Mechanism and Determinants of Amphipathic Helix-Containing Protein Targeting to Lipid Droplets

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Involvement of VAT‐1 in Phosphatidylserine Transfer from the Endoplasmic Reticulum to Mitochondria

Mitochondria receive phosphatidylserine (PS) from the endoplasmic reticulum (ER), but how PS is moved from the ER to mitochondria is unclear. Current models postulate a physical link between the organelles, but no involvement of cytosolic proteins. Here, we have reconstituted PS transport from the ER to mitochondria in vitro using Xenopus egg components. Transport is independent of ER proteins, but is dependent on a cytosolic factor that has a preferential affinity for PS. Crosslinking with a photoactivatable PS analog identified VAT‐1 as a candidate for a cytosolic PS transport protein. Recombinant, purified VAT‐1 stimulated PS transport into mitochondria and depletion of VAT‐1 from Xenopus cytosol with specific antibodies led to a reduction of transport. Our results suggest that cytosolic factors have a role in PS transport from the ER to mitochondria, implicate VAT‐1 in the transport process, and indicate that physical contact between the organelles is not essential.      

Specific Antiserum and Monoclonal Antibodies Against the Taurine Biosynthesis Enzyme Cysteine Sulfinate Decarboxylase: Identity of Brain and Liver Enzyme

Cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme for taurine, was purified 1,800-fold with a 1% yield from rat liver, where it was found to be 20-fold enriched compared with brain. The final fraction was homogeneous, as ascertained through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase HPLC. An antiserum was raised in the rabbit that (a) quantitatively immunoprecipitated CSD activity and (b) immunolabeled only one band (MW = 51,000) on an immunoblot from liver homogenate. Monoclonal antibodies were also raised that recognized the CSD protein and immunolabeled the same 51-kilodalton protein on an immunoblot from liver homogenate. In a brain extract, two CSD activities had been previously found and named CSDI and CSDII, according to their chromatographic elution patterns. We have compared the properties of CSDI from brain--the most likely enzyme involved in the biosynthesis of taurine in the brain, according to previous investigations-and CSD from liver: Both activities (a) were similarly eluted on ion-exchange and hydroxyapatite chromatographies, (b) showed the same elution pattern on gel filtration with an apparent native molecular weight of approximately 63,000, and (c) were immunoprecipitated in a strictly identical manner by the antiserum against liver CSD. Moreover, this antiserum as well as the monoclonal antibodies immunolabeled a single band (51 kilodaltons) on an immunoblot from brain CSD-enriched fraction or liver fraction. All these data show that CSDI from brain and liver CSD are the same monomeric enzyme. They also indicate that a specific antiserum against rat liver CSD has been raised that can be used for immunocytochemical visualization of CSD-containing cells in the brain.     

Phosphatidylinositol 4,5-Bisphosphate Homeostasis Regulated by Nir2 and Nir3 Proteins at Endoplasmic Reticulum-Plasma Membrane Junctions

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP₂) at the plasma membrane (PM) constitutively controls many cellular functions, and its hydrolysis via receptor stimulation governs cell signaling. The PI transfer protein Nir2 is essential for replenishing PM PIP₂ following receptor-induced hydrolysis, but key mechanistic aspects of this process remain elusive. Here, we demonstrate that PI at the membrane of the endoplasmic reticulum (ER) is required for the rapid replenishment of PM PIP₂ mediated by Nir2. Nir2 detects PIP₂ hydrolysis and translocates to ER-PM junctions via binding to phosphatidic acid. With distinct phosphatidic acid binding abilities and PI transfer protein activities, Nir2 and its homolog Nir3 differentially regulate PIP₂ homeostasis in cells during intense receptor stimulation and in the resting state, respectively. Our study reveals that Nir2 and Nir3 work in tandem to achieve different levels of feedback based on the consumption of PM PIP₂ and function at ER-PM junctions to mediate nonvesicular lipid transport between the ER and the PM.     

磷缺失引起小麦叶片中脂含量的变化与脂的合成和磷脂酰甘油的降解有关

对生长在不同磷营养水平条件下小麦(Triticum aestivum var.Zhongyou 9507)叶片中光合膜脂含量变化的原因进行了研究.通过对生长在不同磷营养水平条件下9 d龄和16 d龄小麦叶片中光合膜脂含量的分析,发现在磷缺失培养条件下,小麦光合膜脂的相对含量发生了很大变化,这种变化与小麦叶龄密切相关.在16d龄小麦植株中,第一片叶为老叶,第二片叶为较老叶,而第三片叶为新叶,PG和MGDG在叶片中的相对含量从新叶到老叶逐渐下降,而DGDG和SQDG含量逐渐上升;在磷缺失条件下,16 d龄小麦第一叶片中PG的含量(2.5%)远远低于其在9 d龄第一叶片中的含量(5.5%).以上结果说明,磷缺失引起小麦叶片中脂含量的变化不仅与脂合成有关,而且与PG的降解有关;新生叶片中PG含量减少的主要原因是由于磷供应不足,从而影响了PG的合成;而PG的降解则是老叶中PG含量下降的主要原因.     

磷缺失引起小麦叶片中脂含量的变化与脂的合成和磷脂酰甘油的降解有关(英文)

对生长在不同磷营养水平条件下小麦(Triticum aestivum var.Zhongyou 9507)叶片中光合膜脂含量变化的原因进行了研究。通过对生长在不同磷营养水平条件下9 d龄和16 d龄小麦叶片中光合膜脂含量的分析,发现在磷缺失培养条件下,小麦光合膜脂的相对含量发生了很大变化,这种变化与小麦叶龄密切相关。在16 d龄小麦植株中,第一片叶为老叶,第二片叶为较老叶,而第三片叶为新叶,PG和MGDG在叶片中的相对含量从新叶到老叶逐渐下降,而DGDG和SQDG含量逐渐上升;在磷缺失条件下,16 d龄小麦第一叶片中PG的含量(2.5％)远远低于其在9 d龄第一叶片中的含量(5.5％)。以上结果说明,磷缺失引起小麦叶片中脂含量的变化不仅与脂合成有关,而且与PG的降解有关;新生叶片中PG含量减少的主要原因是由于磷供应不足,从而影响了PG的合成;而PG的降解则是老叶中PG含量下降的主要原因。     

Reconstitution of the Mitochondrial ATP-Dependent Potassium Channel into Bilayer Lipid Membrane1

Electrical properties and regulation of the mitochondrialATP-dependent potassium channel were studied. The channel protein wassolubilized from the mitochondrial membrane using an ethanol/water mixture.Reconstituted into a bilayer lipid membrane BLM), the protein formed aslightly voltage-dependent channel with a conductance of 10 pS in 100 mM KCl.Often, several channels worked simultaneously (clusters) when many channelswere incorporated into the BLM. The elementary channel and the clusters wereboth highly potassium selective. At concentrations of 1 to 10 M, ATPfavors channel opening, while channels become closed at 1–3 mM ATP. GDP(0.5 mM) reactivated the ATP-closed channels without affecting the untreatedchannels. The sulfhydryl-reducing agent ditiothreitol increased the openprobability at concentrations of 1 to 3 mM, but damaged the selectivity ofthe channel.     

Structures of the Mitochondrial CDP-DAG Synthase Tam41 Suggest a Potential Lipid Substrate Pathway from Membrane to the Active Site

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An intimate liaison: spatial organization of the endoplasmic reticulum–mitochondria relationship

Organelle localization is often crucial to properly modulate cellular functions and signalling cascades. For example, the distribution of organelles in axons is crucial for their function and is dysregulated in several diseases. Similarly, relative positioning of two or more organelles is also important to perform certain specialized processes. Perhaps, the best‐known form of interorganellar organization is that between endoplasmic reticulum (ER) and mitochondria. Close communication between these two compartments has been observed for a long time. Recent evidence suggests that this is the basis for a bidirectional communication regulating a number of physiological processes ranging from mitochondrial energy and lipid metabolism to Ca²⁺ signalling and cell death. The recent discovery of some of the molecular mediators of the tethering already allowed to extend the function of this paradigmatic spatial organization to previously unexpected functions, and will foster future research to explore it in cellular signalling cascades as well as in disease.     

Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER

Intracellular antibodies (intrabodies) constitute a potent tool to neutralize the function of target proteins inside specific cell compartments (cytosol, nucleus, mitochondria and ER). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals and complements or replaces knockdown techniques such as antisense-RNA, RNAi and RNA aptamers. This article focuses on intrabodies targeted to the ER. Intracellular anti-bodies expressed and retained inside the ER (ER intrabodies) are shown to be highly efficient in blocking the translocation of secreted and cell surface molecules from the ER to the cell surface.The advantage of ER intrabodies over cytoplasmic intrabodies is that they are correctly folded and easier to select. A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins.The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells.In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing.In the future, endosomal localized receptors involved in bacterial and viral infections, intracellular oncogenic receptors and enzymes involved in glycosylation of tumour antigens might be new targets for ER intrabodies.     

Dual control of formin-nucleated actin assembly by the chromatin and ER in mouse oocytes

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Direct Targeting of Proteins from the Cytosol to Organelles: The ER versus Endosymbiotic Organelles

In eukaryotic cells consisting of many different types of organelles, targeting of organellar proteins is one of the most fundamental cellular processes. Proteins belonging to the endoplasmic reticulum (ER), chloroplasts and mitochondria are targeted individually from the cytosol to their cognate organelles. As the targeting to these organelles occurs in the cytosol during or after translation, the most crucial aspect is how specific targeting to these three organelles can be achieved without interfering with other targeting pathways. For these organelles, multiple mechanisms are used for targeting proteins, but the exact mechanism used depends on the type of protein and organelle, the location of targeting signals in the protein and the location of the protein in the organelle. In this review, we discuss the various mechanisms involved in protein targeting to the ER, chloroplasts and mitochondria, and how the targeting specificity is determined for these organelles in plant cells .     

Transport of Phosphatidylserine from the Endoplasmic Reticulum to the Site of Phosphatidylserine Decarboxylase2 in Yeast

Over the past two decades, most of the genes specifying lipid synthesis and metabolism in yeast have been identified and characterized. Several of these biosynthetic genes and their encoded enzymes have provided valuable tools for the genetic and biochemical dissection of interorganelle lipid transport processes in yeast. One such pathway involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), and its non‐vesicular transport to the site of phosphatidylserine decarboxylase2 (Psd2p) in membranes of the Golgi and endosomal sorting system. In this review, we summarize the identification and characterization of the yeast phosphatidylserine decarboxylases, and examine their role in studies of the transport‐dependent pathways of de novo synthesis of phosphatidylethanolamine (PtdEtn). The emerging picture of the Psd2p‐specific transport pathway is one in which the enzyme and its non‐catalytic N‐terminal domains act as a hub to nucleate the assembly of a multiprotein complex, which facilitates PtdSer transport at membrane contact sites between the ER and Golgi/endosome membranes. After transport to the catalytic site of Psd2p, PtdSer is decarboxylated to form PtdEtn, which is disseminated throughout the cell to support the structural and functional needs of multiple membranes.      

Mechanisms of Lipid Scrambling by the G Protein-Coupled Receptor Opsin

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Mitochondrial apoptosis induced by BH3‐only molecules in the exclusive presence of endoplasmic reticular Bak

Bak and Bax are critical apoptotic mediators that naturally localize to both mitochondria and the endoplasmic reticulum (ER). Although it is generally accepted that mitochondrial expression of Bak or Bax suffices for apoptosis initiated by BH3‐only homologues, it is currently unclear whether their reticular counterparts may have a similar potential. In this study, we show that cells exclusively expressing Bak in endoplasmic membranes undergo cytochrome c mobilization and mitochondrial apoptosis in response to BimEL and Puma, even when these BH3‐only molecules are also targeted to the ER. Surprisingly, calcium was necessary but not sufficient to drive the pathway, despite normal ER calcium levels. We provide evidence that calcium functions coordinately with the ER‐stress surveillance machinery IRE1α/TRAF2 to transmit apoptotic signals from the reticulum to mitochondria. These results indicate that BH3‐only mediators can rely on reticular Bak to activate an ER‐to‐mitochondria signalling route able to induce cytochrome c release and apoptosis independently of the canonical Bak,Bax‐dependent mitochondrial gateway, thus revealing a new layer of complexity in apoptotic regulation.