scholarly journals Cryo–electron microscopy structures of human oligosaccharyltransferase complexes OST-A and OST-B

Science ◽  
2019 ◽  
Vol 366 (6471) ◽  
pp. 1372-1375 ◽  
Author(s):  
Ana S. Ramírez ◽  
Julia Kowal ◽  
Kaspar P. Locher
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Secretory Proteins ◽  
Catalytic Subunits ◽  
Protein Substrates ◽  
Cryo Electron Microscopy ◽  
Structural Differences ◽  
High Mannose ◽  
Equivalent Region ◽  
High Mannose Glycan

Oligosaccharyltransferase (OST) catalyzes the transfer of a high-mannose glycan onto secretory proteins in the endoplasmic reticulum. Mammals express two distinct OST complexes that act in a cotranslational (OST-A) or posttranslocational (OST-B) manner. Here, we present high-resolution cryo–electron microscopy structures of human OST-A and OST-B. Although they have similar overall architectures, structural differences in the catalytic subunits STT3A and STT3B facilitate contacts to distinct OST subunits, DC2 in OST-A and MAGT1 in OST-B. In OST-A, interactions with TMEM258 and STT3A allow ribophorin-I to form a four-helix bundle that can bind to a translating ribosome, whereas the equivalent region is disordered in OST-B. We observed an acceptor peptide and dolichylphosphate bound to STT3B, but only dolichylphosphate in STT3A, suggesting distinct affinities of the two OST complexes for protein substrates.

scholarly journals Structure of the posttranslational Sec protein-translocation channel complex from yeast

Science ◽  
2018 ◽  
Vol 363 (6422) ◽  
pp. 84-87 ◽  
Author(s):  
Samuel Itskanov ◽  
Eunyong Park
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Binding Sites ◽  
Protein Translocation ◽  
Secretory Proteins ◽  
Conducting Channel ◽  
Cryo Electron Microscopy ◽  
Lateral Gate

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo–electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61’s lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.

scholarly journals Opportunities and challenges for assigning cofactors in cryo-EM density maps of chlorophyll-containing proteins

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Christopher J. Gisriel ◽  
Jimin Wang ◽  
Gary W. Brudvig ◽  
Donald A. Bryant
Keyword(s):  
Electron Microscopy ◽  
Electrostatic Potential ◽  
Protein Function ◽  
Protein Complexes ◽  
Chemical Environment ◽  
Cryo Electron Microscopy ◽  
Functional Differences ◽  
Density Maps ◽  
Structural Differences

AbstractThe accurate assignment of cofactors in cryo-electron microscopy maps is crucial in determining protein function. This is particularly true for chlorophylls (Chls), for which small structural differences lead to important functional differences. Recent cryo-electron microscopy structures of Chl-containing protein complexes exemplify the difficulties in distinguishing Chl b and Chl f from Chl a. We use these structures as examples to discuss general issues arising from local resolution differences, properties of electrostatic potential maps, and the chemical environment which must be considered to make accurate assignments. We offer suggestions for how to improve the reliability of such assignments.

scholarly journals Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on aminopeptidase N and sucrase-isomaltase

1982 ◽  
Vol 204 (3) ◽  
pp. 639-645 ◽  
Author(s):  
E M Danielsen
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Plasma Membrane ◽  
Aminopeptidase N ◽  
Secretory Proteins ◽  
Oligosaccharide Structure ◽  
Membrane Flow ◽  
Polypeptide Synthesis ◽  
High Mannose ◽  
Small Intestinal

The biogenesis of two microvillar enzymes, aminopeptidase N (EC 3.4.11.2) and sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. Both enzymes became inserted into the membrane during or immediately after polypeptide synthesis, indicating that translation takes place on ribosomes attached to the rough endoplasmic reticulum. The earliest detectable forms of aminopeptidase and sucrase-isomaltase were polypeptides of Mr 140 000 and 240 000 respectively. These polypeptides were susceptible to treatment with endo-β-N-acetylglucosaminidiase H (EC 3.2.1.96), suggesting that the microvillar enzymes during or immediately after completion of protein synthesis become glycosylated with a ‘high-mannose’ oligosaccharide structure similarly to other plasma-membrane and secretory proteins. After 20-40 min or 60-90 min of chase, respectively, aminopeptidase N and sucrase-isomaltase were reglycosylated to give the polypeptides of Mr 166 000 (aminopeptidase N) and 265 000 (sucrase-isomaltase). These were expressed at the microvillar membrane after 60-90 min. During the entire process of synthesis and transport to the microvillar membrane the enzymes were bound to membranes, indicating that the biogenesis of aminopeptidase N and sucrase-isomaltase occurs in accordance with the membrane flow hypothesis.

scholarly journals Cryo-EM structures of SERCA2b reveal the mechanism of regulation by the luminal extension tail

2020 ◽  
Vol 6 (33) ◽  
pp. eabb0147 ◽  
Author(s):  
Yuxia Zhang ◽  
Michio Inoue ◽  
Akihisa Tsutsumi ◽  
Satoshi Watanabe ◽  
Tomohiro Nishizawa ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Calcium Homeostasis ◽  
Critical Role ◽  
Kinetic Properties ◽  
Cryo Electron Microscopy ◽  
Domain Arrangement ◽  
Tm Domain ◽  
Conformational Regulation ◽  
Multiple Conformations

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ from the cytosol into the ER and maintains the cellular calcium homeostasis. Herein, we present cryo–electron microscopy (cryo-EM) structures of human SERCA2b in E1∙2Ca2+–adenylyl methylenediphosphonate (AMPPCP) and E2-BeF3− states at 2.9- and 2.8-Å resolutions, respectively. The structures revealed that the luminal extension tail (LE) characteristic of SERCA2b runs parallel to the lipid-water boundary near the luminal ends of transmembrane (TM) helices TM10 and TM7 and approaches the luminal loop flanked by TM7 and TM8. While the LE served to stabilize the cytosolic and TM domain arrangement of SERCA2b, deletion of the LE rendered the overall conformation resemble that of SERCA1a and SERCA2a and allowed multiple conformations. Thus, the LE appears to play a critical role in conformational regulation in SERCA2b, which likely explains the different kinetic properties of SERCA2b from those of other isoforms lacking the LE.

scholarly journals Structural differences between yeast and mammalian microtubules revealed by cryo-EM

2017 ◽  
Vol 216 (9) ◽  
pp. 2669-2677 ◽  
Author(s):  
Stuart C. Howes ◽  
Elisabeth A. Geyer ◽  
Benjamin LaFrance ◽  
Rui Zhang ◽  
Elizabeth H. Kellogg ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Size ◽  
Budding Yeast ◽  
Sequence Conservation ◽  
Gtp Hydrolysis ◽  
Allosteric Coupling ◽  
Cryo Electron Microscopy ◽  
Structural Differences

Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end–tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrations used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. Our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.

scholarly journals Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis–driven translocation

Science ◽  
2018 ◽  
Vol 362 (6418) ◽  
pp. eaav0725 ◽  
Author(s):  
Andres H. de la Peña ◽  
Ellen A. Goodall ◽  
Stephanie N. Gates ◽  
Gabriel C. Lander ◽  
Andreas Martin
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Adenosine Triphosphatase ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Phosphate Release ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Cryo Electron Microscopy ◽  
Aaa Atpases

The 26S proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo–electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.

ATP-Dependent Conformational Changes and Translocation of Substrates in Clpap Protease as Revealed by Cryo-Electron Microscopy

2000 ◽  
Vol 6 (S2) ◽  
pp. 260-261
Author(s):  
Takashi Ishikawa ◽  
Fabienne Beuron ◽  
Martin Kessel ◽  
Sue Wickner ◽  
Michael R. Maurizi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Layered Structure ◽  
Bacteriophage P1 ◽  
E Coli ◽  
Protein Substrates ◽  
Cryo Electron Microscopy ◽  
Clpap Protease ◽  
Image Averaging

ClpAP, an ATP-dependent protease of E. coli, recognizes and unfolds protein substrates via ClpA, its chaperonelike ATPase component, and digests them in ClpP, its protease component . ClpA forms hexameric rings with a two-layered structure, and stacks axially on either face of the double heptameric rings of ClpP. Protein substrates can bind to ClpAP in the presence of ATPγS, which is not hydrolyzed by ClpA, but are not degraded unless ATP is added. This property makes it possible to synchronize degradation in vitro by forming enzymesubstrate complexes in the presence of ATPγS and then adding ATP to trigger subsequent steps. We have used image averaging of electron micrographs of frozen hydrated and negatively stained specimens to characterize interactions of ClpA and ClpAP complexes with the model substrate, bacteriophage P1 protein, RepA.

scholarly journals Substrate and product complexes reveal mechanisms of Hedgehog acylation by HHAT

Science ◽  
2021 ◽  
Vol 372 (6547) ◽  
pp. 1215-1219
Author(s):  
Yiyang Jiang ◽  
Thomas L. Benz ◽  
Stephen B. Long
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Active Site ◽  
Endoplasmic Reticulum Membrane ◽  
Physiochemical Properties ◽  
Amino Terminal ◽  
Cryo Electron Microscopy ◽  
Precursor Proteins ◽  
Hedgehog Proteins ◽  
Hedgehog Acyltransferase

Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). We present cryo–electron microscopy structures of human HHAT in complex with its palmitoyl–coenzyme A substrate and of a product complex with a palmitoylated Hedgehog peptide at resolutions of 2.7 and 3.2 angstroms, respectively. The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. These principles are relevant to related enzymes that catalyze the acylation of Wnt and of the appetite-stimulating hormone ghrelin. The structural and mechanistic insights may advance the development of inhibitors for cancer.

Modification of Pineal Ultrastructure by Exogenous Hormones

1967 ◽  
Vol 25 ◽  
pp. 170-171
Author(s):  
R. A. Turner ◽  
A. E. Rodin ◽  
D. K. Roberts
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Estradiol Benzoate ◽  
Female Rats ◽  
List Type ◽  
Type Number ◽  
Pregnant Rats ◽  
Gonadal Atrophy ◽  
Pineal Ultrastructure

There have been many reports which establish a relationship between the pineal and sexual structures, including gonadal hypertrophy after pinealectomy, and gonadal atrophy after injection of pineal homogenates or of melatonin. In order to further delineate this relationship the pineals from 5 groups of female rats were studied by electron microscopy:ControlsPregnant ratsAfter 4 weekly injections of 0.1 mg. estradiol benzoate.After 8 daily injections of 150 mcgm. melatonin (pineal hormone).After 8 daily injections of 3 mg. serotonin (melatonin precursor).No ultrastructural differences were evident between the control, and the pregnancy and melatonin groups. However, the estradiol injected animals exhibited a marked increase in the amount and size of rough endoplasmic reticulum within the pineal cells.

Copper Localization in Cirrhotic Rat Liver by Scanning Electron Microscopy

1969 ◽  
Vol 27 ◽  
pp. 38-39 ◽  
Author(s):  
J. C. Russ ◽  
E. McNatt
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Copper Acetate ◽  
Lead Citrate ◽  
Dense Bodies ◽  
Transmission Electron ◽  
Electron Mode ◽  
Scanning Electron

In order to study the retention of copper in cirrhotic liver, rats were made cirrhotic by carbon tetrachloride inhalation twice weekly for three months and fed 0.2% copper acetate ad libidum in drinking water for one month. The liver tissue was fixed in osmium, sectioned approximately 2000 Å thick, and stained with lead citrate. The section was examined in a scanning electron microscope (JEOLCO JSM-2) in the transmission electron mode.Figure 1 shows a typical area that includes a red blood cell in a sinusoid, a disse, and a portion of the cytoplasm of a hepatocyte which contains several mitochondria, peribiliary dense bodies, glycogen granules, and endoplasmic reticulum.

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