Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation

The relationship between protein synthesis, folding and disulfide formation within the endoplasmic reticulum (ER) is poorly understood. Previous studies have suggested pre-existing disulfide links are absolutely required to allow protein folding and, conversely, that protein folding occurs prior to disulfide formation. To address the question of what happens first within the ER; that is, protein folding or disulfide formation, we studied folding events at the early stages of polypeptide chain translocation into the mammalian ER using stalled translation intermediates. Our results demonstrate that polypeptide folding can occur without complete domain translocation. Protein disulfide isomerase (PDI) interacts with these early intermediates, but disulfide formation does not occur unless the entire sequence of the protein domain is translocated. This is the first evidence that folding of the polypeptide chain precedes disulfide formation within a cellular context and highlights key differences between protein folding in the ER and refolding of purified proteins

Mechanisms of disulfide bond formation in nascent polypeptides entering the secretory pathway

Disulfide bonds are an abundant feature of proteins across all domains of life that are important for structure, stability, and function. In eukaryotic cells, a major site of disulfide bond formation is the endoplasmic reticulum (ER). How cysteines correctly pair during polypeptide folding to form the native disulfide bond pattern is a complex problem that is not fully understood. In this paper, the evidence for different folding mechanisms involved in ER-localised disulfide bond formation is reviewed with emphasis on events that occur during ER entry. Disulfide formation in nascent polypeptides is discussed with focus on (i) its mechanistic relationship with conformational folding, (ii) evidence for its occurrence at the co-translational stage during ER entry, and (iii) the role of protein disulfide isomerase (PDI) family members. This review highlights the complex array of cellular processes that influence disulfide bond formation and identifies key questions that need to be addressed to further understand this fundamental process

Post-translational membrane insertion of an endogenous YidC substrate

AbstractMembrane protein insertion is controlled by proteinaceous factors embedded in the lipid bilayer. Bacterial inner membrane proteins utilise the Sec translocon as the major facilitator of insertion; however some proteins are Sec independent and instead require only YidC. A common feature of YidC substrates is the exposure of a signal anchor sequence when translation is close to completion; this allows minimal time for targeting and favours a post-translational insertion mechanism. Despite this there is little evidence of YidC's post-translational activity. Here we develop an experimental system that uncouples translation and insertion of the endogenous YidC substrate F0c (subunit c of the F0F1 ATP synthase). In this process we (i) develop a novel one step purification method for YidC, including an on column membrane reconstitution, (ii) isolate a soluble form of F0c and (iii) show that incubation of F0c with YidC proteoliposomes results in a high level of membrane integration. Conformational analyses of inserted F0c through Blue Native PAGE and fluorescence quenching reveal a native, oligomerised structure. These data show that YidC can act as a post-translational insertase, a finding which could explain the absence of a ribosome binding domain on YidC. This correlates with the post-translational activity of other YidC family members lacking the ribosome binding domain

Adaptive multi-scale retinex algorithm for contrast enhancement of real world scenes

Contrast enhancement is a classic image restoration technique that traditionally has been performed using forms of histogram equalization. While effective these techniques often introduce unrealistic tonal rendition in real-world scenes. This paper explores the use of Retinex theory to perform contrast enhancement of real-world scenes. We propose an improvement to the Multi-Scale Retinex algorithm which enhances its ability to perform dynamic range compression while not introducing halo artifacts and greying. The algorithm is well suited to be implemented on the GPU and by doing so real-time processing speeds are achieved

In vitro clonal multiplication of Cardiospermum halicacabum L.

A simple and efficient protocol for in vitro multiplication of mature plants of Cardiospermum halicacabum using nodal and shoot segments has been successfully developed. The stem of C. halicacabum being soft and delicate is very sensitive to physical handling and sterilization. In case of C. halicacabum extra care must be taken while selecting the explant and surface sterilizing it. Three to four shoots were initiated per auxiliary meristems on Murashige and Skoog (MS) medium supplemented with 2.0 mgl/1 BAP and 0.5mgl/1 IAA within two weeks, while less numbers of shoots produced on MS medium augmented with Kinetin (KIN). Repeated transfer of the initial explants for up to five passages on MS medium with 0.5 mgl/1 BAP and KIN + 0.5 mgl/1 IAA yielded maximum numbers of shoots. Healthy and elongated shoots were rooted on 1/2 MS medium + 2.0 mgl/1 Indole-3 butyric acid (IBA). The plantlets thus obtained were successfully hardened in green house and transferred to the field

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Somatic embryogenesis and plant regeneration from cotyledon tissue of Arachis hypogaea L.

Tissue culture techniques are useful for ex situ conservation of rare, endemic orthreatened plant species. This report describes a protocol for somatic embryogenesis ofpeanut (Arachis hypogaea L.) through cotyledon explants. Only 10% of the explants werecontaminated by using this material to start the in vitro culture. The highest frequency(35.4%) of embryogenic calli induction was observed on MS medium supplemented with0.50 mg/l KIN in combination with 0.10 mg/l 2,4,-D. the percentage response for embryogenicproliferation increased in the medium supplemented with GA3(0.20 mg/l) and 1.0 mg/l BA.The regenerated embryos were successfully transferred to the embryo development mediumand transferred to the trays after the development of secondary and tertiary rootdevelopment

Hiding in the Shadows II: Collisional Dust as Exoplanet Markers

Observations of the youngest planets ($\sim$1-10 Myr for a transitional disk) will increase the accuracy of our planet formation models. Unfortunately, observations of such planets are challenging and time-consuming to undertake even in ideal circumstances. Therefore, we propose the determination of a set of markers that can pre-select promising exoplanet-hosting candidate disks. To this end, N-body simulations were conducted to investigate the effect of an embedded Jupiter mass planet on the dynamics of the surrounding planetesimal disk and the resulting creation of second generation collisional dust. We use a new collision model that allows fragmentation and erosion of planetesimals, and dust-sized fragments are simulated in a post process step including non-gravitational forces due to stellar radiation and a gaseous protoplanetary disk. Synthetic images from our numerical simulations show a bright double ring at 850 $\mu$m for a low eccentricity planet, whereas a high eccentricity planet would produce a characteristic inner ring with asymmetries in the disk. In the presence of first generation primordial dust these markers would be difficult to detect far from the orbit of the embedded planet, but would be detectable inside a gap of planetary origin in a transitional disk.Comment: Accepted for publication in Ap

Protein secondary structure determines the temporal relationship between folding and disulfide formation

How and when disulfides bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship, the folded precursor model, in which a nascent structure forms before disulfides do and the quasi-stochastic model where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, β2-microglobulin (β2M), prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize (i) the timing of disulfide formation relative to translocation into the endoplasmic reticulum and (ii) the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to non-native disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during co-translational folding

Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway