Subcellular Localization of the S Locus F-box Protein AhSLF-S2 in Pollen and Pollen Tubes of Self-Incompatible Antirrhinum

The distribution of the S locus F-box (SLF) protein was examined by immunocytochemistry and Western blot techniques using an antibody against the C-terminal part of AhSLF-S2 in self-incompatible lines of Antirrhinum. Abundant gold particles were found where pollen tubes emerge in vitro. With the elongation of pollen tubes, binding sites for the antibody were found in the cytoplasm of the pollen tubes,including the peripheral part of the endoplasmic reticulum. After germination in vitro for 16 h, the product of AhSLF-S2 and possibly its allelic products could still be detectable, implying that the SLF protein has a role in the elongating process of pollen tubes. The present study provides evidence at the protein level that the SLF protein is present in pollen cytoplasm during pollen tube growth. These findings are discussed, as is their potential role in the self-incompatible response in Antirrhinum.     

家蚕耐氟性差异的细胞化学研究

对不同蚕品种的耐氟性、ACPase的氟敏感性、蚕品种耐氟性机理的研究表明,在供试蚕品种中以浙农1号的耐氟性最强,杭 8的耐氟性最弱;家蚕Bombyx mori血淋巴ACPase活性与蚕品种的耐氟性无明显关系;氟对蚕的血淋巴和中肠组织细胞的ACPase活性都有抑制作用,并随着氟添食浓度的增加ACPase活性降低,但超过一定浓度的氟添食,血淋巴ACPase活性反而有一个回升的过程,这个转折点出现可能的浓度及回升的幅度与蚕品种的耐氟性有关;细胞化学研究发现此转折点的出现是由于高浓度氟引起细胞结构的破坏而导致蚕体组织细胞内的ACPase大量向血腔释放的结果;氟敏感性蚕品种杭 8在很低氟量添食即可引起中肠组织细胞的ACPase大量向血腔释放,使血淋巴中的ACPase活性大幅度上升,随后ACPase活性受到完全的抑制;耐氟性较强的蚕品种浙农1号则在较高的氟含量添食时才向血腔释放ACPase,且血淋巴中ACPase增高的幅度小,在很高的氟量添食时全面抑制中肠ACPase活性。氟对不同品种ACPase活性影响的差异被认为是家蚕品种耐氟性差异机理之一。     

Activities,Quantitative Changes and Subcellular Localization of |á-Amylase During Development of Apple Fruit

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. α-Amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was often shown extrachloroplastic in living cells. The present experiment showed that α-amylase activity was progressively increasing concomitantly with the decreasing starch concentrations during the development of apple (Malus domestica Borkh cv. Starkrimson) fruit. The apparent amount of α-amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The enzyme subcellular-localization studies via immunogold electron-microscopy technique showed that α-amylase visualized by gold particles was predominantly located in plastids, but the gold particles were scarcely found in other subcellular compartments. A high density of the enzyme was observed at the periphery of starch granules during the middle and late developmental stages. These data proved that the enzyme is compartmented in its functional sites in the living cells of the fruit. The predominantly plastid-distributed pattern of α-amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (α-amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that α-amylase is involved in starch hydrolysis in plastids of the fruit cells.     

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.     

Ubiquitination Regulates the Proteasomal Degradation and Nuclear Translocation of the Fat Mass and Obesity-Associated (FTO) Protein

Genetic polymorphisms in the fat mass and obesity-associated (FTO) gene have been strongly associated with obesity in humans. The cellular level of FTO is tightly regulated, with alterations in its expression influencing energy metabolism, food intake and body weight. Although the proteasome system is involved, the cellular mechanism underlying FTO protein turnover remains unknown. Here, we report that FTO undergoes post-translational ubiquitination on Lys-216. Knock-in HeLa cells harboring the ubiquitin-deficient K216R mutation displayed a slower rate of FTO turnover, resulting in an increase in the level of FTO as well as enhanced phosphorylation of the ribosomal S6 kinase. Surprisingly, we also found that K216R mutation reduced the level of nuclear FTO and completely abolished the nuclear translocation of FTO in response to amino acid starvation. Collectively, our results reveal the functional importance of ubiquitination in controlling FTO expression and localization, which may be crucial for determining body mass and composition.     

The cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on the LDLR

Mutations in the proprotein convertase PCSK9 gene are associated with autosomal dominant familial hyper- or hypocholesterolemia. These phenotypes are caused by a gain or loss of function of proprotein convertase subtilisin kexin 9 (PCSK9) to elicit the degradation of the low-density lipoprotein receptor (LDLR) protein. Herein, we asked whether the subcellular localization of wild-type PCSK9 or mutants of PCSK9 and the LDLR would provide insight into the mechanism of PCSK9-dependent LDLR degradation. We show that the LDLR is the dominant partner in regulating the cellular trafficking of PCSK9. In cells lacking the LDLR, PCSK9 localized in the endoplasmic reticulum (ER). In cells expressing the LDLR, PCSK9 sorted to post-ER compartments (i.e. endosomes in cell lines and Golgi apparatus in primary hepatocytes), where it colocalized with the LDLR. In cell lines, PCSK9 also colocalized with the LDLR at the cell surface, requiring the presence of the C-terminal Cys/His-rich domain of PCSK9. We provide evidence that PCSK9 promotes the degradation of the LDLR by an endocytic mechanism, as small interfering RNA-mediated knockdown of the clathrin heavy chain reduced the functional activity of PCSK9. We also compared the subcellular localization of natural mutants of PCSK9 with that of the wild-type enzyme in human hepatic (HuH7) cells. Whereas the mutants associated with hypercholesterolemia (S127R, F216L and R218S) localized to endosomes/lysosomes, those associated with hypocholesterolemia did not reach this compartment. We conclude that the sorting of PCSK9 to the cell surface and endosomes is required for PCSK9 to fully promote LDLR degradation and that retention in the ER prevents this activity. Mutations that affect this transport can lead to hyper- or hypocholesterolemia.     

Strategies for the production of cell wall‐deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production

Microbial cell wall‐deconstructing enzymes are widely used in the food, wine, pulp and paper, textile, and detergent industries and will be heavily utilized by cellulosic biorefineries in the production of fuels and chemicals. Due to their ability to use freely available solar energy, genetically engineered bioenergy crops provide an attractive alternative to microbial bioreactors for the production of cell wall‐deconstructing enzymes. This review article summarizes the efforts made within the last decade on the production of cell wall‐deconstructing enzymes in planta for use in the deconstruction of lignocellulosic biomass. A number of strategies have been employed to increase enzyme yields and limit negative impacts on plant growth and development including targeting heterologous enzymes into specific subcellular compartments using signal peptides, using tissue‐specific or inducible promoters to limit the expression of enzymes to certain portions of the plant or certain times, and fusion of amplification sequences upstream of the coding region to enhance expression. We also summarize methods that have been used to access and maintain activity of plant‐generated enzymes when used in conjunction with thermochemical pretreatments for the production of lignocellulosic biofuels.     

Dihydrodipicolinate synthase ofnicotiana sylvestris,a chloroplast-localized enzyme of the lysine pathway

The first enzyme of the lysine-biosynthesis pathway, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has been purified and characterized inNicotiana sylvestris Speggazini et Comes. A purification scheme was developed for the native DHDPS that subsequently led to the purification to homogeneity of its subunits using two-dimensional gel electrophoresis. Subsequent elution of the purified polypeptide has opened the way for the production of rabbit polyclonal anti-DHDPS sera. The molecular weight of the enzyme was determined to be 164000 daltons (Da) by an electrophoretic method. By labeling with [¹⁴C]pyruvate, the enzyme was shown to be composed of four identical subunits of 38500 Da. Pyruvate acts as a stabilizing agent and contributes to the preservation of the tetrameric structure of the enzyme. The enzyme ofN. sylvestris is strongly inhibited by lysine with anI _0.5 of 15 μM; S-(2-aminoethyl)L-cysteine and γ-hydroxylysine, two lysine analogs, were found to be only weak inhibitors. An analog of pyruvate, 2-oxobutyrate, competitively inhibited the enzyme and was found to act at the level of the pyruvate-binding site. Dihydrodipicolinate synthase was localized in the chloroplast and identified as a soluble stromal enzyme by enzymatic and immunological methods. Its properties are compared with those known for other plant and bacterial DHDPS enzymes.     

Filipin-labelled complexes are polarized in their distribution in the cytoplasm of meiotically mature mouse eggs

Unfertilized (germinal vesicle [GV] stage, superovulated and naturally ovulated) and fertilized mouse eggs were treated with the polyene antibiotic filipin, which complexes with unesterified sterols; specimens were observed by fluorescence microscopy and scanning electron microscopy (SEM). In all oocytes examined, filipin fluorescence was localized to the plasma membrane and to subcellular structures of various sizes. In the unfertilized oocyte, polarity was observed both in the plasma membrane stain and in the pattern formed by the subcellular structures. SEM of filipin-treated oocytes had several characteristic features including a specific distribution of heterogeneous microvilli that appears to have a spatial relationship with the fluorescent pattern of the filipin-positive subcellular structures. In GV stage and fertilized eggs the filipin-positive subcellular structures were associated with the germinal vesicle and in fertilized eggs they were associated with the site of polar body abstriction.     

Subcellular distribution and chemical forms of Cd in Bougainvillea spectabilis Willd. as an ornamental phytostabilizer: An integrated consideration

The effects of Cd stress on the growth and Cd accumulation of Bougainvillea spectabilis Willd. as an ornamental plant and the related mechanisms were investigated in the study. We studied the impact of Cd on the plant ultrastructure, examined the cellular distribution of Cd, explored the Cd chemical forms and transformation, and determined the organic acid secretion in the plants. The results showed that B. spectabilis could grow well in the Cd treatment groups, and the roots could accumulate high concentration of Cd. The soluble fraction (primarily in the vacuole) as the form of citrate in leaves of B. spectabilis was the major compartment for Cd storage. The citric acid secreted by B. spectabilis played an important role in the detoxification of Cd, as well as the growth of plants and Cd accumulation. As an ornamental plant, B. spectabilis has the potential to be used in the phytostabilization of Cd-contaminated soils and can beautify the environment at the same time.