Degradation of the Endoplasmic Reticulum by Autophagy during Endoplasmic Reticulum Stress in Arabidopsis

In this article, we show that the endoplasmic reticulum (ER) in Arabidopsis thaliana undergoes morphological changes in structure during ER stress that can be attributed to autophagy. ER stress agents trigger autophagy as demonstrated by increased production of autophagosomes. In response to ER stress, a soluble ER marker localizes to autophagosomes and accumulates in the vacuole upon inhibition of vacuolar proteases. Membrane lamellae decorated with ribosomes were observed inside autophagic bodies, demonstrating that portions of the ER are delivered to the vacuole by autophagy during ER stress. In addition, an ER stress sensor, INOSITOL-REQUIRING ENZYME-1b (IRE1b), was found to be required for ER stress–induced autophagy. However, the IRE1b splicing target, bZIP60, did not seem to be involved, suggesting the existence of an undiscovered signaling pathway to regulate ER stress–induced autophagy in plants. Together, these results suggest that autophagy serves as a pathway for the turnover of ER membrane and its contents in response to ER stress in plants.     

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.      

Mitofusin-2 Independent Juxtaposition of Endoplasmic Reticulum and Mitochondria: An Ultrastructural Study

Besides its role in controlling the morphology of mitochondria, mitofusin-2 has been proposed to tether mitochondria to the endoplasmic reticulum (ER), based largely on light microscopic analysis. In this study we have examined by electron microscopy the organization of ER and mitochondria in cells expressing or not mitofusin-2. Contrary to previous studies, we observed that loss of mitofusin-2 increased ER-mitochondria juxtaposition. These results suggest that mitofusin-2 does not play a critical role in the juxtapostion of ER and mitochondria, and highlight the essential role of ultrastructural analysis to visualize and measure contact between two intracellular compartments.     

HIV-1 Protein Nef Inhibits Activity of ATP-binding Cassette Transporter A1 by Targeting Endoplasmic Reticulum Chaperone Calnexin

HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism.     

Subcellular Partitioning of Protein Tyrosine Phosphatase 1B to the Endoplasmic Reticulum and Mitochondria Depends Sensitively on the Composition of Its Tail Anchor

The canonical protein tyrosine phosphatase PTP1B is an important regulator of diverse cellular signaling networks. PTP1B has long been thought to exert its influence solely from its perch on the endoplasmic reticulum (ER); however, an additional subpopulation of PTP1B has recently been detected in mitochondria extracted from rat brain tissue. Here, we show that PTP1B’s mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor. Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor. We additionally examined the mechanism of PTP1B’s insertion into the ER membrane through heterologous expression of PTP1B’s tail anchor in wild-type yeast and yeast mutants of major conserved ER insertion pathways: In none of these yeast strains was ER targeting significantly impeded, providing in vivo support for the hypothesis of spontaneous membrane insertion (as previously demonstrated in vitro). Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states.     

Intracellular Sorting Signals for Sequential Trafficking of Human Cytomegalovirus UL37 Proteins to the Endoplasmic Reticulum and Mitochondria

Human cytomegalovirus UL37 antiapoptotic proteins, including the predominant UL37 exon 1 protein (pUL37x1), traffic sequentially from the endoplasmic reticulum (ER) through the mitochondrion-associated membrane compartment to the mitochondrial outer membrane (OMM), where they inactivate the proapoptotic activity of Bax. We found that widespread mitochondrial distribution occurs within 1 h of pUL37x1 synthesis. The pUL37x1 mitochondrial targeting signal (MTS) spans its first antiapoptotic domain (residues 5 to 34) and consists of a weak hydrophobicity leader (MTSα) and proximal downstream residues (MTSβ). This MTS arrangement of a hydrophobic leader and downstream proximal basic residues is similar to that of the translocase of the OMM 20, Tom20. We examined whether the UL37 MTS functions analogously to Tom20 leader. Surprisingly, lowered hydropathy of the UL37x1 MTSα, predicted to block ER translocation, still allowed dual targeting of mutant to the ER and OMM. However, increased hydropathy of the MTS leader caused exclusion of the UL37x1 high-hydropathy mutant from mitochondrial import. Conversely, UL37 MTSα replacement with the Tom20 leader did not retarget pUL37x1 exclusively to the OMM; rather, the UL37x1-Tom20 chimera retained dual trafficking. Moreover, replacement of the UL37 MTSβ basic residues did not reduce OMM import. Ablation of the MTSα posttranslational modification site or of the downstream MTS proline-rich domain (PRD) increased mitochondrial import. Our results suggest that pUL37x1 sequential ER to mitochondrial trafficking requires a weakly hydrophobic leader and is regulated by MTSβ sequences. Thus, HCMV pUL37x1 uses a mitochondrial importation pathway that is genetically distinguishable from that of known OMM proteins.During infection of permissive cells, the human cytomegalovirus (HCMV) UL37 immediate-early locus encodes multiple UL37 isoforms (4, 11, 16, 22, 24, 25) (Fig. ​(Fig.1A).1A). The predominant isoform, the UL37 exon 1 protein (pUL37x1), or the viral mitochondrial inhibitor of apoptosis (vMIA), is an essential HCMV gene product required for its growth in humans (17) and in cell culture (14, 20, 36, 47). pUL37x1 induces calcium efflux from the endoplasmic reticulum (ER) (40), regulates viral early gene expression (6, 12), disrupts the F-actin cytoskeleton (35, 40), binds and inactivates Bax at the mitochondrial outer membrane (OMM) (5, 32-34), and inhibits mitochondrial serine protease at late times of infection (27).Open in a separate windowFIG. 1.(A) HCMV UL37 isoforms. UL37 proteins share N-terminal UL37x1 MTS, including a moderately hydrophobic MTSα leader (aa 1 to 22, cylinder), MTSβ proximal basic residues (aa 23 to 29, ++++), downstream acidic (aa 81 to 108, —) and basic (aa 134 to 151, +++) domains. The unique C-terminal sequences encoded by UL37 exon 3 contain an N-glycosylation domain (aa 206 to 391, branches) as well as two additional TM domains (aa 178 to 196 and aa 433 to 459, cylinders). The fusion proteins carrying the full length (pUL37x1 wt_1-163) or MTS (wt_1-36-YFP) with C-terminal fluorophores are represented below. The two UL37x1 antiapoptotic domains are also shown (17). (B) Kinetics of pUL37x1 mitochondrial importation. HFFs were cotransfected with plasmids encoding pUL37x1 wt_1-163-YFP and DsRed1-mito (Clontech). After 2 h, anisomycin (70 μM) was added to the medium. After 12 h, the cells were either fixed with 100% methanol (0 min) or washed with 1× PBS and overlaid with fresh, anisomycin-free medium. The cells were incubated for the indicated times before methanol fixation and confocal imaging. The images were obtained by using comparable settings of aperture and laser power. (C) Colocalization of newly synthesized pUL37x1 with a mitochondrial marker. HFFs transiently transfected with pUL37x1 wt_1-163-YFP (green) were treated with anisomycin-containing medium as in panel B for 12 h. Inhibitor-containing medium was removed, and the cells were washed and overlaid with fresh, anisomycin-free medium for 45 min. At that time, 50 nM MitoTracker Red CMXRos (red, Invitrogen) was added to the medium, followed by incubation for 15 min at 37°C, prior to methanol fixation. The cells were then imaged by confocal microscopy. The panels on the left and center are grayscale. The panel on the right is the color merge of both channels. The small insets are enlargements of the indicated region of interest in the cell. (D) UL37x1 MTS is sufficient for mitochondrial import. HFFs were transiently transfected with expression vectors for wt_1-36-YFP and treated with MitoTracker Red (top row) as described above or for wt_1-163-YFP and DsRed1-mito (bottom). Cells were harvested 24 h later and imaged by confocal microscopy. The left and center panels are grayscale. The panels on the right show merged images of both channels.To accomplish their multiple functions in the cell, HCMV UL37 proteins sequentially traffic from the ER to mitochondria (4, 9, 17, 24-26, 45). The amino-terminal UL37x1 antiapoptotic domain serves as a mitochondrial targeting sequence (MTS) (16, 17, 24, 26). UL37 proteins first translocate into the ER, traffic through the mitochondria-associated membrane (MAM) subcompartment of the ER, and then to the OMM (9, 11, 24-26, 45). The MAM is a lipid-rich subdomain of the ER, which directly contacts mitochondria, allowing for the transfer of lipids from the ER to the OMM and the inner mitochondrial membrane (41), and functionally provides microdomains for efficient coupling of ER to mitochondria calcium transfer (37, 42).The HCMV UL37x1 bipartite MTS includes a weakly hydrophobic leader (MTSα, amino acids [aa] 1 to 22) that is required for ER translocation and mitochondrial import, as well as downstream sequences (MTSβ, aa 23 to 34) that are additionally required for its OMM importation (24) (Fig. ​(Fig.2A).2A). The HCMV UL37 MTS is conserved in the homologous primate CMV UL37x1 genes (28).Open in a separate windowFIG. 2.(A) Conservation of UL37x1 MTS among the primate cytomegaloviruses. The sequences of HCMV, chimpanzee CMV (CCMV), rhesus monkey CMV (RhCMV), and African green monkey (AgmCMV) are shown (top). The boxed areas enclose MTSα, the predicted alpha-helical domain, based upon HMMTOP analysis, within each leader. The MTSβ spans downstream residues 23 to 36. The boldfacing and filled circles indicate identity among primate CMV UL37x1 genes. The HCMV UL37x1 hydrophobic leader was mutated to lowered hydrophobicity by replacement of nonconserved residues V4G, L8G, and L14G while maintaining the same length of the TM in the LH mutant (bottom). The predicted hydrophobicity scores (grand average of hydropathicity, GRAVY, Kyte-Doolittle scale) were calculated for the boxed residues of the wt and LH mutant using ProtParam application on the ExPASy Proteomics Server. (B) Colocalization of UL37x1 LH_1-36-YFP with MitoTracker. HFFs transiently transfected with a vector expressing pUL37x1 LH_1-36-YFP for 24 h were treated with 50 nM MitoTracker as described above and imaged by confocal microscopy. Shown on the left and middle panels are the grayscale images, while the panel on the right is the overlay both channels. The small insets are enlargements of the indicated regions of interest. (C) ER translocation and mitochondrial import of pUL37x1 LH_1-36-YFP and LH_1-163-YFP. HeLa cells were transfected with expression vectors of wt_1-36-YFP, LH_1-36-YFP, or YFP vector alone (top) or wt_1-163-YFP, LH_1-163-YFP, or YFP vector alone (bottom). ER and mitochondrial fractions were isolated as described previously (8, 9). (Top) 10 μg (wt_1-36-YFP and YFP vector alone) or 40 μg (LH_1-36-YFP) of each fraction was analyzed by Westerns with anti-GFP (1:200) antibody. (Bottom) 20 μg of each fraction was analyzed by Western analysis with anti-UL37x1 (DC35, 1:2,500) or Grp75 (1:1,000) antibodies.In contrast, most signal-anchored proteins of the OMM are synthesized in the cytosol as precursors with NH₂-terminal sequences that directly target them to mitochondria (31). Signal-anchored OMM proteins, such as the translocase of the OMM subunits, Tom20 and Tom70 (43, 46), are similar in topology to pUL37x1 and the NH₂-terminal cleavage product, pUL37_NH2, of the UL37 glycoprotein (gpUL37) (26). Tom20 and Tom70 are anchored to the OMM by short NH₂-terminal transmembrane (TM) domains with the bulk of the polypeptides exposed to the cytosol in a type I orientation (21). The important structural elements of their signal anchor sequences are (i) moderate hydrophobicity of the TM domain and (ii) positively charged amino acids in its flanking domain (21, 43). Tom20 is targeted from the cytosol to the OMM by a moderately hydrophobic NH₂-terminal leader (score = 1.826) with a minimal requirement for a net basic charge within one to five residues downstream of the leader (21). The juxtaposed basic residues release the Tom20 hydrophobic leader from the ER-targeting signal recognition particle (SRP) and allow for its direct targeting to the OMM. This arrangement of the Tom20 intracellular sorting signals (20, 41) is similar to that of the MTS of pUL37x1 (22), whose leader, while lower in hydropathy (score = 1.289), is nonetheless ER translocated rather than imported from the cytosol directly into the OMM (24, 26).Our studies were undertaken to define the sequence requirements for pUL37x1 sequential targeting to the ER and to the OMM and to determine whether these signals are distinct from those of other OMM proteins. We examined the potential role of conventional OMM targeting signals (leader hydrophobicity and proximal basic residues) as well as sequences conserved in the homologues of primate CMVs. Unpredictably, UL37x1 MTSβ (aa 23 to 36) did not act analogously to the Tom20 mitochondrial targeting leader. Rather, HCMV UL37x1 sequences retargeted the Tom20 hydrophobic leader to sequential ER to OMM import. Moreover, mutation of conventional mitochondrial targeting basic residues did not markedly alter pUL37x1 mitochondrial import. Similarly, UL37x1 lowered hydrophobicity MTSα mutants dually trafficked to the ER and mitochondria. Conversely, pUL37x1 trafficking was altered by increased hydropathy, which effectively blocked mitochondrial import. From these studies, we conclude that weak hydrophobicity of the pUL37x1 MTSα and downstream residues play a role in directing translocation but involve more complex interplay than previously appreciated. Importantly, two previously unrecognized MTS signals, the consensus MTSα posttranslational modification (PTM) site (²¹SY) and a downstream MTSβ proline-rich domain (PRD, aa 33 to 36), regulated pUL37x1 mitochondrial import.(These studies were performed by C.D.W. in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)     

Translocation of Bax and Bid to Mitochondria, Endoplasmic Reticulum and Nuclear Envelope: Possible Control Points in Apoptosis

The cross-talk between endoplasmic reticulum (ER) and mitochondria was investigated during apoptosis in a breast cancer cell line (MCF-7) in culture. The effect of camptothecin, an inducer of apoptosis and a specific inhibitor of topoisomerase I, was investigated by morphological, immunocytochemical and histochemical techniques for electron microscopy. Our ultrastructural morphological data demonstrate alterations in ER configuration and communication with neighbouring mitochondria early after stimulation by camptothecin. Immunoelectron studies have demonstrated that Bax and Bid translocate from cytoplasm to mitochondria where they initiate mitochondrial dysfunction and cytochrome c release. Bax and Bid were also localized in ER and nuclear envelope. Since ER and mitochondria function as intracellular Ca2+ storage, we hypothesize that Bax and Bid are involved in the emptying of ER Ca2+ pool, triggers secondary changes in mitochondrial Ca2+ levels that contribute to cytochrome c release and cell death.     

A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria

Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER) to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC) has decreased phosphatidylserine (PS) transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE). Cells lacking EMC proteins and the ER–mitochondria tethering complex called ERMES (the ER–mitochondria encounter structure) are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER–mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM) complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.     

Comparative Model Analysis of Calcium Exchange between the Cytosol and Stores of Mitochondria or Endoplasmic Reticulum

The objects of the study were single-compartment mathematical models corresponding to a fragment of the dendrite of a cerebellar Purkinje neuron containing the mitochondria (model 1) or a cistern of the endoplasmic reticulum, ER, (model 2) as the calcium stores. We investigated the dependence of the intracellular Ca²⁺ dynamics on geometrical sizes of calcium exchanging parts of the intracellular space and the difference between the kinetic characteristics of storing in two types of stores occupying different portions of the compartment volume. The plasma membrane of the compartment bore the ion channels, particularly those conducting excitatory synaptic current, and the calcium pump typical of this neuron type. The model equations took into account Ca²⁺ exchange between the cytosol, extracellular medium, organelle stores, non-organelle endogenous buffers, and an exogenous buffer (fluorescent dye), and also the diffusion of Са²⁺ into adjacent regions of the dendrite. In model 1, the mitochondria exchanged Са²⁺ with the cytosol via the uniporter and sodium/calcium exchanger; mitochondrial processes, such as the tricarboxylic acid cycle and aerobic cellular respiration, were also taken into account. In model 2, the ER membrane contained the calcium pump, channels of passive leak, and channels of calcium-induced and inositol-3-phosphate-dependent release of Са²⁺. Increases in the portion of the stores in the total volume of the compartment from 1 to 36% led to a proportional increase in the peak values of the cytosolic calcium concentration ([Ca²⁺] _i ); the concentration of Са²⁺ in the mitochondria ([Ca²⁺]_mit) or ER ([Ca²⁺]_ER) increased correspondingly. During generation of bell-shaped cytosolic calcium signals of equal intensity and duration, the ER (due to a greater rate of storing, as compared with that in the mitochondria) was able to uptake several times more Са²⁺ (four times at 36% filling of the volume by the organelles). It is suggested that the revealed different kinetic characteristics of Са²⁺ storing by different organelles are determined by the rates of binding to transport molecules present in the store membrane and, therefore, are defined by concentrations (surface densities) of these molecules and their saturation at certain levels of [Ca²⁺]i. It has been shown that the occupancy of the intracellular volume by organelle stores of any type is a structural factor, which is able to essentially modulate the values of Ca²⁺ concentration.     

Statolith Sedimentation Kinetics and Force Transduction to the Cortical Endoplasmic Reticulum in Gravity-Sensing Arabidopsis Columella Cells

The starch statolith hypothesis of gravity sensing in plants postulates that the sedimentation of statoliths in specialized statocytes (columella cells) provides the means for converting the gravitational potential energy into a biochemical signal. We have analyzed the sedimentation kinetics of statoliths in the central S2 columella cells of Arabidopsis thaliana. The statoliths can form compact aggregates with gap sizes between statoliths approaching <30 nm. Significant intra-aggregate sliding motions of individual statoliths suggest a contribution of hydrodynamic forces to the motion of statoliths. The reorientation of the columella cells accelerates the statoliths toward the central cytoplasm within <1 s of reorientation. During the subsequent sedimentation phase, the statoliths tend to move at a distance to the cortical endoplasmic reticulum (ER) boundary and interact only transiently with the ER. Statoliths moved by laser tweezers against the ER boundary experience an elastic lift force upon release from the optical trap. High-resolution electron tomography analysis of statolith-to-ER contact sites indicate that the weight of statoliths is sufficient to locally deform the ER membranes that can potentially activate mechanosensitive ion channels. We suggest that in root columella cells, the transduction of the kinetic energy of sedimenting statoliths into a biochemical signal involves a combination of statolith-driven motion of the cytosol, statolith-induced deformation of the ER membranes, and a rapid release of kinetic energy from the ER during reorientation to activate mechanosensitive sites within the central columella cells.     

Roles of the Mitochondria and Endoplasmic Reticulum in Calcium Elevation during Osmotic Shock in Adrenocortical Cells

Steroid hormones participate in various metabolic processes, and dysfunction of the adrenocortical system leads to numerous pathologies in humans. One of the factors that can influence the secretory properties of adrenocorticocytes is changes in the cell volume observed during osmotic shock. In our study, we tested the hypothesis that osmotic stress modifies intracellular Ca²⁺ signalling and in such a way can influence the secretion of steroids by adrenocorticocytes. The effects of hyperosmotic stress on the cytosolic Ca²⁺ concentration ([Ca] _i ) in cultured adrenocortical cells from the zona fasciculata of the rat adrenals were investigated using the indicator fura-2 technique. Our experiments have shown that exposure of the cells to a hyperosmotic solution caused a decrease in the cell volume, as well as a reversible rise in the [Ca] _i . Calcium-free media partly eliminated [Ca] _i responses. Pretreatment of the cells with thapsigargin or CCCP (blockers of internal calcium stores) significantly decreased the magnitude of responses induced by osmotic stress. These findings indicate that osmotic shock causes an increase in the [Ca] _i in adrenocortical cells, mostly due to depletion of the intracellular stores, and may in such a way stimulate steroidogenesis.     

Molecular Cloning the Gene of Small Heat Shock Protein in the Mitochondria and Endoplasmic Reticulum of Tomato

A cDNA library was constructed with the heat shocked tomato (Lycopersicon esculentum Mill.) flowers and then was screened with the probes of mitochondrial and endoplasmic reticulum conservative regions that were cloned by using RT-PCR. The complete cDNAs of mitochondrial and endoplasmic reticulum small heat shock protein ( shsp ) were selected out from the cDNA library. Furthermore, the temperature responses of these shsp genes were determined. Northern hybridization showed that the heat response temperatures of both genes in tomato flower were lower than that in leaf and that mitochondria shsp in leaf was cold-inducible. In this paper, the molecular features of the cloned genes, the causes of the uncommon heat response temperatures of sHSP in flower and the cold inducible character of mitochondria shsp gene in leaf were discussed.      

番茄线粒体和内质网小分子热激蛋白基因的分子克隆

以热激处理的番茄（Lycopersicon esculentum Mill．）花为实验材料,构建了cDNA库,运用ＲＴ－ＰＣＲ方法克隆番茄粒体和内质网小分子热激蛋白cDNA,利用这两个保守区片段为探针,筛选cDNA库,获得线粒体和内质网小分子热激蛋白全序列cDNA。;通过分析线粒体和内质网小分子热激蛋白基因对温度的反应,发现小分子热激蛋白基因在番茄花中的热激应答温度低于它们在叶片中的热激应答温度,并且番茄叶片中的线粒体小分子热激蛋白基因还具有低温应答特性。对线粒体和内质网小分子热激蛋白基因的分子结构特点,小分子热激蛋白基因在番茄花中的特别热激应答温度的调控机理以及线粒体小分子热激蛋白的基因在中片中的低温度应答成因进行了讨论。     

ER-Phagy: Selective Autophagy of the Endoplasmic Reticulum

Throughout their life, cells must maintain homeostasis while facing constantly fluctuating demands on their different organelles. A major mechanism for the homeostatic control of organelle function is the unfolded protein response (UPR), a signaling pathway that triggers a comprehensive remodeling of the endoplasmic reticulum (ER) and the biosynthetic pathway according to need. We discovered that activation of the UPR in yeast also induces a new branch of macroautophagy that selectively targets the ER. We term this process “ER-phagy”, in analogy to pexophagy and mitophagy, the two other known forms of organelle-specific marcoautophagy. ER-phagy involves the generation of autophagosomes that selectively include ER membranes and whose delimiting double membranes also derive, at least in part, from the ER. This finding provides direct evidence that the ER can serve as a membrane source for autophagosome formation and indicates that ER-phagy entails engulfment of the ER by itself. ER-phagy could remove damaged or redundant parts of the ER and thus represent an important degradative functionality of the UPR that helps to afford homeostatic control.

Addendum to:

Autophagy Counterbalances Endoplasmic Reticulum Expansion during the Unfolded Protein Response

S. Bernales, K.L. McDonald and P. Walter

PLoS Biol 2006; 4: e423     

An Association between Mitochondria and the Endoplasmic Reticulum in Cells of the Pseudobranch Gland of a Teleost

An elaborate and apparently unique specialization of the endoplasmic reticulum having the form of tubules and a precise orientation with respect to the mitochondria has been described for the specific cell of the pseudobranch gland. The tubules also are concentrated near the vascular border of the cell where they show continuity with the plasma membrane and open directly against the basement membrane. On the other side of the basement membrane, the endothelial cells of the sinusoid show openings or discontinuities characteristically associated with secretory cells. The pseudobranch gland is presumed to have carbonic anhydrase as one of its primary products, if not its only one, and the elaborate ultrastructure is thought to be associated with the special problems of secreting this enzyme.