Functional studies of CpSRP54 in diatoms show that the mechanism of thylakoid protein insertion differs from plants and green algae

AbstractIn green algae and plants, state transitions serve as a short-term light acclimation process to regulate light harvesting capacity of photosystems I and II (PSI and PSII). During the process, a portion of the light-harvesting complexes II (LHCII) are phosphorylated, dissociate from PSII and bind PSI to form PSI-LHCI-LHCII supercomplex. Here we report high-resolution structures of PSI-LHCI-LHCII supercomplex from Chlamydomonas reinhardtii, revealing the mechanism of assembly between PSI-LHCI complex and two phosphorylated LHCII trimers containing all four types of LhcbM proteins. Two specific LhcbM isoforms, namely LhcbM1 and LhcbM5, directly interact with the PSI core through their phosphorylated amino-terminal regions. Furthermore, biochemical and functional studies on mutant strains lacking either LhcbM1 or LhcbM5 indicate that only LhcbM5 is indispensable in the supercomplex formation. The results unraveled the specific interactions and potential excitation energy transfer routes between green algal PSI and two phosphorylated LHCIIs.

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BamA β16C strand and periplasmic turns are critical for outer membrane protein insertion and assembly

Biochemical Journal ◽

10.1042/bcj20170636 ◽

2017 ◽

Vol 474 (23) ◽

pp. 3951-3961 ◽

Cited By ~ 8

Author(s):

Yinghong Gu ◽

Yi Zeng ◽

Zhongshan Wang ◽

Changjiang Dong

Keyword(s):

Outer Membrane ◽

Outer Membrane Proteins ◽

Membrane Insertion ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Protein Insertion ◽

E Coli ◽

Functional Studies ◽

Functional Assays ◽

Membrane Protein Insertion

Outer membrane (OM) β-barrel proteins play important roles in importing nutrients, exporting wastes and conducting signals in Gram-negative bacteria, mitochondria and chloroplasts. The outer membrane proteins (OMPs) are inserted and assembled into the OM by OMP85 family proteins. In Escherichia coli, the β-barrel assembly machinery (BAM) contains four lipoproteins such as BamB, BamC, BamD and BamE, and one OMP BamA, forming a ‘top hat’-like structure. Structural and functional studies of the E. coli BAM machinery have revealed that the rotation of periplasmic ring may trigger the barrel β1C–β6C scissor-like movement that promote the unfolded OMP insertion without using ATP. Here, we report the BamA C-terminal barrel structure of Salmonella enterica Typhimurium str. LT2 and functional assays, which reveal that the BamA's C-terminal residue Trp, the β16C strand of the barrel and the periplasmic turns are critical for the functionality of BamA. These findings indicate that the unique β16C strand and the periplasmic turns of BamA are important for the outer membrane insertion and assembly. The periplasmic turns might mediate the rotation of the periplasmic ring to the scissor-like movement of BamA β1C–β6C, triggering the OMP insertion. These results are important for understanding the OMP insertion in Gram-negative bacteria, as well as in mitochondria and chloroplasts.

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Freeze-Fracture Replication in the Ultrastructure of Blue-Green Algae

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060386 ◽

1967 ◽

Vol 25 ◽

pp. 74-75

Author(s):

L. V. Leak

Keyword(s):

Cell Wall ◽

Electron Microscope ◽

Green Algae ◽

Three Dimensional ◽

Freeze Fracture ◽

Electron Microscopic ◽

Photosynthetic Membranes ◽

Blue Green Algae ◽

Cell Biol ◽

Cellular Components

Electron microscopic observations of freeze-fracture replicas of Anabaena cells obtained by the procedures described by Bullivant and Ames (J. Cell Biol., 1966) indicate that the frozen cells are fractured in many different planes. This fracturing or cleaving along various planes allows one to gain a three dimensional relation of the cellular components as a result of such a manipulation. When replicas that are obtained by the freeze-fracture method are observed in the electron microscope, cross fractures of the cell wall and membranes that comprise the photosynthetic lamellae are apparent as demonstrated in Figures 1 & 2.A large portion of the Anabaena cell is composed of undulating layers of cytoplasm that are bounded by unit membranes that comprise the photosynthetic membranes. The adjoining layers of cytoplasm are closely apposed to each other to form the photosynthetic lamellae. Occassionally the adjacent layers of cytoplasm are separated by an interspace that may vary in widths of up to several 100 mu to form intralamellar vesicles.

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The low-resolution 3D-structure of porin, a channel-forming protein in E. coliouter membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100075932 ◽

1983 ◽

Vol 41 ◽

pp. 440-441

Author(s):

A. Engel ◽

D.L. Dorset ◽

A. Massalski ◽

J.P. Rosenbusch

Keyword(s):

Crystal Packing ◽

3D Structure ◽

Gram Negative Bacteria ◽

Protein Ratio ◽

Crystal Forms ◽

Large Molecules ◽

Functional Studies ◽

Voltage Dependent ◽

Planar Membranes ◽

Hexagonal Arrays

Porins represent a group of channel forming proteins that facilitate diffusion of small solutes across the outer membrane of Gram-negative bacteria, while excluding large molecules (>650 Da). Planar membranes reconstituted from purified matrix porin (OmpF protein) trimers and phospholipids have allowed quantitative functional studies of the voltage-dependent channels and revealed concerted activation of triplets. Under the same reconstitution conditions but using high protein concentrations porin aggregated to 2D lattices suitable for electron microscopy and image processing. Depending on the lipid-to- protein ratio three different crystal packing arrangements were observed: a large (a = 93 Å) and a small (a = 79 Å) hexagonal and a rectangular (a = 79 Å b = 139 Å) form with p3 symmetry for the hexagonal arrays. In all crystal forms distinct stain filled triplet indentations could be seen and were found to be morphologically identical within a resolution of (22 Å). It is tempting to correlate stain triplets with triple channels, but the proof of this hypothesis requires an analysis of the structure in 3 dimensions.

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Ionic homeostasis and subcellular element compartmentation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010013434x ◽

1990 ◽

Vol 48 (2) ◽

pp. 150-151

Author(s):

Ann LeFurgey ◽

Peter Ingram ◽

J.J. Blum ◽

M.C. Carney ◽

L.A. Hawkey ◽

...

Keyword(s):

Leishmania Major ◽

Ionic Homeostasis ◽

X Ray ◽

Functional Studies ◽

Cell Compartments ◽

X Ray Imaging ◽

Resolution Electron ◽

Multiple Cell ◽

Role Of Zn

Subcellular compartments commonly identified and analyzed by high resolution electron probe x-ray microanalysis (EPXMA) include mitochondria, cytoplasm and endoplasmic or sarcoplasmic reticulum. These organelles and cell regions are of primary importance in regulation of cell ionic homeostasis. Correlative structural-functional studies, based on the static probe method of EPXMA combined with biochemical and electrophysiological techniques, have focused on the role of these organelles, for example, in maintaining cell calcium homeostasis or in control of excitation-contraction coupling. New methods of real time quantitative x-ray imaging permit simultaneous examination of multiple cell compartments, especially those areas for which both membrane transport properties and element content are less well defined, e.g. nuclei including euchromatin and heterochromatin, lysosomes, mucous granules, storage vacuoles, microvilli. Investigations currently in progress have examined the role of Zn-containing polyphosphate vacuoles in the metabolism of Leishmania major, the distribution of Na, K, S and other elements during anoxia in kidney cell nuclel and lysosomes; the content and distribution of S and Ca in mucous granules of cystic fibrosis (CF) nasal epithelia; the uptake of cationic probes by mltochondria in cultured heart ceils; and the junctional sarcoplasmic retlculum (JSR) in frog skeletal muscle.

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A Scanning Electron Microscopic Study of Pro-Vascular Cellular Morphology in Chaetophora Incressata

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100119880 ◽

1985 ◽

Vol 43 ◽

pp. 638-639

Author(s):

A. E. Hotchkiss ◽

A. T. Hotchkiss ◽

R. P. Apkarian

Keyword(s):

Green Algae ◽

Vascular Plants ◽

Vascular System ◽

Higher Plants ◽

Wall Structure ◽

Electron Microscopic ◽

Physiological Processes ◽

Scanning Electron Microscopic Study ◽

Scanning Electron Microscopic ◽

Scanning Electron

Multicellular green algae may be an ancestral form of the vascular plants. These algae exhibit cell wall structure, chlorophyll pigmentation, and physiological processes similar to those of higher plants. The presence of a vascular system which provides water, minerals, and nutrients to remote tissues in higher plants was believed unnecessary for the algae. Among the green algae, the Chaetophorales are complex highly branched forms that might require some means of nutrient transport. The Chaetophorales do possess apical meristematic groups of cells that have growth orientations suggestive of stem and root positions. Branches of Chaetophora incressata were examined by the scanning electron microscope (SEM) for ultrastructural evidence of pro-vascular transport.

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Phytochemical and functional studies on the roots of Armoracia rusticana

10.1055/s-0037-1608402 ◽

2017 ◽

Author(s):

E Jimenez Negro ◽

J Sendker ◽

B Scharf ◽

M Kleinwächter ◽

B Lipowicz ◽

...

Keyword(s):

Armoracia Rusticana ◽

Functional Studies

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Functional Characterization of a Variant Factor XII (F XII Locarno) in a Cross Reacting Material Positive F XII Deficient Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648416 ◽

1992 ◽

Vol 67 (02) ◽

pp. 219-225 ◽

Cited By ~ 6

Author(s):

Walter A Wuillemin ◽

Miha Furlan ◽

Hans Stricker ◽

Bernhard Lämmle

Keyword(s):

Dextran Sulfate ◽

Functional Characterization ◽

Healthy Woman ◽

Chain Molecule ◽

Plasma Kallikrein ◽

Factor Xii ◽

Single Chain ◽

Sulfate Adsorption ◽

Prolonged Incubation ◽

Functional Studies

SummaryThe plasma of a healthy woman was found to contain half normal factor XII (FXII) antigen level (0.46 U/ml) without any FXII clotting activity (<0.01 U/ml). The variant FXII in this plasma, denoted as FXII Locarno, was partially characterized by immunological and functional studies on the proposita’s plasma. FXII Locarno is a single chain molecule with the same size (M r = 80 kDa) as normal FXII. Isoelectric focusing suggested an excess of negative charge in the variant FXII as compared to normal FXII. In contrast to FXII in normal plasma, FXII Locarno was not proteolytically cleaved upon prolonged incubation of proposita’s plasma with dextran sulfate. Adsorption to kaolin was similar for both, abnormal and normal FXII. Incubation of the proposita’s plasma with dextran sulfate and exogenous plasma kallikrein showed normal cleavage of FXII Locarno outside of the tentative disulfide loop Cys340-Cys467, but only partial cleavage within this disulfide loop. Furthermore, plasma kallikrein-cleaved abnormal FXII showed neither amidolytic activity nor proteolytic activity against factor XI and plasma prekallikrein.These results suggest a structural alteration of FXII Locarno, affecting the plasma kallikrein cleavage site Arg353-Val354 and thus formation of activated FXII (a-FXIIa).

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Identification of new ARMC5 missense mutations in Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) and their functional studies in vitro

Endocrine Abstracts ◽

10.1530/endoabs.56.gp36 ◽

2018 ◽

Author(s):

Anna Vaczlavik ◽

Stephanie Espiard ◽

Marie-Odile North ◽

Ludivine Drougat ◽

Marthe Rizk-Rabin ◽

...

Keyword(s):

Missense Mutations ◽

Adrenal Hyperplasia ◽

Functional Studies

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Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability

Reproduction ◽

10.1530/reprod/118.1.145 ◽

2000 ◽

pp. 145-152 ◽

Cited By ~ 38

Author(s):

B Pintado ◽

J de la Fuente ◽

ER Roldan

Keyword(s):

Nucleic Acid ◽

Propidium Iodide ◽

Hoechst 33258 ◽

Experimental Conditions ◽

Functional Studies ◽

Viable Cells ◽

Direct Microscopic Examination ◽

Acrosomal Exocytosis ◽

Viable Sperm ◽

Boar Spermatozoa

This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.

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