Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O- and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphosphorylceramide levels were drastically reduced.     

p37 is a p97 adaptor required for Golgi and ER biogenesis in interphase and at the end of mitosis

We previously reported that p97/p47-assisted membrane fusion is important for the reassembly of organelles at the end of mitosis, but not for their maintenance during interphase. We have now identified a p97 adaptor protein, p37, which forms a complex with p97 in the cytosol and localizes to the Golgi and ER. siRNA experiments revealed that p37 is required for Golgi and ER biogenesis. Injection of anti-p37 antibodies into cells at different cell cycle stages showed that p37 plays an important role in both Golgi and ER maintenance during interphase as well as in their reassembly at the end of mitosis. In an in vitro Golgi reassembly assay, the p97/p37 complex has membrane fusion activity. In contrast to the p97/p47 pathway, this pathway requires p115-GM130 tethering and SNARE GS15, but not syntaxin5. Interestingly, although VCIP135 is also required, its deubiquitinating activity is unnecessary for p97/p37-mediated activities.     

The heme-oxygenase family required for phytochrome chromophore biosynthesis is necessary for proper photomorphogenesis in higher plants

The committed step in the biosynthesis of the phytochrome chromophore phytochromobilin involves the oxidative cleavage of heme by a heme oxygenase (HO) to form biliverdin IXalpha. Through positional cloning of the photomorphogenic mutant hy1, the Arabidopsis HO (designated AtHO1) responsible for much of phytochromobilin synthesis recently was identified. Using the AtHO1 sequence, we identified families of HO genes in a number of plants that cluster into two subfamilies (HO1- and HO2-like). The tomato (Lycopersicon esculentum) yg-2 and Nicotiana plumbaginifolia pew1 photomorphogenic mutants are defective in specific HO genes. Phenotypic analysis of a T-DNA insertion mutant of Arabidopsis HO2 revealed that the second HO subfamily also contributes to phytochromobilin synthesis. Homozygous ho2-1 plants show decreased chlorophyll accumulation, reduced growth rate, accelerated flowering time, and reduced de-etiolation. A mixture of apo- and holo-phyA was detected in etiolated ho2-1 seedlings, suggesting that phytochromobilin is limiting in this mutant, even in the presence of functional AtHO1. The patterns of Arabidopsis HO1 and HO2 expression suggest that the products of both genes overlap temporally and spatially. Taken together, the family of HOs is important for phytochrome-mediated development in a number of plants and that each family member may uniquely contribute to the phytochromobilin pool needed to assemble holo-phytochromes.     

Defining components required for transport from the ER to the Golgi complex in yeast

Several complementary approaches have been fruitful in the study of transport from the ER to the Golgi complex in yeast. Mutational analysis has led to the identification of genes required for this process, many of which are now being studied at the molecular and biochemical level. In the case of SEC18, DNA sequence analysis has demonstrated homology to a factor needed for transport in mammalian in vitro systems. In addition, the events that take place at this stage of the secretory pathway have been reconstituted in vitro.     

Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and reproduction in Arabidopsis

Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss‐of‐function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen‐specific rescue construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid‐mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short‐chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. This study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important roles for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status.     

Regulation of sphingolipid and glycosphingolipid metabolism in extrahepatic tissues by endotoxin

The host response to infection and inflammation is associated with multiple alterations in lipid metabolism. We have shown that endotoxin [lipopolysaccharide (LPS)] stimulates hepatic sphingolipid synthesis and increases ceramide and glucosylceramide (GlcCer) content in circulating lipoproteins in Syrian hamsters. LPS also increases the activity and mRNA levels of serine palmitoyltransferase (SPT) and GlcCer synthase, the committed enzymes in sphingolipid and glycosphingolipid (GSL) synthesis, respectively, in the liver. To determine whether sphingolipid and GSL metabolism are regulated in other tissues during the host response to infection, we examined the effect of LPS on the regulation of SPT and GlcCer synthase in extrahepatic tissues in Syrian hamsters. LPS significantly increased SPT activity in spleen and kidney after 16 h of treatment, but had no effect on SPT activity in lung and brain, suggesting that the effect of LPS on sphingolipid metabolism is tissue specific. LPS also increased SPT mRNA levels in spleen and kidney by approximately 3-fold, suggesting that the increase in SPT activity is due to an increase in SPT mRNA expression. LPS significantly increased GlcCer synthase activity in spleen and kidney, and produced 4- and 15-fold increases in GlcCer synthase mRNA levels in spleen and kidney, respectively. LPS treatment increased GlcCer content by 1.3-fold in spleen and by 6.2-fold in kidney. LPS also increased the content of ceramide trihexoside by 1.7-fold in spleen. These results suggest that LPS regulates sphingolipid and GSL metabolism in spleen and kidney. An increase in GSL metabolites in spleen and kidney during the host response to infection and inflammation may be required for modulation of immune responses and regulation of cell growth. -- Memon, R. A., W. M. Holleran, Y. Uchida, A. H. Moser, C. Grunfeld, and K. R. Feingold. Regulation of sphingolipid and glycosphingolipid metabolism in extrahepatic tissues by endotoxin. J. Lipid Res. 2001. 42: 452--459.     

Syntaxin 17 cycles between the ER and ERGIC and is required to maintain the architecture of ERGIC and Golgi

Background information. Syntaxin 17 is a SNARE (soluble N‐ethylmaleimide‐sensitive‐factor‐attachment protein receptor) protein that predominantly localizes to the ER (endoplasmic reticulum) and to some extent in the ERGIC (ER—Golgi intermediate compartment). Syntaxin 17 has been suggested to function as a receptor at the ER membrane that mediates trafficking between the ER and post‐ER compartments. It has a unique 33 amino acid luminal tail whose function is not known. Here we have investigated the structural requirements for localization of syntaxin 17 to the ERGIC and its role in trafficking. Results. Deletion analysis showed that syntaxin 17 required its cytoplasmic domain to exit the ER and localize to the ERGIC. Mutation of a conserved tyrosine residue in the cytoplasmic domain resulted in reduced localization of syntaxin 17 in the ERGIC and ER‐exit sites, suggesting the presence of a tyrosine‐based ER export motif. Syntaxin 17 also required its C‐terminal tail to localize to the ERES (ER exit sites) and ERGIC. Knockdown of syntaxin 17 destabilized the ERGIC organization and also caused fragmentation of the Golgi complex. Syntaxin 17 showed direct interaction with transmembrane proteins p23 and p25 (cargo receptors that cycle between the ER and Golgi) with the help of its C‐terminal tail. Overexpression of syntaxin 17 redistributed β‐COP (β‐coatomer protein) which required its C‐terminal tail. Overexpression of syntaxin 17 also blocked the anterograde transport of VSVG (vesicular stomatitis virus G‐protein) in the ERGIC. Conclusions. We show that syntaxin 17 has a tyrosine‐based motif which is required for its incorporation into COPII (coatomer protein II) vesicles, exit from the ER and localization to the ERGIC. Our results suggest that syntaxin 17 cycles between the ER and ERGIC through classical trafficking pathways involving COPII and COPI (coatomer protein I) vesicles, which requires its unique C‐terminal tail. We also show that syntaxin 17 is essential for maintaining the architecture of ERGIC and Golgi.     

A long chain spin label for glycosphingolipid studies: transbilayer fatty acid interdigitation of lactosyl ceramide

16-Carbon and 18-carbon fatty acids with covalently attached nitroxide free radicals have seen wide usage in membrane studies of phospholipid dynamics, orientation, and associations. However, they are inadequate for dealing with some very important questions that relate to glycosphingolipids. We report here the synthesis of a long chain (24-carbon) spin-labelled fatty acid designed for such problems. We have used both the new 24-carbon and the more conventional 18-carbon spin-labelled fatty acids to replace the natural fatty acid of lactosyl ceramide so that we may begin to compare short and long chain derivatives to analyse the molecular basis of their functional differences. Spectra seen are consistent with the view that in a bilayer host matrix the methyl end of the long fatty acid crosses the hydrophobic membrane center and interdigitates with fatty acids of phospholipids of the opposing monolayer.     

Synthetic, non-natural sphingolipid analogs inhibit the biosynthesis of cellular sphingolipids, elevate ceramide and induce apoptotic cell death

Numerous studies have demonstrated the participation of sphingolipids in signal transduction and regulation of cell growth. Several cellular stress agents have been shown to elevate ceramide, the basic precursor of all sphingolipids, initiating a cascade of events leading to arrest of the cell cycle, apoptosis and cell death. Aiming at inhibiting metabolic pathways of sphingolipid metabolism that might lead to an increase of cellular ceramide, we have synthesized non-natural analogs of ceramide, sphingosine and trimethylsphingosine. When the respective analogs were applied to HL60 human myeloid leukemic cells they inhibited the biosynthesis of sphingomyelin (SPM) and glycosphingolipids and induced apoptosis that led to cell death. A fluorescent procedure which has been developed for quantifying the biosynthesis of cellular ceramide indicated an increase in the ceramide content following an incubation with the synthetic analogs. These results suggest that the newly synthesized sphingolipid analogs might be valuable for potential application as a therapeutic modality in leukemia and other malignancies.     

Polo-like kinase is required for Golgi and bilobe biogenesis in Trypanosoma brucei

A bilobed structure marked by TbCentrin2 regulates Golgi duplication in the protozoan parasite Trypanosoma brucei. This structure must itself duplicate during the cell cycle for Golgi inheritance to proceed normally. We show here that duplication of the bilobed structure is dependent on the single polo-like kinase (PLK) homologue in T. brucei (TbPLK). Depletion of TbPLK leads to malformed bilobed structures, which is consistent with an inhibition of duplication and an increase in the number of dispersed Golgi structures with associated endoplasmic reticulum exit sites. These data suggest that the bilobe may act as a scaffold for the controlled assembly of the duplicating Golgi.     

Golgi apparatus-localized synaptotagmin 2 is required for unconventional secretion in Arabidopsis

Background

Most secretory proteins contain signal peptides that direct their sorting to the ER and secreted via the conventional ER/Golgi transport pathway, while some signal-peptide-lacking proteins have been shown to export through ER/Golgi independent secretory pathways. Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus that is active against both prokaryotic and eukaryotic cells. The hygromycin phosphotransferase (HYG^R) can phosphorylate and inactivate the hygromycin B, and has been widely used as a positive selective marker in the construction of transgenic plants. However, the localization and trafficking of HYG^R in plant cells remain unknown. Synaptotagmins (SYTs) are involved in controlling vesicle endocytosis and exocytosis as calcium sensors in animal cells, while their functions in plant cells are largely unclear.

Methodology/Principal Findings

We found Arabidopsis synaptotagmin SYT2 was localized on the Golgi apparatus by immunofluorescence and immunogold labeling. Surprisingly, co-expression of SYT2 and HYG^R caused hypersensitivity of the transgenic Arabidopsis plants to hygromycin B. HYG^R, which lacks a signal sequence, was present in the cytoplasm as well as in the extracellular space in HYG^R-GFP transgenic Arabidopsis plants and its secretion is not sensitive to brefeldin A treatment, suggesting it is not secreted via the conventional secretory pathway. Furthermore, we found that HYG^R-GFP was truncated at carboxyl terminus of HYG^R shortly after its synthesis, and the cells deficient SYT2 failed to efficiently truncate HYG^R-GFP,resulting in HYG^R-GFP accumulated in prevacuoles/vacuoles, indicating that SYT2 was involved in HYG^R-GFP trafficking and secretion.

Conclusion/Significance

These findings reveal for the first time that SYT2 is localized on the Golgi apparatus and regulates HYG^R-GFP secretion via the unconventional protein transport from the cytosol to the extracelluar matrix in plant cells.     

Golgi manganese transport is required for rapamycin signaling in Saccharomyces cerevisiae

The Pmr1 Golgi Ca2+/Mn2+ ATPase negatively regulates target of rapamycin complex (TORC1) signaling, the rapamycin-sensitive TOR complex in Saccharomyces cerevisiae. Since pmr1 causes resistance to rapamycin and tor1 causes hypersensitivity, we looked for genetic interactions of pmr1 with tor1. Deletion of TOR1 restored two wild-type phenotypes. Loss of TOR1 restored the ability of the pmr1 strain to grow on media containing 2 mm MnCl2 and conferred wild type as well as the wild-type sensitivity to rapamycin. Mn2+ additions to media partially suppressed rapamycin resistance of wild type and pmr1 tor1, suggesting that Tor1 and Tor2 are regulated by manganese. We parsed the roles of Ca2+ and Mn2+ transport and the compartments in rapamycin response using separation-of-function mutants available for Pmr1. A strain containing the D53A mutant (Mn2+ transporting) of Pmr1 is rapamycin sensitive, but the Q783A mutant (Ca2+ transporting) strain is rapamycin resistant. Mn2+ transport into the Golgi lumen appears to be required for rapamycin sensitivity. Overexpression of Ca2+ pump SERCA1, Ca2+/H+ antiporter Vcx1, or a Mn2+ transporting mutant of Vcx1 (Vcx1-M1) failed to restore rapamycin sensitivity, and loss of Pmr1 but not other transporters of Ca2+ or Mn2+ results in rapamycin resistance. Overexpression of Ccc1, a Fe2+ and Mn2+ transporter that has been localized to Golgi and the vacuole, does restore rapamycin sensitivity to pmr1Delta. We conclude that Mn2+ in the Golgi inhibits TORC1 signaling.     

Egghead and brainiac are essential for glycosphingolipid biosynthesis in vivo

The Drosophila genes, brainiac and egghead, encode glycosyltransferases predicted to act sequentially in early steps of glycosphingolipid biosynthesis, and both genes are required for development in Drosophila. egghead encodes a beta4-mannosyltransferase, and brainiac encodes a beta3-N-acetylglucosaminyltransferase predicted by in vitro analysis to control synthesis of the glycosphingolipid core structure, GlcNAcbeta1-3Manbeta1-4Glcbeta1-Cer, found widely in invertebrates but not vertebrates. In this report we present direct in vivo evidence for this hypothesis. egghead and brainiac mutants lack elongated glycosphingolipids and exhibit accumulation of the truncated precursor glycosphingolipids. Furthermore, we demonstrate that despite fundamental differences in the core structure of mammalian and Drosophila glycosphingolipids, the Drosophila egghead mutant can be rescued by introduction of the mammalian lactosylceramide glycosphingolipid biosynthetic pathway (Galbeta1-4Glcbeta1-Cer) using a human beta4-galactosyltransferase (beta4Gal-T6) transgene. Conversely, introduction of egghead in vertebrate cells (Chinese hamster ovary) resulted in near complete blockage of biosynthesis of glycosphingolipids and accumulation of Manbeta1-4Glcbeta1-Cer. The study demonstrates that glycosphingolipids are essential for development of complex organisms and suggests that the function of the Drosophila glycosphingolipids in development does not depend on the core structure.     

Carboxylated lysine is required for higher activities in Hydantoinases

Hydantoinases are industrial enzymes with varying degree of activities on variable substrates to form different products. Although, few of the hydantoinase structures were known recently, the functional details and active site mechanism were not clearly understood yet. In a structure determination effort we reported that Bacillus sp. AR9 hydantoinase contains uncarboxylated lysine in the active site, whereas all the other hydantoinases have a carboxylated active site lysine. Here we describe the importance of carboxylated lysine for differential activities by making lysine mutations as well as carboxylating the lysine in a D-hydantoinase from Bacillus sp. AR9. The lysine to alanine and lysine to arginine mutations showed reduced activities whereas carboxylation of the lysine has enhanced the activity. Theoretical studies involving the calculation of electrostatic potentials for the hydroxide ion between the two metal ions present in the active site suggest that the presence of carboxylated lysine increases the nucleophilicity of the hydroxide.     

Progranulin is required for proper ER stress response and inhibits ER stress-mediated apoptosis through TNFR2

Progranulin (PGRN) was reported to be a stress-response factor in response to hypoxia and acidosis. Here we present evidences demonstrating that PGRN is also an endoplasmic reticulum (ER) stress responsive factor: PGRN expression was induced and its activation of Erk1/2 and Akt signaling enhanced in response to ER stress; Normal ER stress response was lost in PGRN deficient cells and PGRN deficient cells became hypersusceptible to ER stress-induced apoptosis; additionally, recombinant PGRN could rescue the defects in ER-stress responses seen in PGRN deficient cells. Mechanistic studies indicated that PGRN/TNFR2 was critical for PGRN mediated regulation of ER stress response: similar to PGRN, the expression of TNFR2, but not TNFR1, was also induced in the course of ER stress; in addition, the association between PGRN and TNFR2 was markedly enhanced following ER stress; More importantly, PGRN protection of ER stress induced apoptosis was abolished when TNFR2 signaling was blocked. In addition, the 2nd and 3rd cysteine-rich domains (CRD) in the extracellular portion of TNFR2 (CRD2CRD3), known to directly bind to PGRN, disturbed the interaction of PGRN with TNFR2, and in turn abolished PGRN-mediated activation of Erk1/2 and Akt signaling and protection against apoptosis in response to ER-stress. Collectively, PGRN plays an important role in ER stress and regulates ER stress response through interacting with TNFR2. This study provides new insight into PGRN regulation of stress response and may also present PGRN as a potential molecular target for treating stress-associated disorders.     

Inheritance of cortical ER in yeast is required for normal septin organization

How cells monitor the distribution of organelles is largely unknown. In budding yeast, the largest subdomain of the endoplasmic reticulum (ER) is a network of cortical ER (cER) that adheres to the plasma membrane. Delivery of cER from mother cells to buds, which is termed cER inheritance, occurs as an orderly process early in budding. We find that cER inheritance is defective in cells lacking Scs2, a yeast homologue of the integral ER membrane protein VAP (vesicle-associated membrane protein-associated protein) conserved in all eukaryotes. Scs2 and human VAP both target yeast bud tips, suggesting a conserved action of VAP in attaching ER to sites of polarized growth. In addition, the loss of either Scs2 or Ice2 (another protein involved in cER inheritance) perturbs septin assembly at the bud neck. This perturbation leads to a delay in the transition through G2, activating the Saccharomyces wee1 kinase (Swe1) and the morphogenesis checkpoint. Thus, we identify a mechanism involved in sensing the distribution of ER.     

Alpha-glucosidase I is required for cellulose biosynthesis and morphogenesis in Arabidopsis

Novel mutations in the RSW1 and KNOPF genes were identified in a large-scale screen for mutations that affect cell expansion in early Arabidopsis embryos. Embryos from both types of mutants were radially swollen with greatly reduced levels of crystalline cellulose, the principal structural component of the cell wall. Because RSW1 was previously shown to encode a catalytic subunit of cellulose synthase, the similar morphology of knf and rsw1-2 embryos suggests that the radially swollen phenotype of knf mutants is largely due to their cellulose deficiency. Map-based cloning of the KNF gene and enzyme assays of knf embryos demonstrated that KNF encodes alpha-glucosidase I, the enzyme that catalyzes the first step in N-linked glycan processing. The strongly reduced cellulose content of knf mutants indicates that N-linked glycans are required for cellulose biosynthesis. Because cellulose synthase catalytic subunits do not appear to be N glycosylated, the N-glycan requirement apparently resides in other component(s) of the cellulose synthase machinery. Remarkably, cellular processes other than extracellular matrix biosynthesis and the formation of protein storage vacuoles appear unaffected in knf embryos. Thus in Arabidopsis cells, like yeast, N-glycan trimming is apparently required for the function of only a small subset of N-glycoproteins.     

Myt1 protein kinase is essential for Golgi and ER assembly during mitotic exit

Myt1 was originally identified as an inhibitory kinase for Cdc2 (Cdk1), the master engine of mitosis, and has been thought to function, together with Wee1, as a negative regulator of mitotic entry. In this study, we report an unexpected finding that Myt1 is essential for Golgi and endoplasmic reticulum (ER) assembly during telophase in mammalian cells. Our analyses reveal that both cyclin B1 and cyclin B2 serve as targets of Myt1 for proper Golgi and ER assembly to occur. Thus, our results show that Myt1-mediated suppression of Cdc2 activity is not indispensable for the regulation of a broad range of mitotic events but is specifically required for the control of intracellular membrane dynamics during mitosis.