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Plant Science : An International... Nov 2023In vascular plants, the thylakoid architecture is dominated by the highly structured multiple membrane layers known as grana. The structural diversity of the thylakoid...
In vascular plants, the thylakoid architecture is dominated by the highly structured multiple membrane layers known as grana. The structural diversity of the thylakoid system among plant species is mainly determined by the adaptation to the growth light regime, according to a paradigm stating that shade-tolerant species are featured by a high membrane extension with an enhanced number of thylakoid layers per granum. In this study, the thylakoid system was analysed in Selaginella martensii Spring, a shade-adapted rainforest species belonging to lycophytes, a diminutive plant lineage, sister clade of all other vascular plants (euphyllophytes, including ferns and seed plants). The species is characterized by giant cup-shaped chloroplasts in the upper epidermis and, quantitatively less important, disk-shaped chloroplasts in the mesophyll and lower epidermis. The study aimed at the quantitative assessment of the thylakoid appression exploiting a combination of complementary methods, including electron microscopy, selective thylakoid solubilisation, electron paramagnetic resonance, and simultaneous analysis of fast chlorophyll a fluorescence and P700 redox state. With a chlorophyll a/b ratio of 2.6 and PSI/PSII ratio of 0.31, the plant confirmed two typical hallmarks of shade-adaptation. The morphometric analysis of electron micrographs revealed a 33% fraction of non-appressed thylakoid domains. However, contrasting with the structural paradigm of thylakoid shade-adaptation in angiosperms, S. martensii privileges the increase in the granum diameter in place of the increase in the number of layers building the granum. The very wide grana diameter, 727 nm on average, largely overcame the threshold of 500 nm currently hypothesized to allow an effective diffusion of long-range electron carriers. The fraction of non-appressed membranes based on the selective solubilisation of thylakoids with digitonin was 26%, lower than the morphometric determination, indicating the presence of non-appressed domains inaccessible to the detergent, most probably because of the high three-dimensional complexity of the thylakoid system in S. martensii. Particularly, strong irregularity of grana stacks is determined by assembling thylakoid layers of variable width that tend to slide apart from each other as the number of stacked layers increases.
PubMed: 37595894
DOI: 10.1016/j.plantsci.2023.111833 -
Cell Structure and Function Jan 2020The polytopic plasma membrane protein Rim21 senses both the elevation of ambient pH and alterations in plasma membrane lipid asymmetry in the Rim101 pathway in budding...
The polytopic plasma membrane protein Rim21 senses both the elevation of ambient pH and alterations in plasma membrane lipid asymmetry in the Rim101 pathway in budding yeast. Rim21 is known to undergo N-glycosylation, but the site and function of the glycosylation modification is not known. Using a systematic mutation analysis, we found that Rim21 is N-glycosylated at an unconventional motif located in the N-terminal extracellular region. The Rim21 mutant protein that failed to receive N-glycosylation showed prolonged protein lifetime compared to that of WT Rim21 protein. Although both the WT and mutant Rim21 localized to the plasma membrane, they exhibited different biochemical fractionation profiles. The mutant Rim21, but not WT Rim21, was mainly fractionated into the heavy membrane fraction. Further, compared to WT Rim21, mutant Rim21 was more easily solubilized with digitonin but was conversely more resistant to solubilization with Triton X-100. Despite these different biochemical properties from WT Rim21, mutant Rim21 protein could still activate the Rim101 pathway in response to external alkalization. Collectively, N-glycosylation of Rim21 is not indispensable for its activity as a sensor protein, but modulates the residence of Rim21 protein to some microdomains within the plasma membrane with distinct lipid conditions, thereby affecting its turnover.Key words: plasma membrane, lipid asymmetry, N-linked glycosylation, microdomain, Saccharomyces cerevisiae.
Topics: Cell Membrane; Glycosylation; Membrane Proteins; Receptors, Cell Surface; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31787665
DOI: 10.1247/csf.19021 -
International Journal of Biological... Nov 2020The mosquito Aedes aegypti L. is a vector transmitting diseases such as dengue, chikungunya and Zika virus fever. The water-soluble lectin from Moringa oleifera Lam....
The mosquito Aedes aegypti L. is a vector transmitting diseases such as dengue, chikungunya and Zika virus fever. The water-soluble lectin from Moringa oleifera Lam. seeds (WSMoL) is larvicidal, ovicidal and can stimulate oviposition in A. aegypti. This study aimed to investigate whether WSMoL could bind to membrane proteins from A. aegypti legs. Initially, proteins from the legs were extracted using sodium deoxycholate, digitonin, dodecyl sodium sulfate (SDS) or Triton X-100. The protein concentration was found to be higher in the extract obtained using Triton X-100, which was applied to a WSMoL-Sepharose column. The adsorbed proteins were evaluated using gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE) in presence of SDS. The similarity in the sequences of adsorbed proteins with those available in databases was determined. The proteins adsorbed on the matrix were eluted forming a single peak. Gel filtration chromatography and SDS-PAGE revealed the presence of proteins with molecular masses of approximately 20 kDa and polypeptide bands of 17.0 and 23.7 kDa, respectively. MS/MS analysis indicated similarity between these proteins and ABC carriers, which are expressed in the legs of mosquitos. WSMoL could bind to membrane proteins in the legs of A. aegypti females and induce oviposition through these interactions.
Topics: Aedes; Animals; Insect Proteins; Membrane Proteins; Moringa oleifera; Oviposition; Plant Lectins
PubMed: 32585265
DOI: 10.1016/j.ijbiomac.2020.06.189 -
Journal of Colloid and Interface Science Mar 2020The present paper discusses the use of monolayers of lipid mixtures mimicking the composition of biological membranes of bacteria, erythrocyte and yeast in the context...
The present paper discusses the use of monolayers of lipid mixtures mimicking the composition of biological membranes of bacteria, erythrocyte and yeast in the context of the anti-bacterial, hemolytic and anti-fungal activity of saponins. Saponins are plant-produced glycosidic biosurfactants with either steroidal or triterpenoidal aglycone. In the present study we used digitonin as a representative steroidal saponin (extracted from Digitalis purpurea) and a mixture of triterpenoid saponins from Quillaja saponaria Molina. The effect of saponins was studied first on monolayers consisting of single lipids characteristic for the given type of biological membrane, and then - on model mixed lipid monolayers. Finally, the monolayers were formed from total lipid extracts of natural cell membranes (E. coli and S. cerevisiae) to verify the results obtained in the simplified models. The effect of saponins on monolayers was studied by a combination of surface pressure relaxation, infrared reflection - absorption spectroscopy (IRRAS) and fluorescence microscopy. In line with expectations, sterols (cholesterol and ergosterol) play a major role in the saponin-lipid interactions in monolayers, which may explain especially the hemolytic and antifungal properties of saponins. In contrast, bacterial membranes are devoid of sterols, although the presence of similar compounds may be responsible for their affinity to saponins. Nevertheless, the effect of saponins on bacterial models is less pronounced than for the erythrocyte or fungal ones.
Topics: Anti-Bacterial Agents; Antifungal Agents; Cell Membrane; Erythrocytes; Escherichia coli; Humans; Microbial Sensitivity Tests; Microscopy, Fluorescence; Molecular Structure; Particle Size; Saccharomyces cerevisiae; Saponins; Steroids; Surface Properties; Triterpenes
PubMed: 31874308
DOI: 10.1016/j.jcis.2019.12.014 -
Plant Methods Jun 2024PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin...
PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin transport while the endoplasmic reticulum (ER) localized PIN5 and PIN8 maintain the intracellular auxin homeostasis. Although an antagonistic activity of PIN5 and PIN8 proteins in regulating the intracellular auxin homeostasis and other developmental events have been reported, the membrane topology of these proteins, which might be a basis for their antagonistic function, is poorly understood. In this study we optimized digitonin based PM-permeabilizing protocols coupled with immunocytochemistry labeling to map the membrane topology of PIN5 and PIN8 in Arabidopsis thaliana root cells. Our results indicate that, except for the similarities in the orientation of the N-terminus, PIN5 and PIN8 have an opposite orientation of the central hydrophilic loop and the C-terminus, as well as an unequal number of transmembrane domains (TMDs). PIN8 has ten TMDs with groups of five alpha-helices separated by the central hydrophilic loop (HL) residing in the ER lumen, and its N- and C-terminals are positioned in the cytoplasm. However, the topology of PIN5 comprises nine TMDs. Its N-terminal end and the central HL face the cytoplasm while its C-terminus resides in the ER lumen. Overall, this study shows that PIN5 and PIN8 proteins have a divergent membrane topology while introducing a toolkit of methods for studying membrane topology of integral proteins including those localized at the ER membrane.
PubMed: 38825682
DOI: 10.1186/s13007-024-01182-7 -
Frontiers in Microbiology 2019The twin-arginine translocation (Tat) system transports folded proteins across membranes of prokaryotes, plant plastids, and some mitochondria. According to blue-native...
The twin-arginine translocation (Tat) system transports folded proteins across membranes of prokaryotes, plant plastids, and some mitochondria. According to blue-native polyacrylamide gel electrophoresis after solubilization with digitonin, distinct interactions between the components TatA, TatB, and TatC result in two major TatBC-containing complexes in that can bind protein substrates. We now report the first detection of a TatABC complex that likely represents the state at which transport occurs. This complex was initially found when the photo cross-linking amino acid -benzoyl-l-phenylalanine (Bpa) was introduced at position I50 on the periplasmic side of the first trans-membrane domain of TatC. Cross-linking of TatC resulted in TatC-TatC-cross-links, indicating a close proximity to neighboring TatC in the complex. However, the new complex was not caused by cross-links but rather by non-covalent side chain interactions, as it was also detectable without UV-cross-linking or with an I50Y exchange. The new complex did not contain any detectable substrate. It was slightly upshifted relative to previously reported substrate-containing TatABC complexes. In the absence of TatA, an inactive TatBC complex was formed of the size of wild-type substrate-containing TatABC complexes, suggesting that TatB occupies TatA-binding sites at TatC. When substrate binding was abolished by point mutations, this TatBC complex shifted analogously to active TatABC complexes, indicating that a defect substrate-binding site further enhances TatB association to TatA-binding sites. Only TatA could shift the complex with an intact substrate-binding site, which explains the TatA requirement for substrate transport by TatABC systems.
PubMed: 31354642
DOI: 10.3389/fmicb.2019.01482 -
Biochimica Et Biophysica Acta.... Aug 2019Light drives photosynthesis. In plants it is absorbed by light-harvesting antenna complexes associated with Photosystem I (PSI) and photosystem II (PSII). As PSI and...
Light drives photosynthesis. In plants it is absorbed by light-harvesting antenna complexes associated with Photosystem I (PSI) and photosystem II (PSII). As PSI and PSII work in series, it is important that the excitation pressure on the two photosystems is balanced. When plants are exposed to illumination that overexcites PSII, a special pool of the major light-harvesting complex LHCII is phosphorylated and moves from PSII to PSI (state 2). If instead PSI is over-excited the LHCII complex is dephosphorylated and moves back to PSII (state 1). Recent findings have suggested that LHCII might also transfer energy to PSI in state 1. In this work we used a combination of biochemistry and (time-resolved) fluorescence spectroscopy to investigate the PSI antenna size in state 1 and state 2 for Arabidopsis thaliana. Our data shows that 0.7 ± 0.1 unphosphorylated LHCII trimers per PSI are present in the stroma lamellae of state-1 plants. Upon transition to state 2 the antenna size of PSI in the stroma membrane increases with phosphorylated LHCIIs to a total of 1.2 ± 0.1 LHCII trimers per PSI. Both phosphorylated and unphosphorylated LHCII function as highly efficient PSI antenna.
Topics: Arabidopsis; Digitonin; Energy Transfer; Light; Light-Harvesting Protein Complexes; Phosphorylation; Photosystem I Protein Complex; Photosystem II Protein Complex; Spectrometry, Fluorescence
PubMed: 31299182
DOI: 10.1016/j.bbabio.2019.07.001