SecYEG
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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.
- 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
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.
- 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
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.
- 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 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
Autosomal-dominant tubulo-interstitial kidney disease (ADTKD) encompasses a group of disorders characterized by renal tubular and interstitial abnormalities, leading to slow progressive loss of kidney function requiring dialysis and kidney transplantation. Mutations in UMOD, MUC1, and REN are responsible for many, but not all, cases of ADTKD. We report on two families with ADTKD and congenital anemia accompanied by either intrauterine growth retardation or neutropenia. Ultrasound and kidney biopsy revealed small dysplastic kidneys with cysts and tubular atrophy with secondary glomerular sclerosis, respectively. Exclusion of known ADTKD genes coupled with linkage analysis, whole-exome sequencing, and targeted re-sequencing identified heterozygous missense variants in SEC61A1-c.553A>G (p.Thr185Ala) and c.200T>G (p.Val67Gly)-both affecting functionally important and conserved residues in SEC61. Both transiently expressed SEC6A1A variants are delocalized to the Golgi, a finding confirmed in a renal biopsy from an affected individual. Suppression or CRISPR-mediated deletions of sec61al2 in zebrafish embryos induced convolution defects of the pronephric tubules but not the pronephric ducts, consistent with the tubular atrophy observed in the affected individuals. Human mRNA encoding either of the two pathogenic alleles failed to rescue this phenotype as opposed to a complete rescue by human wild-type mRNA. Taken together, these findings provide a mechanism by which mutations in SEC61A1 lead to an autosomal-dominant syndromic form of progressive chronic kidney disease. We highlight protein translocation defects across the endoplasmic reticulum membrane, the principal role of the SEC61 complex, as a contributory pathogenic mechanism for ADTKD.
- MeSH
- alely MeSH
- anemie genetika MeSH
- biopsie MeSH
- chronická nemoc MeSH
- dánio pruhované embryologie genetika MeSH
- dítě MeSH
- dominantní geny MeSH
- dospělí MeSH
- endoplazmatické retikulum metabolismus MeSH
- exom genetika MeSH
- fenotyp MeSH
- Golgiho aparát metabolismus MeSH
- heterozygot * MeSH
- lidé středního věku MeSH
- lidé MeSH
- messenger RNA analýza genetika MeSH
- missense mutace genetika MeSH
- mladý dospělý MeSH
- molekulární modely MeSH
- mutace * MeSH
- nemoci ledvin genetika patologie MeSH
- neutropenie genetika MeSH
- novorozenec MeSH
- progrese nemoci MeSH
- rodokmen MeSH
- růstová retardace plodu genetika MeSH
- sekvence aminokyselin MeSH
- senioři MeSH
- syndrom MeSH
- translokační kanály SEC chemie genetika MeSH
- zvířata MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
The human Sec61 complex is a widely distributed and abundant molecular machine. It resides in the membrane of the endoplasmic reticulum to channel two types of cargo: protein substrates and calcium ions. The SEC61A1 gene encodes for the pore-forming Sec61α subunit of the Sec61 complex. Despite their ubiquitous expression, the idiopathic SEC61A1 missense mutations p.V67G and p.T185A trigger a localized disease pattern diagnosed as autosomal dominant tubulointerstitial kidney disease (ADTKD-SEC61A1). Using cellular disease models for ADTKD-SEC61A1, we identified an impaired protein transport of the renal secretory protein renin and a reduced abundance of regulatory calcium transporters, including SERCA2. Treatment with the molecular chaperone phenylbutyrate reversed the defective protein transport of renin and the imbalanced calcium homeostasis. Signal peptide substitution experiments pointed at targeting sequences as the cause for the substrate-specific impairment of protein transport in the presence of the V67G or T185A mutations. Similarly, dominant mutations in the signal peptide of renin also cause ADTKD and point to impaired transport of this renal hormone as important pathogenic feature for ADTKD-SEC61A1 patients as well.
- MeSH
- endoplazmatické retikulum metabolismus MeSH
- fenylbutyráty metabolismus farmakologie MeSH
- HEK293 buňky MeSH
- lidé MeSH
- missense mutace MeSH
- molekulární chaperony metabolismus MeSH
- nemoci ledvin patofyziologie MeSH
- polycystická choroba ledvin MeSH
- renin genetika metabolismus MeSH
- sarkoplazmatická Ca2+-ATPáza metabolismus MeSH
- translokační kanály SEC chemie genetika metabolismus MeSH
- transport proteinů genetika MeSH
- vápník metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Zkřížená prezentace antigenů je proces, kdy dendritické buňky předkládají antigeny zvenčí CD8+ T lymfocytům na MHC I glykoproteinech. Jako zkřížená se označuje proto, že oproti cestám klasické prezentace vede vskutku křížem, protože vnější antigeny jsou zpravidla prezentovány na MHC II a vnitřní na MHC I glykoproteinech. Molekulární mechanismus zkřížené prezentace doposud není uspokojivě objasněn. Uvažují se dvě hlavní cesty – vakuolární a cytosolická. Vakuolární cesta předpokládá, že pohlcené antigeny jsou naštěpeny v endosomu pomocí proteas a následně navázány na MHC I. Cytosolická cesta předpokládá, že pohlcený antigen proniká z váčku do cytosolu, kde je naštěpen v proteazomu. Odtud putuje buď do endoplasmatického retikula (ER), kde dochází k vazbě peptidu na MHC I i při klasické prezentaci antigenu, nebo zpět do endosomu, kam se mašinérie vázající peptid na MHC I přesouvá. Procesu se účastní proteiny z ER, včetně těch, které spolupracují na mechanismu ERAD, Rab GTPasy regulující váčkový transport a struktury podílející se na maturaci endosomů. Zkřížená prezentace má svůj význam z medicínského hlediska, jelikož aktivuje CD8+ T lymfocyty proti intracelulárním patogenům a rakovinným buňkám a také navozuje toleranci na periferii.
Antigen cross‑presentation is a process, when dendritic cells present exogenous antigens in context of MHC I to CD8+ T lymphocytes. Unlike classical antigen presentation, this one goes crosswise, because exogenous antigens are otherwise usually presented on MHC‑II and endogenous antigens on MHC‑I glycoproteins. Molecular mechanism of cross‑presentation has not been well established yet. Two major pathways are considered – vacuolar and cytosolic. In the vacuolar pathway, the internalised antigens are cleaved in the endosome by proteases and then loaded onto MHC I. In the cytosolic pathway, the internalised antigens leave the endosome to be cleaved by the proteasome in the cytosol. They are then imported into the endoplasmic reticulum (ER) to by loaded onto MHC I as in classical antigen presentation, or they go back into the endosome where the MHC‑I loading machinery is trafficked. This process is mediated by ER proteins including those participating in ERAD, by Rab GTPases regulating vesicular transport, and by structures important for endosome maturation. Cross presentation is important in medicine, because it ensures activation of CD8+ T lymphocytes against intracellular pathogens and cancer cells, and induction of tolerance at the periphery.
- Klíčová slova
- zkřížená prezentace, retrotranslokony, vnitrobuněčný transport,
- MeSH
- antigen prezentující buňky MeSH
- antigeny MeSH
- bakteriální infekce imunologie MeSH
- CD8-pozitivní T-lymfocyty MeSH
- cytosol MeSH
- degradace proteinů v endoplasmatickém retikulu MeSH
- dendritické buňky imunologie MeSH
- geny MHC třídy I MeSH
- geny MHC třídy II MeSH
- HLA antigeny MeSH
- hlavní histokompatibilní komplex MeSH
- imunitní systém - jevy MeSH
- nádory imunologie MeSH
- prezentace antigenu * imunologie MeSH
- rab proteiny vázající GTP MeSH
- translokační kanály SEC MeSH
- ubikvitinligasy MeSH
- vakuoly MeSH
- virové nemoci imunologie MeSH
- Publikační typ
- práce podpořená grantem MeSH
Chlorophyll (Chl) is an essential component of the photosynthetic apparatus. Embedded into Chl-binding proteins, Chl molecules play a central role in light harvesting and charge separation within the photosystems. It is critical for the photosynthetic cell to not only ensure the synthesis of a sufficient amount of new Chl-binding proteins but also avoids any misbalance between apoprotein synthesis and the formation of potentially phototoxic Chl molecules. According to the available data, Chl-binding proteins are translated on membrane bound ribosomes and their integration into the membrane is provided by the SecYEG/Alb3 translocon machinery. It appears that the insertion of Chl molecules into growing polypeptide is a prerequisite for the correct folding and finishing of Chl-binding protein synthesis. Although the Chl biosynthetic pathway is fairly well-described on the level of enzymatic steps, a link between Chl biosynthesis and the synthesis of apoproteins remains elusive. In this review, I summarize the current knowledge about this issue putting emphasis on protein-protein interactions. I present a model of the Chl biosynthetic pathway organized into a multi-enzymatic complex and physically attached to the SecYEG/Alb3 translocon. Localization of this hypothetical large biosynthetic centre in the cyanobacterial cell is also discussed as well as regulatory mechanisms coordinating the rate of Chl and apoprotein synthesis.
The stringent response enables bacteria to respond to nutrient limitation and other stress conditions through production of the nucleotide-based second messengers ppGpp and pppGpp, collectively known as (p)ppGpp. Here, we report that (p)ppGpp inhibits the signal recognition particle (SRP)-dependent protein targeting pathway, which is essential for membrane protein biogenesis and protein secretion. More specifically, (p)ppGpp binds to the SRP GTPases Ffh and FtsY, and inhibits the formation of the SRP receptor-targeting complex, which is central for the coordinated binding of the translating ribosome to the SecYEG translocon. Cryo-EM analysis of SRP bound to translating ribosomes suggests that (p)ppGpp may induce a distinct conformational stabilization of the NG domain of Ffh and FtsY in Bacillus subtilis but not in E. coli.
- MeSH
- bakteriální proteiny metabolismus MeSH
- Escherichia coli metabolismus MeSH
- guanosinpentafosfát metabolismus MeSH
- proteiny z Escherichia coli * metabolismus MeSH
- receptory cytoplazmatické a nukleární metabolismus MeSH
- signál-rozpoznávající částice * metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
