Efficient mitochondrial targeting relies on co-operation of multiple protein signals in plants

To date, the most prevalent model for transport of pre-proteins to plant mitochondria is based on the activity of an N-terminal extension serving as a targeting peptide. Whether the efficient delivery of proteins to mitochondria is based exclusively on the action of the N-terminal extension or also on that of other protein determinants has yet to be defined. A novel mechanism is reported here for the targeting of a plant protein, named MITS1, to mitochondria. It was found that MITS1 contains an N-terminal extension that is responsible for mitochondrial targeting. Functional dissection of this extension shows the existence of a cryptic signal for protein targeting to the secretory pathway. The first 11 amino acids of the N-terminal extension are necessary to overcome the activity of this signal sequence and target the protein to the mitochondria. These data suggest that co-operation of multiple determinants within the N-terminal extension of mitochondrial proteins may be necessary for efficient mitochondrial targeting. It was also established that the presence of a tryptophan residue toward the C-terminus of the protein is crucial for mitochondrial targeting, as mutation of this residue results in a redistribution of MITS1 to the endoplasmic reticulum and Golgi apparatus. These data suggest a novel targeting model whereby protein traffic to plant mitochondria is influenced by domains in the full-length protein as well as the N-terminal extension

Nuclear Mitochondrial DNA Activates Replication in Saccharomyces cerevisiae

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in

Arabidopsis Qc‑SNARE genes BET11 and BET12 are required for fertility and pollen tube elongation

ORCID IDs: 0000-0003-1729-0561 (P.B.-V.); 0000-0003-3459-1331 (G.-Y.J.)Pollen tubes are rapidly growing specialized structures that elongate in a polar manner. They play a crucial role in the delivery of sperm cells through the stylar tissues of the flower and into the embryo sac, where the sperm cells are released to fuse with the egg cell and the central cell to give rise to the embryo and the endosperm. Polar growth at the pollen tube tip is believed to result from secretion of materials by membrane trafficking mechanisms. In this study, we report the functional characterization of Arabidopsis BET11 and BET12, two genes that may code for Qc-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). Double mutants (bet11/bet12) in a homozygous/heterozygous background showed reduced transmission of the mutant alleles, reduced fertilization of seeds, defective embryo development, reduced pollen tube lengths and formation of secondary pollen tubes. Both BET11 and BET12 are required for fertility and development of pollen tubes in Arabidopsis. More experiments are required to dissect the mechanisms involved.Academia Sinica (Taiwan)National Science and Technology Program for Agricultural Biotechnology (NSTP/AB, 098S0030055-AA), TaiwanNational Science Council (NSF; 99-2321-B-001-036-MY3), TaiwanUniversidad de Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Estación Experimental Agrícola Fabio Baudrit Moreno (EEAFBM

Le métier de chercheur. Atelier. Lycée Dumont D'Urville

CERVOXYNational audienc

De Novo Formation of Plant Endoplasmic Reticulum Export Sites Is Membrane Cargo Induced and Signal Mediated

The plant endoplasmic reticulum (ER) contains functionally distinct subdomains at which cargo molecules are packed into transport carriers. To study these ER export sites (ERES), we used tobacco (Nicotiana tabacum) leaf epidermis as a model system and tested whether increased cargo dosage leads to their de novo formation. We have followed the subcellular distribution of the known ERES marker based on a yellow fluorescent protein (YFP) fusion of the Sec24 COPII coat component (YFP-Sec24), which, differently from the previously described ERES marker, tobacco Sar1-YFP, is visibly recruited at ERES in both the presence and absence of overexpressed membrane cargo. This allowed us to quantify variation in the ERES number and in the recruitment of Sec24 to ERES upon expression of cargo. We show that increased synthesis of membrane cargo leads to an increase in the number of ERES and induces the recruitment of Sec24 to these ER subdomains. Soluble proteins that are passively secreted were found to leave the ER with no apparent up-regulation of either the ERES number or the COPII marker, showing that bulk flow transport has spare capacity in vivo. However, de novo ERES formation, as well as increased recruitment of Sec24 to ERES, was found to be dependent on the presence of the diacidic ER export motif in the cytosolic domain of the membrane cargo. Our data suggest that the plant ER can adapt to a sudden increase in membrane cargo-stimulated secretory activity by signal-mediated recruitment of COPII machinery onto existing ERES, accompanied by de novo generation of new ERES

Windowless in situ Water Condensation on NaCl nanocubes in Environmental TEM. - VIRTUEL

SSCI-VIDE+ATARI:MEME+FCA:EEH:CLH:LMA:TEPInternational audienceObserving liquids in a Transmission Electron Microscope (TEM) has long been impossible owing to basic thermodynamic limitations due to the need for a high vacuum, typically 10-5 mbar or better, within the column of the instrument, making it impossible to maintain a liquid state at room temperature. With the development of dedicated sealed liquid cells mounted on specific specimen holders, Liquid Cell TEM (LCTEM) has become possible about a decade ago, opening a huge range of possible applications in the field of biology, crystal growth or electrochemistry [1]. In parallel, Environmental TEM (ETEM) was also developed [2]; here a partial pressure can be maintained in the pole-pieces gap where the tip of the sample holder, including the sample itself, is inserted, allowing to perform observations under gas without any sealing membranes as needed with the close-cell technology. Such an ‘open-cell’ approach was also developed in Scanning EM (ESEM, e.g. [3]). With these dedicated ETEM or ESEM configurations, observing liquid such as water layers is possible under a partial pressure of a few mbar if the temperature is cooled down close to the dew point in order to insure a thermodynamic equilibrium between the solid, gas and liquid states: for water, the liquid state can effectively been stabilized in a temperature and pressure range of typically 0 to 11°C and 6 to 15 mbar respectively [4], which are conditions easily accessible in ETEM and ESEM. While LCTEM in a close-cell permits to reach atmospheric pressure, thus allowing to observe water at room temperature, it has the drawback of its advantage: the presence of top and bottom sealing membranes makes it very difficult to perform water condensation from a humid atmosphere and to control water vapor states. Such experiments are possible in ‘open-cell’ ESEM [5] and ETEM [6] and enhance our understanding of the hygroscopic behavior of atmospheric aerosol particles that are known to act as cloud condensation nuclei [7]. Hygroscopic growth, deliquescence and efflorescence of model and real) atmospheric nanoparticles can be directly visualized by these techniques. The present contribution aims at establishing conditions under which aerosols can be adequately observed in a Titan ETEM (FEI/TFS). We use a Gatan liquid-nitrogen (LN2) cryo-holder to cool down the specimen around 0°C. We adjust the temperature by mixing LN2 with a controlled quantity of ethanol. For the purpose of this preliminary investigation, we use NaCl nanoparticles, obtained by vaporizing a salt solution onto a classical holey carbon TEM grid, as a model aerosols. Observations were performed at 300 kV under a humid atmosphere generated by pumping a small sealed water reservoir connected to one of the input lines of the ETEM, the pumping being insured by the molecular turbopumps of its vacuum system. The presence of water (vapor) was controlled by the residual gas analyzer equipping the microscope and by Electron Energy-Loss Spectroscopy (EELS). In a first step, and considering the high voltage at which experiments were performed, a control of the electron flux and dose was realized using different illumination settings in order to define safe imaging conditions avoiding noticeable irradiation damage of the nanocrystals (Fig. 1). Then, both water condensation and evaporation have been performed to follow the evolution of NaCl cubes (Fig. 2). Results will be discussed in terms of relationships between percentage of relative humidity and water uptake of the NaCl particles as a function of T and P [8]. References:[1] FM Ross (Ed.), Liquid Cell Electron Microscopy (Advances in Microscopy and Microanalysis), Cambridge University Press, Cambridge (2017), 524 p.[2] TW Hansen, J.B. Wagner (Ed.), Controlled Atmosphere TEM, Springer, New York, (2016), 332 p.[3] A Bogner et al. Micron, 38 (2007) 390. [4] DJ Stokes. Principles and Practice of Variable Pressure/Environmental Scanning Electron Microscopy (VP-ESEM), (2008), John Wiley & Sons Ltd., 221 p.[5] RC Hoffman et al. J. Aerosol Science, 35 7 (2004) 869. [6] ME Wise et al. Aerosol Science & Technology, 39:9 (2005) 849; C Cassidy et al. Plos One, 12 11 (2017) e0186899; BDA Levin et al. Microscopy and Microanal. (2020) 1.[7] U Lohmann et al. An Introduction to Clouds: From the Microscale to Climate, Cambridge University Press, Cambridge, UK (2016), 391 p.[8] The authors acknowledge the Consortium Lyon – St-Etienne de Microscopie (CLYM, www.clym.fr), the Centre Technologique des Microstructures (http://microscopies.univ-lyon1.fr/) for practical assistance and the French National Research Agency (ANR, www.anr.fr) for supporting project ANR-20-CE42-0008

Windowless in situ Water Condensation on NaCl nanocubes in Environmental TEM. - VIRTUEL

SSCI-VIDE+ATARI:MEME+FCA:EEH:CLH:LMA:TEPInternational audienceObserving liquids in a Transmission Electron Microscope (TEM) has long been impossible owing to basic thermodynamic limitations due to the need for a high vacuum, typically 10-5 mbar or better, within the column of the instrument, making it impossible to maintain a liquid state at room temperature. With the development of dedicated sealed liquid cells mounted on specific specimen holders, Liquid Cell TEM (LCTEM) has become possible about a decade ago, opening a huge range of possible applications in the field of biology, crystal growth or electrochemistry [1]. In parallel, Environmental TEM (ETEM) was also developed [2]; here a partial pressure can be maintained in the pole-pieces gap where the tip of the sample holder, including the sample itself, is inserted, allowing to perform observations under gas without any sealing membranes as needed with the close-cell technology. Such an ‘open-cell’ approach was also developed in Scanning EM (ESEM, e.g. [3]). With these dedicated ETEM or ESEM configurations, observing liquid such as water layers is possible under a partial pressure of a few mbar if the temperature is cooled down close to the dew point in order to insure a thermodynamic equilibrium between the solid, gas and liquid states: for water, the liquid state can effectively been stabilized in a temperature and pressure range of typically 0 to 11°C and 6 to 15 mbar respectively [4], which are conditions easily accessible in ETEM and ESEM. While LCTEM in a close-cell permits to reach atmospheric pressure, thus allowing to observe water at room temperature, it has the drawback of its advantage: the presence of top and bottom sealing membranes makes it very difficult to perform water condensation from a humid atmosphere and to control water vapor states. Such experiments are possible in ‘open-cell’ ESEM [5] and ETEM [6] and enhance our understanding of the hygroscopic behavior of atmospheric aerosol particles that are known to act as cloud condensation nuclei [7]. Hygroscopic growth, deliquescence and efflorescence of model and real) atmospheric nanoparticles can be directly visualized by these techniques. The present contribution aims at establishing conditions under which aerosols can be adequately observed in a Titan ETEM (FEI/TFS). We use a Gatan liquid-nitrogen (LN2) cryo-holder to cool down the specimen around 0°C. We adjust the temperature by mixing LN2 with a controlled quantity of ethanol. For the purpose of this preliminary investigation, we use NaCl nanoparticles, obtained by vaporizing a salt solution onto a classical holey carbon TEM grid, as a model aerosols. Observations were performed at 300 kV under a humid atmosphere generated by pumping a small sealed water reservoir connected to one of the input lines of the ETEM, the pumping being insured by the molecular turbopumps of its vacuum system. The presence of water (vapor) was controlled by the residual gas analyzer equipping the microscope and by Electron Energy-Loss Spectroscopy (EELS). In a first step, and considering the high voltage at which experiments were performed, a control of the electron flux and dose was realized using different illumination settings in order to define safe imaging conditions avoiding noticeable irradiation damage of the nanocrystals (Fig. 1). Then, both water condensation and evaporation have been performed to follow the evolution of NaCl cubes (Fig. 2). Results will be discussed in terms of relationships between percentage of relative humidity and water uptake of the NaCl particles as a function of T and P [8]. References:[1] FM Ross (Ed.), Liquid Cell Electron Microscopy (Advances in Microscopy and Microanalysis), Cambridge University Press, Cambridge (2017), 524 p.[2] TW Hansen, J.B. Wagner (Ed.), Controlled Atmosphere TEM, Springer, New York, (2016), 332 p.[3] A Bogner et al. Micron, 38 (2007) 390. [4] DJ Stokes. Principles and Practice of Variable Pressure/Environmental Scanning Electron Microscopy (VP-ESEM), (2008), John Wiley & Sons Ltd., 221 p.[5] RC Hoffman et al. J. Aerosol Science, 35 7 (2004) 869. [6] ME Wise et al. Aerosol Science & Technology, 39:9 (2005) 849; C Cassidy et al. Plos One, 12 11 (2017) e0186899; BDA Levin et al. Microscopy and Microanal. (2020) 1.[7] U Lohmann et al. An Introduction to Clouds: From the Microscale to Climate, Cambridge University Press, Cambridge, UK (2016), 391 p.[8] The authors acknowledge the Consortium Lyon – St-Etienne de Microscopie (CLYM, www.clym.fr), the Centre Technologique des Microstructures (http://microscopies.univ-lyon1.fr/) for practical assistance and the French National Research Agency (ANR, www.anr.fr) for supporting project ANR-20-CE42-0008

Diacidic Motifs Influence the Export of Transmembrane Proteins from the Endoplasmic Reticulum in Plant Cells

In yeast and mammals, amino acid motifs in the cytosolic tails of transmembrane domains play a role in protein trafficking by facilitating export from the endoplasmic reticulum (ER). However, little is known about ER export signals of membrane proteins in plants. Therefore, we investigated the role of diacidic motifs in the ER export of Golgi-localized membrane proteins. We show that diacidic motifs perform a significant function in the export of transmembrane proteins to the Golgi apparatus, as mutations of these signals impede the efficient anterograde transport of multispanning, type II, and type I proteins. Furthermore, we demonstrate that diacidic motifs instigate the export of proteins that reside in the ER due to the lengths of their transmembrane domains. However, not all of the diacidic motifs in the cytosolic tails of the proteins studied were equally important in ER export. Transport of Golgi proteins was disrupted only by mutagenesis of specific diacidic signals, suggesting that the protein environment of these signals affects their function. Our findings indicate that diacidic ER export motifs are present and functional in plant membrane proteins and that they are dominant over transmembrane domain length in determining the export of proteins from the ER in plant cells