Nuclear Fusion in Yeast and Plant Reproduction.

Nuclear fusion is essential for the sexual reproduction of various organisms, including plants, animals, and fungi. During the life cycle of flowering plants, nuclear fusion occurs three times: once during female gametogenesis and twice during double fertilization, when two sperm cells fertilize the egg and the central cell. Haploid nuclei migrate in an actin filament-dependent manner to become in close contact and, then, two nuclei fuse.

Expression of GEX1 Orthologs of and Rescued the Nuclear Fusion Defect of the Arabidopsis GEX1 Mutant.

Nuclear fusion is required for the sexual reproduction of various organisms, including angiosperms. During the life cycle of angiosperms, nuclear fusion occurs three times: once during female gametogenesis, when the two polar nuclei fuse in the central cell, and twice during double fertilization. Nuclear fusion in plant reproduction is achieved by sequential nuclear fusion events: outer and inner nuclear membrane fusion.

GEX1 Is a Nuclear Membrane Protein of Gametes Required for Nuclear Fusion During Reproduction.

During the life cycle of flowering plants, nuclear fusion, or karyogamy, occurs three times: once during female gametogenesis, when the two polar nuclei fuse in the central cell, and twice during double fertilization. In , nuclear fusion events during sexual reproduction proceed without the breakdown of the nuclear envelope, indicating that nuclear membrane fusion is essential for the completion of this process. gamete expressed 1 (GEX1) is a membrane protein that is conserved among plant species.

ERdj3B-Mediated Quality Control Maintains Anther Development at High Temperatures.

Pollen development is highly sensitive to heat stress, which impairs cellular proteostasis by causing misfolded proteins to accumulate. Therefore, each cellular compartment possesses a dedicated protein quality control system. An elaborate quality control system involving molecular chaperones, including immunoglobulin-binding protein (BiP), heat shock protein70, and regulatory J domain-containing cochaperones (J proteins), allows the endoplasmic reticulum (ER) to withstand a large influx of proteins.

Fertilization-Coupled Sperm Nuclear Fusion Is Required for Normal Endosperm Nuclear Proliferation.

Angiosperms exhibit double fertilization, a process in which one of the sperm cells released from the pollen tube fertilizes the egg, while the other sperm cell fertilizes the central cell, giving rise to the embryo and endosperm, respectively. We have previously reported two polar nuclear fusion-defective double knockout mutants of Arabidopsis thaliana immunoglobulin binding protein (BiP), a molecular chaperone of the heat shock protein 70 (Hsp70) localized in the endoplasmic reticulum (ER), (bip1 bip2) and its partner ER-resident J-proteins, ERdj3A and P58IPK (erdj3a p58ipk). These mutants are defective in the fusion of outer nuclear membrane and exhibit characteristic seed developmental defects after fertilization with wild-type pollen, which are accompanied by aberrant endosperm nuclear proliferation.

Development of a Heat-Inducible Gene Expression System Using Female Gametophytes of Arabidopsis thaliana.

Female gametophyte (FG) is crucial for reproduction in flowering plants. Arabidopsis thaliana produces Polygonum-type FGs, which consist of an egg cell, two synergid cells, three antipodal cells and a central cell. Egg cell and central cell are the two female gametes that give rise to the embryo and surrounding endosperm, respectively, after fertilization.

The ribosomal stalk protein is crucial for the action of the conserved ATPase ABCE1.

The ATP-binding cassette (ABC) protein ABCE1 is an essential factor in ribosome recycling during translation. However, the detailed mechanochemistry of its recruitment to the ribosome, ATPase activation and subunit dissociation remain to be elucidated. Here, we show that the ribosomal stalk protein, which is known to participate in the actions of translational GTPase factors, plays an important role in these events.

Quality control of nonstop membrane proteins at the ER membrane and in the cytosol.

Since messenger RNAs without a stop codon (nonstop mRNAs) for organelle-targeted proteins and their translation products (nonstop proteins) generate clogged translocon channels as well as stalled ribosomes, cells have mechanisms to degrade nonstop mRNAs and nonstop proteins and to clear the translocons (e.g. the Sec61 complex) by release of nonstop proteins into the organellar lumen.

BiP3 supports the early stages of female gametogenesis in the absence of BiP1 and BiP2 in Arabidopsis thaliana.

Immunoglobulin binding protein (BiP) is an essential heat shock protein 70 (Hsp70) in the endoplasmic reticulum (ER) that functions in various processes including protein translocation, protein folding and quality control. Arabidopsis thaliana harbors ubiquitously expressed genes BIP1 and BIP2, as well as BIP3, which is induced only by ER stress. Recently, we reported that these BIP genes are expressed in male gametophytes and cooperate with each other to support male gametogenesis and pollen competitiveness.

Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism.

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey).

Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis.

Multiple BiP genes of Arabidopsis thaliana are required for male gametogenesis and pollen competitiveness.

Immunoglobulin-binding protein (BiP) is a molecular chaperone of the heat shock protein 70 (Hsp70) family. BiP is localized in the endoplasmic reticulum (ER) and plays key roles in protein translocation, protein folding and quality control in the ER. The genomes of flowering plants contain multiple BiP genes.

The Plant Organelles Database 3 (PODB3) update 2014: integrating electron micrographs and new options for plant organelle research.

The Plant Organelles Database 2 (PODB2), which was first launched in 2006 as PODB, provides static image and movie data of plant organelles, protocols for plant organelle research and external links to relevant websites. PODB2 has facilitated plant organellar research and the understanding of plant organelle dynamics. To provide comprehensive information on plant organelles in more detail, PODB2 was updated to PODB3 (http://podb.

Analyses of protein-protein interactions by in vivo photocrosslinking in budding yeast.

Recent development of methods for genetic incorporation of unnatural amino acids into proteins in live cells enables us to analyze protein interactions by site-specific photocrosslinking. Here we describe a method to incorporate p-benzoyl-L-phenylalanine (pBpa), a photoreactive unnatural amino acid, into defined positions of a target protein in living yeast cells. Photocrosslinking using the pBpa-incorporated proteins has been proven to be a powerful method for analyzing protein-protein interactions at the spatial resolution of amino-acid residues.

Tam41 is a CDP-diacylglycerol synthase required for cardiolipin biosynthesis in mitochondria.

CDP-diacylglycerol (CDP-DAG) is central to the phospholipid biosynthesis pathways in cells. A prevailing view is that only one CDP-DAG synthase named Cds1 is present in both the endoplasmic reticulum (ER) and mitochondrial inner membrane (IM) and mediates generation of CDP-DAG from phosphatidic acid (PA) and CTP. However, we demonstrate here by using yeast Saccharomyces cerevisiae as a model organism that Cds1 resides in the ER but not in mitochondria, and that Tam41, a highly conserved mitochondrial maintenance protein, directly catalyzes the formation of CDP-DAG from PA in the mitochondrial IM.

Roles of dom34:hbs1 in nonstop protein clearance from translocators for normal organelle protein influx.

Because messenger RNAs without a stop codon (nonstop mRNAs) generate stalled ribosomes, cells have developed a mechanism allowing degradation of nonstop mRNAs and their translation products (nonstop proteins) in the cytosol. Here, we observe the fate of nonstop proteins destined for organelles such as the endoplasmic reticulum (ER) and mitochondria. Nonstop mRNAs for secretory-pathway proteins in yeast generate nonstop proteins that become stuck in the translocator, the Sec61 complex, in the ER membrane.

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation.

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency.

The Plant Organelles Database 2 (PODB2): an updated resource containing movie data of plant organelle dynamics.

The Plant Organelles Database (PODB) was launched in 2006 and provides imaging data of plant organelles, protocols for plant organelle research and external links to relevant websites. To provide comprehensive information on plant organelle dynamics and accommodate movie files that contain time-lapse images and 3D structure rotations, PODB was updated to the next version, PODB2 (http://podb.nibb.

LAP5 and LAP6 encode anther-specific proteins with similarity to chalcone synthase essential for pollen exine development in Arabidopsis.

Pollen grains of land plants have evolved remarkably strong outer walls referred to as exine that protect pollen and interact with female stigma cells. Exine is composed of sporopollenin, and while the composition and synthesis of this biopolymer are not well understood, both fatty acids and phenolics are likely components. Here, we describe mutations in the Arabidopsis (Arabidopsis thaliana) LESS ADHESIVE POLLEN (LAP5) and LAP6 that affect exine development.

BiP-mediated polar nuclei fusion is essential for the regulation of endosperm nuclei proliferation in Arabidopsis thaliana.

Nuclear fusion is an essential process in the sexual reproduction of animals and plants. In flowering plants, nuclear fusion occurs three times: once during female gametogenesis, when the two polar nuclei fuse to produce the diploid central cell nucleus, and twice during double fertilization. The yeast Ig binding protein (BiP) is a molecular chaperone Hsp70 in the endoplasmic reticulum that regulates nuclear membrane fusion during mating.

LAP3, a novel plant protein required for pollen development, is essential for proper exine formation.

We isolated lap3-1 and lap3-2 mutants in a screen for pollen that displays abnormal stigma binding. Unlike wild-type pollen, lap3-1 and lap3-2 pollen exine is thinner, weaker, and is missing some connections between their roof-like tectum structures. We describe the mapping and identification of LAP3 as a novel gene that contains a repetitive motif found in beta-propeller enzymes.

Roles of Tom70 in import of presequence-containing mitochondrial proteins.

Mitochondrial protein traffic requires precise recognition of the mitochondrial targeting signals by the import receptors on the mitochondrial surface including a general import receptor Tom20 and a receptor for presequence-less proteins, Tom70. Here we took a proteome-wide approach of mitochondrial protein import in vitro to find a set of presequence-containing precursor proteins for recognition by Tom70. The presequences of the Tom70-dependent precursor proteins were recognized by Tom20, whereas their mature parts exhibited Tom70-dependent import when attached to the presequence of Tom70-independent precursor proteins.

Seeing is believing: on the use of image databases for visually exploring plant organelle dynamics.

Organelle dynamics vary dramatically depending on cell type, developmental stage and environmental stimuli, so that various parameters, such as size, number and behavior, are required for the description of the dynamics of each organelle. Imaging techniques are superior to other techniques for describing organelle dynamics because these parameters are visually exhibited. Therefore, as the results can be seen immediately, investigators can more easily grasp organelle dynamics.

Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space.

The mitochondrial intermembrane space (IMS) contains many small cysteine-bearing proteins, and their passage across the outer membrane and subsequent folding require recognition and disulfide bond transfer by an oxidative translocator Tim40/Mia40 in the inner membrane facing the IMS. Here we determined the crystal structure of the core domain of yeast Mia40 (Mia40C4) as a fusion protein with maltose-binding protein at a resolution of 3 A. The overall structure of Mia40C4 is a fruit-dish-like shape with a hydrophobic concave region, which accommodates a linker segment of the fusion protein in a helical conformation, likely mimicking a bound substrate.