenylate cyclase toxin (CyaA) binds the alpha(M)beta(2) integrin (CD11b/CD18, Mac-1, or CR3) of myeloid phagocytes and delivers into their cytosol an adenylate cyclase (AC) enzyme that converts ATP into the key signaling molecule cAMP. We show that penetration of the AC domain across cell membrane proceeds in two steps. It starts by membrane insertion of a toxin 'translocation intermediate', which can be 'locked' in the membrane by the 3D1 antibody blocking AC domain translocation. Insertion of the 'intermediate' permeabilizes cells for influx of extracellular calcium ions and thus activates calpain-mediated cleavage of the talin tether. Recruitment of the integrin-CyaA complex into lipid rafts follows and the cholesterol-rich lipid environment promotes translocation of the AC domain across cell membrane. AC translocation into cells was inhibited upon raft disruption by cholesterol depletion, or when CyaA mobilization into rafts was blocked by inhibition of talin processing. Furthermore, CyaA mutants unable to mobilize calcium into cells failed to relocate into lipid rafts, and failed to translocate the AC domain across cell membrane, unless rescued by Ca(2+) influx promoted in trans by ionomycin or another CyaA protein. Hence, by mobilizing calcium ions into phagocytes, the 'translocation intermediate' promotes toxin piggybacking on integrin into lipid rafts and enables AC enzyme delivery into host cytosol.

• MeSH
• adenylátcyklasový toxin chemie   metabolismus   MeSH
• antigeny CD11b metabolismus   MeSH
• antigeny CD18 metabolismus   MeSH
• Bordetella enzymologie   MeSH
• buněčná membrána enzymologie   mikrobiologie   MeSH
• cholesterol metabolismus   MeSH
• cytosol enzymologie   MeSH
• extracelulární prostor metabolismus   MeSH
• lidé MeSH
• makrofágový antigen 1 metabolismus   MeSH
• makrofágy metabolismus   mikrobiologie   MeSH
• membránové mikrodomény enzymologie   mikrobiologie   MeSH
• myši MeSH
• talin metabolismus   MeSH
• terciární struktura proteinů MeSH
• U937 buňky MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• antigeny CD11b MeSH
• antigeny CD18 MeSH
• cholesterol MeSH
• ITGAM protein, human MeSH Prohlížeč
• makrofágový antigen 1 MeSH
• talin MeSH
• vápník MeSH

Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein complexes on timescales spanning sub-milliseconds to minutes (10-9-102 s).

• Klíčová slova
• FRET, Lipid, Liposome, Protein export, SecA, SecY, Translocation,
• MeSH
• fluorescence * MeSH
• konformace proteinů MeSH
• lidé MeSH
• membránové proteiny analýza   chemie   metabolismus   MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH

Despite ongoing research on antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs), their precise translocation mechanism remains elusive. This includes Buforin 2 (BF2), a well-known AMP, for which spontaneous translocation across the membrane has been proposed but a high barrier has been calculated. Here, we used computer simulations to investigate the effect of a nonequilibrium situation where the peptides are adsorbed on one side of the lipid bilayer, mimicking experimental conditions. We demonstrated that the asymmetric membrane adsorption of BF2 enhances its translocation across the lipid bilayer by lowering the energy barrier by tens of kJ mol-1. We showed that asymmetric membrane adsorption also reduced the free energy barrier of lipid flip-flop but remained unlikely even at BF2 surface saturation. These results provide insight into the driving forces behind membrane translocation of cell-penetrating peptides in nonequilibrium conditions, mimicking experiments.

• MeSH
• adsorpce MeSH
• antimikrobiální peptidy chemie   farmakologie   MeSH
• buněčná membrána metabolismus   chemie   MeSH
• kationické antimikrobiální peptidy chemie   farmakologie   metabolismus   MeSH
• lipidové dvojvrstvy * chemie   metabolismus   MeSH
• penetrační peptidy chemie   metabolismus   MeSH
• proteiny MeSH
• simulace molekulární dynamiky MeSH
• termodynamika MeSH
• terpeny chemie   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antimikrobiální peptidy MeSH
• buforin II MeSH Prohlížeč
• kationické antimikrobiální peptidy MeSH
• lipidové dvojvrstvy * MeSH
• penetrační peptidy MeSH
• proteiny MeSH
• terpeny MeSH