scholarly journals Observing the nonvectorial yet cotranslational folding of a multidomain protein, LDL receptor, in the ER of mammalian cells

2020 ◽  
Vol 117 (28) ◽  
pp. 16401-16408 ◽  
Author(s):  
Hiroshi Kadokura ◽  
Yui Dazai ◽  
Yo Fukuda ◽  
Naoya Hirai ◽  
Orie Nakamura ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Mammalian Cells ◽  
Protein Translocation ◽  
Large Fraction ◽  
Ldl Receptor ◽  
Current View ◽  
Multidomain Proteins ◽  
Multidomain Protein ◽  
Cotranslational Folding ◽  
2D Gel

Proteins have evolved by incorporating several structural units within a single polypeptide. As a result, multidomain proteins constitute a large fraction of all proteomes. Their domains often fold to their native structures individually and vectorially as each domain emerges from the ribosome or the protein translocation channel, leading to the decreased risk of interdomain misfolding. However, some multidomain proteins fold in the endoplasmic reticulum (ER) nonvectorially via intermediates with nonnative disulfide bonds, which were believed to be shuffled to native ones slowly after synthesis. Yet, the mechanism by which they fold nonvectorially remains unclear. Using two-dimensional (2D) gel electrophoresis and a conformation-specific antibody that recognizes a correctly folded domain, we show here that shuffling of nonnative disulfide bonds to native ones in the most N-terminal region of LDL receptor (LDLR) started at a specific timing during synthesis. Deletion analysis identified a region on LDLR that assisted with disulfide shuffling in the upstream domain, thereby promoting its cotranslational folding. Thus, a plasma membrane-bound multidomain protein has evolved a sequence that promotes the nonvectorial folding of its upstream domains. These findings demonstrate that nonvectorial folding of a multidomain protein in the ER of mammalian cells is more coordinated and elaborated than previously thought. Thus, our findings alter our current view of how a multidomain protein folds nonvectorially in the ER of living cells.

Pyrimidine dimers block simian virus 40 replication forks

1986 ◽  
Vol 6 (10) ◽  
pp. 3443-3450
Author(s):  
C A Berger ◽  
H J Edenberg
Keyword(s):  
Mammalian Cells ◽  
Replication Fork ◽  
Uv Light ◽  
Large Fraction ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Replication Forks ◽  
Pyrimidine Dimers ◽  
Leading Strand ◽  
Uv Lesions

UV light produces lesions, predominantly pyrimidine dimers, which inhibit DNA replication in mammalian cells. The mechanism of inhibition is controversial: is synthesis of a daughter strand halted at a lesion while the replication fork moves on and reinitiates downstream, or is fork progression itself blocked for some time at the site of a lesion? We directly addressed this question by using electron microscopy to examine the distances of replication forks from the origin in unirradiated and UV-irradiated simian virus 40 chromosomes. If UV lesions block replication fork progression, the forks should be asymmetrically located in a large fraction of the irradiated molecules; if replication forks move rapidly past lesions, the forks should be symmetrically located. A large fraction of the simian virus 40 replication forks in irradiated molecules were asymmetrically located, demonstrating that UV lesions present at the frequency of pyrimidine dimers block replication forks. As a mechanism for this fork blockage, we propose that polymerization of the leading strand makes a significant contribution to the energetics of fork movement, so any lesion in the template for the leading strand which blocks polymerization should also block fork movement.

scholarly journals Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Valentina Peleh ◽  
Emmanuelle Cordat ◽  
Johannes M Herrmann
Keyword(s):  
Disulfide Bonds ◽  
Protein Import ◽  
Protein Translocation ◽  
Hydrophobic Interactions ◽  
Substrate Binding ◽  
Intermembrane Space ◽  
Cysteine Motif ◽  
Mitochondrial Intermembrane Space ◽  
Necessary And Sufficient ◽  
Yeast Mutants

Many proteins of the mitochondrial IMS contain conserved cysteines that are oxidized to disulfide bonds during their import. The conserved IMS protein Mia40 is essential for the oxidation and import of these proteins. Mia40 consists of two functional elements: an N-terminal cysteine-proline-cysteine motif conferring substrate oxidation, and a C-terminal hydrophobic pocket for substrate binding. In this study, we generated yeast mutants to dissect both Mia40 activities genetically and biochemically. Thereby we show that the substrate-binding domain of Mia40 is both necessary and sufficient to promote protein import, indicating that trapping by Mia40 drives protein translocation. An oxidase-deficient Mia40 mutant is inviable, but can be partially rescued by the addition of the chemical oxidant diamide. Our results indicate that Mia40 predominantly serves as a trans-site receptor of mitochondria that binds incoming proteins via hydrophobic interactions thereby mediating protein translocation across the outer membrane by a ‘holding trap’ rather than a ‘folding trap’ mechanism.

scholarly journals Disruption of the three cytoskeletal networks in mammalian cells does not affect transcription, translation, or protein translocation changes induced by heat shock.

1985 ◽  
Vol 5 (7) ◽  
pp. 1571-1581 ◽  
Author(s):  
W J Welch ◽  
J R Feramisco
Keyword(s):  
Heat Shock ◽  
Heat Shock Response ◽  
Mammalian Cells ◽  
Stress Proteins ◽  
Protein Translocation ◽  
Shock Response ◽  
Cytochalasin E ◽  
Cytoskeletal Networks ◽  
Shock Proteins ◽  
Cytoskeletal Elements

Mammalian cells show a complex series of transcriptional and translational switching events in response to heat shock treatment which ultimately lead to the production and accumulation of a small number of proteins, the so-called heat shock (or stress) proteins. We investigated the heat shock response in both qualitative and quantitative ways in cells that were pretreated with drugs that specifically disrupt one or more of the three major cytoskeletal networks. (These drugs alone, cytochalasin E and colcemid, do not result in induction of the heat shock response.) Our results indicated that disruption of the actin microfilaments, the vimentin-containing intermediate filaments, or the microtubules in living cells does not hinder the ability of the cell to undergo an apparently normal heat shock response. Even when all three networks were simultaneously disrupted (resulting in a loose, baglike appearance of the cells), the cells still underwent a complete heat shock response as assayed by the appearance of the heat shock proteins. In addition, the major induced 72-kilodalton heat shock protein was efficiently translocated from the cytoplasm into its proper location in the nucleus and nucleolus irrespective of the condition of the three cytoskeletal elements.

scholarly journals The refolding activity of the yeast heat shock proteins Ssa1 and Ssa2 defines their role in protein translocation.

1996 ◽  
Vol 135 (5) ◽  
pp. 1229-1237 ◽  
Author(s):  
G L Bush ◽  
D I Meyer
Keyword(s):  
Protein Translocation ◽  
In Vitro Assay ◽  
Secretory Proteins ◽  
Vitro Assay ◽  
Chaperone Function ◽  
Cotranslational Folding ◽  
Yeast Lysate ◽  
Shock Proteins ◽  
First Time

Ssa1/2p, members of one of the yeast cytosolic hsp70 subfamilies, have been implicated in the translocation of secretory proteins into the lumen of the ER. The involvement of these hsp70s in translocation was tested directly by examining the effect of immunodepleting Ssa1/2p from yeast cytosol and subsequently testing the cytosol for its ability to support co- and post-translational translocation of prepro-alpha-factor. Depletion of Ssa1/2p had no effect on the efficiency of translocation in this in vitro assay. The system was used to examine the effect of the absence of Ssa1/2p on two other putative hsp70 functions: cotranslational folding of nascent luciferase and refolding of denatured luciferase. Depletion of Ssa1/2p had no effect on the ability of the yeast lysate to synthesize enzymatically active luciferase, but had a dramatic effect on the ability of the lysate to refold chemically denatured luciferase. These results demonstrate, for the first time, the refolding activity of Ssa1/2p in the context of the yeast cytosol, and define refolding activity as a chaperone function specific to Ssa1/2p, aprt from other cytosolic hsp70s. They also suggest that Ssa1/2p do not play a significant role in chaperoning the folding of nascent polypeptides. The implications of these findings for Ssa1/2p activity on their proposed role in the process of translocation are discussed.

scholarly journals Minireview: NAD+, a Circadian Metabolite with an Epigenetic Twist

Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Paolo Sassone-Corsi
Keyword(s):  
Circadian Clock ◽  
Nicotinamide Adenine Dinucleotide ◽  
Mammalian Cells ◽  
Feedback Loop ◽  
Large Fraction ◽  
Sirtuin 1 ◽  
Nicotinamide Adenine ◽  
Dynamic Changes ◽  
Metabolic Functions ◽  
Transcriptional Feedback

Abstract A wide variety of endocrine, physiological, and metabolic functions follow daily oscillations. Most of these regulations are controlled at the level of gene expression by the circadian clock and, a remarkably coordinated transcription-translation machinery that exerts its function in virtually all mammalian cells. A large fraction of the genome is under control of the circadian clock, a regulation that is achieved through dynamic changes in chromatin states. Recent findings have demonstrated intimate connections between the circadian clock and epigenetic control. The case of nicotinamide adenine dinucleotide, which modulates the circadian activity of the deacetylase sirtuin 1, constitutes a paradigmatic example of the link between cyclic cellular metabolism and chromatin remodeling. Indeed, the clock transcriptional feedback loop is interlocked with the enzymatic loop of the nicotinamide adenine dinucleotide salvage pathway.

Dynamics of autophagosome formation: a pulse and a sequence of waves

2014 ◽  
Vol 42 (5) ◽  
pp. 1389-1395 ◽  
Author(s):  
Nicholas T. Ktistakis ◽  
Eleftherios Karanasios ◽  
Maria Manifava
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Stress Conditions ◽  
Present Review ◽  
Eukaryotic Cells ◽  
Current View ◽  
Protein Assemblies ◽  
Autophagosome Formation ◽  
Flat Membrane ◽  
A Current

Autophagosomes form in eukaryotic cells in response to starvation or to other stress conditions brought about by the unwanted presence in the cytosol of pathogens, damaged organelles or aggregated protein assemblies. The uniqueness of autophagosomes is that they form de novo and that they are the only double-membraned vesicles known in cells, having arisen from flat membrane sheets which have expanded and self-closed. The various steps describing their formation as well as most of the protein and lipid components involved have been identified. Furthermore, the hierarchical relationships among the components are well documented, and the mechanistic rationale for some of these hierarchies has been revealed. In the present review, we try to provide a current view of the process of autophagosome formation in mammalian cells, emphasizing along the way gaps in our knowledge that need additional work.

scholarly journals Maturation of the Human Papillomavirus 16 Capsid

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Giovanni Cardone ◽  
Adam L. Moyer ◽  
Naiqian Cheng ◽  
Cynthia D. Thompson ◽  
Israel Dvoretzky ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Disulfide Bonds ◽  
Mammalian Cells ◽  
Structural Integrity ◽  
Regular Structure ◽  
Human Papillomaviruses ◽  
Cryo Electron Microscopy ◽  
Cross Links ◽  
Risk Of Cancer

ABSTRACTPapillomaviruses are a family of nonenveloped DNA viruses that infect the skin or mucosa of their vertebrate hosts. The viral life cycle is closely tied to the differentiation of infected keratinocytes. Papillomavirus virions are released into the environment through a process known as desquamation, in which keratinocytes lose structural integrity prior to being shed from the surface of the skin. During this process, virions are exposed to an increasingly oxidative environment, leading to their stabilization through the formation of disulfide cross-links between neighboring molecules of the major capsid protein, L1. We used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV16 capsids assembled in mammalian cells and exposed to an oxidizing environment after cell lysis. Initially, the virion is a loosely connected procapsid that, underin vitroconditions, condenses over several hours into the more familiar 60-nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by ~5% in diameter, its pentameric capsomers change in structure (most markedly in the axial region), and the interaction surfaces between adjacent capsomers are consolidated. A C175S mutant that cannot achieve normal inter-L1 disulfide cross-links shows maturation-related shrinkage but does not achieve the fully condensed 60-nm form. Pseudoatomic modeling based on a 9-Å resolution reconstruction of fully mature capsids revealed C-terminal disulfide-stabilized “suspended bridges” that form intercapsomeric cross-links. The data suggest a model in which procapsids exist in a range of dynamic intermediates that can be locked into increasingly mature configurations by disulfide cross-linking, possibly through a Brownian ratchet mechanism.IMPORTANCEHuman papillomaviruses (HPVs) cause nearly all cases of cervical cancer, a major fraction of cancers of the penis, vagina/vulva, anus, and tonsils, and genital and nongenital warts. HPV types associated with a high risk of cancer, such as HPV16, are generally transmitted via sexual contact. The nonenveloped virion of HPVs shows a high degree of stability, allowing the virus to persist in an infectious form in environmental fomites. In this study, we used cryo-electron microscopy to elucidate the structure of the HPV16 capsid at different stages of maturation. The fully mature capsid adopts a rigid, highly regular structure stabilized by intermolecular disulfide bonds. The availability of a pseudoatomic model of the fully mature HPV16 virion should help guide understanding of antibody responses elicited by HPV capsid-based vaccines.

scholarly journals Versatile multi-transgene expression using improved BAC TG-EMBED toolkit, novel BAC episomes, and BAC-MAGIC

2019 ◽  
Author(s):  
Binhui Zhao ◽  
Pankaj Chaturvedi ◽  
David L. Zimmerman ◽  
Andrew S. Belmont
Keyword(s):  
Mammalian Cells ◽  
Transgene Expression ◽  
Large Fraction ◽  
Fine Tuning ◽  
Cmv Promoter ◽  
Chromosome Position ◽  
High Level ◽  
Genomic Regions ◽  
Expression In Mammalian Cells

ABSTRACTAchieving reproducible, stable, and high-level transgene expression in mammalian cells remains problematic. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC containing the mouseMsh3-Dhfrlocus (DHFR BAC). Here we extend this “BAC TG-EMBED” approach. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01-5 fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes expressed on a single BAC. Second, we show small variability in both the expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making choice of BACs more flexible. Third, we describe an intriguing phenomenon in which BAC transgenes are maintained as episomes in a large fraction of stably selected clones. Finally, we demonstrate the utility of BAC TG-EMBED by simultaneously labeling three nuclear compartments in 94% of stable clones using a multi-reporter DHFR BAC, constructed with a combination of synthetic biology and BAC recombineering tools. Our extended BAC TG-EMBED method provides a versatile platform for achieving reproducible, stable simultaneous expression of multiple transgenes maintained either as episomes or stably integrated copies.

scholarly journals Mandipropamid as a chemical inducer of proximity for in vivo applications

2021 ◽  
Author(s):  
Michael J. Ziegler ◽  
Klaus Yserentant ◽  
Valentin Dunsing ◽  
Volker Middel ◽  
Antoni J. Gralak ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Plant Hormone ◽  
Protein Translocation ◽  
Cell Permeability ◽  
Chemical Inducers ◽  
Chemically Induced ◽  
Endogenous Proteins ◽  
Living Organisms

AbstractDirect control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins.

Current views on the source of the autophagosome membrane

2013 ◽  
Vol 55 ◽  
pp. 29-38 ◽  
Author(s):  
Sharon A. Tooze
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Nucleation Site ◽  
Morphological Characterization ◽  
Significant Advance ◽  
Current View ◽  
Yeast Saccharomyces Cerevisiae ◽  
Molecular Control ◽  
Atg Proteins ◽  
Electron Microscopes

Autophagy was discovered in the late 1950s when scientists using the first electron microscopes saw membrane-bound structures in cells that contained cytoplasmic organelles, including mitochondria. Pursuant to further morphological characterization it was recognized that these vesicles, now called autophagosomes, are found in all eukaryotic cells and undergo changes in morphology from a double-membraned vesicle with recognizable content, i.e. sequestered organelles, to a uniformly dense core autolysosome. Genetic screens in the yeast Saccharomyces cerevisiae in the 1990s provided a molecule framework for the next era of discovery during which the interest in, and research into, autophagy has rapidly expanded into many areas of human biology and disease. A relatively small cohort of approximately 36 proteins, called Atgs (autophagy-related proteins), orchestrate the formation of the autophagosome, and these are now being studied and functionally characterized. Although the function of these proteins is being elucidated, the underlying molecular mechanisms of how autophagosomes form are still not completely understood. Recent advances have, however, provided a significant advance in both our understanding of the molecular control of the Atg proteins and the source of the membranes. A consensus view is emerging from these advances that the endoplasmic reticulum is the nucleation site for the autophagosome, and that contributions from other compartments (Golgi, endosomes and plasma membrane) are required. In the present chapter, I review the data from the pre-molecular decades, and discuss the most recent publications to give an overview of the current view of where, and how, autophagosomes form in mammalian cells.

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