The Experts below are selected from a list of 68514 Experts worldwide ranked by ideXlab platform
Gert Kreibich - One of the best experts on this subject based on the ideXlab platform.
-
active translocon complexes labeled with gfp DAD1 diffuse slowly as large polysome arrays in the endoplasmic reticulum
Journal of Cell Biology, 2002Co-Authors: Andrei V. Nikonov, Erik L. Snapp, Jennifer Lippincottschwartz, Gert KreibichAbstract:In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP–DAD1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where DAD1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP–DAD1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP–DAD1 in the ER membranes, but to a level that is still lower than that of free GFP–DAD1. This suggests that GFP–DAD1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains.
-
Active translocon complexes labeled with GFP–DAD1 diffuse slowly as large polysome arrays in the endoplasmic reticulum
The Journal of cell biology, 2002Co-Authors: Andrei V. Nikonov, Erik L. Snapp, Jennifer Lippincott-schwartz, Gert KreibichAbstract:In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP–DAD1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where DAD1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP–DAD1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP–DAD1 in the ER membranes, but to a level that is still lower than that of free GFP–DAD1. This suggests that GFP–DAD1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains.
-
DAD1 Is Required for the Function and the Structural Integrity of the Oligosaccharyltransferase Complex
The Journal of biological chemistry, 1998Co-Authors: Archana Sanjay, Gert KreibichAbstract:Asparagine-linked glycosylation is a highly conserved protein modification reaction that occurs in all eukaryotic organisms. The oligosaccharyltransferase (OST), which has its active site exposed on the luminal face of the endoplasmic reticulum (ER), catalyzes the transfer of preassembled high mannose oligosaccharides onto certain asparagine residues of nascent polypeptides. The mammalian OST complex was initially thought to be composed of three transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Most recently, a small integral membrane protein of 12 kDa called DAD1 has been identified as an additional member of the mammalian OST complex. A point mutation in the DAD1 gene is responsible for the temperature-sensitive phenotype of a baby hamster kidney-derived cell line (tsBN7) that undergoes apoptosis at the non-permissive temperature. Furthermore, the mutant protein DAD1 is not detectable in tsBN7 cells 6 h after shifting the cells to the non-permissive temperature. This temperature-sensitive cell line offered unique opportunities to study the effects caused by the loss of one OST subunit on the other three subunits and also on N-linked glycosylation. Western blot analysis of cell lysates showed that after 6 h at the non-permissive temperature, steady-state levels of the ribophorins were reduced by about 50%, and OST48 was barely detectable. On the other hand, steady-state levels of other components of the rough ER, such as the alpha-subunits of the TRAP (translocon-associated membrane protein) and the Sec61 complex, which are components of the translocation apparatus, are not affected by the instability of the OST subunits. Furthermore, N-glycosylation of the ribophorins was seriously affected 6 h after shifting the cells to the non-permissive temperature, and after 12 h they were synthesized only in the non-glycosylated form. As may be expected, this defect in the OST complex at the non-permissive temperature caused also the underglycosylation of a secretory glycoprotein. We concluded that degradation of DAD1 at the non-permissive temperature not only affects the stability of OST48 and the ribophorins but also results in the functional inactivation of the OST complex.
-
INTERACTIONS AMONG SUBUNITS OF THE OLIGOSACCHARYLTRANSFERASE COMPLEX
The Journal of biological chemistry, 1997Co-Authors: Mindong Ren, Gert KreibichAbstract:The mammalian oligosaccharyltransferase (OST) is an oligomeric complex composed of three membrane proteins of the endoplasmic reticulum: ribophorin I (RI), ribophorin II (RII), and OST48. In addition, sequence homology between the Ost2 subunit of the yeast OST complex and DAD1 (defender against apoptotic death) suggests that DAD1 may represent a fourth subunit of the mammalian OST complex. In attempts to elucidate the structural organization of this complex, we have studied the interactions among its subunits. Using the yeast two-hybrid system, we have shown that the luminal domains of RI and RII (RIL and RIIL, respectively) interacted with the luminal domain of OST48 (OST48L), but no direct interaction was observed between RIL and RIIL. These results were confirmed by biochemical assays. Deletion analyses using the yeast two-hybrid system showed that subdomain of RIL or RIIL adjacent to the respective transmembrane domains interacted with OST48L. Of the three equal length subdomains of OST48L, the one at the N terminus and the one next to the transmembrane domain interacted with RIL. None of these three subdomains of OST48L interacted with RIIL. The yeast two-hybrid assay also revealed affinity between the cytoplasmically located N-terminal region of DAD1 and the short cytoplasmic tail of OST48, thus placing DAD1 firmly into the OST complex. In addition, we found a homotypic interaction between the cytoplasmic domains of RI, which may play a role in the formation of the oligomeric array formed by components of the translocation machinery.
Andrei V. Nikonov - One of the best experts on this subject based on the ideXlab platform.
-
Active translocon complexes labeled with GFP–DAD1 diffuse slowly as large polysome arrays in the endoplasmic reticulum
The Journal of cell biology, 2002Co-Authors: Andrei V. Nikonov, Erik L. Snapp, Jennifer Lippincott-schwartz, Gert KreibichAbstract:In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP–DAD1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where DAD1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP–DAD1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP–DAD1 in the ER membranes, but to a level that is still lower than that of free GFP–DAD1. This suggests that GFP–DAD1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains.
-
active translocon complexes labeled with gfp DAD1 diffuse slowly as large polysome arrays in the endoplasmic reticulum
Journal of Cell Biology, 2002Co-Authors: Andrei V. Nikonov, Erik L. Snapp, Jennifer Lippincottschwartz, Gert KreibichAbstract:In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP–DAD1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where DAD1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP–DAD1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP–DAD1 in the ER membranes, but to a level that is still lower than that of free GFP–DAD1. This suggests that GFP–DAD1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains.
Ines Ingeborg Kubigsteltig - One of the best experts on this subject based on the ideXlab platform.
-
Involvement of DAD1-like lipases in response to salt and osmotic stress in Arabidopsis thaliana.
Plant signaling & behavior, 2010Co-Authors: Dorothea Ellinger, Ines Ingeborg KubigsteltigAbstract:Acyl hydrolases remodel biological membranes and release signaling molecules in response to a variety of biotic and abiotic stresses. After wounding or pathogen treatment lipases are necessary to release fatty acids as substrate for jasmonate biosynthesis. In osmotic stressed tissue they maintain integrity and functionality of membranes and during senescence lipases destroy and recycle membranes. Recently the role of several acyl hydrolases including DEFECTIVE IN ANTHER DEHISCENCE (DAD1) and DAD1-like lipase, e.g. DONGLE (DGL) and the phospholipase A (PLA) PLA-Iγ1 in jasmonate biosynthesis after wounding were investigated and functional redundancy within this family has been stated. Here we report necessity of diverse DAD1-like lipases in response to salt and sorbitol treatment. The lipase PLA-Iγ1 and PLA-Iβ2, which were both impaired in wound response, were also affected in response to osmotic stress in seed germination assays. Based on our observations and interpretations of transcription analyses gener...
-
dongle and defective in anther dehiscence1 lipases are not essential for wound and pathogen induced jasmonate biosynthesis redundant lipases contribute to jasmonate formation
Plant Physiology, 2010Co-Authors: Dorothea Ellinger, Nadja Stingl, Ines Ingeborg Kubigsteltig, Thomas Bals, Melanie Juenger, Stephan Pollmann, Susanne Berger, Danja Schuenemann, Martin J MuellerAbstract:Lipases are involved in the generation of jasmonates, which regulate responses to biotic and abiotic stresses. Two sn-1-specific acyl hydrolases, DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) and DONGLE (DGL), have been reported to be localized in plastids and to be essential and sufficient for jasmonate biosynthesis in Arabidopsis (Arabidopsis thaliana) leaves. Here, we show that levels of 12-oxo-phytodienoic acid (OPDA) and jasmonic acid in three different DGL RNA interference lines and the DAD1 mutant were similar to wild-type levels during the early wound response as well as after Pseudomonas infection. Due to the lack of sn-2 substrate specificity, synthesis of dinor OPDA was not expected and also not found to be affected in DGL knockdown and DGL-overexpressing lines. As reported, DAD1 participates in jasmonate formation only in the late wound response. In addition, DGL protein was found to be localized in lipid bodies and not in plastids. Furthermore, jasmonate levels in 16 additional mutants defective in the expression of lipases with predicted chloroplast localization did not show strong differences from wild-type levels after wounding, except for a phospholipase A (PLA) PLA-Iγ1 (At1g06800) mutant line that displayed diminished wound-induced dinor OPDA, OPDA, and jasmonic acid levels. A quadruple mutant defective in four DAD1-like lipases displayed similar jasmonate levels as the mutant line of PLA-Iγ1 after wounding. Hence, we identify PLA-Iγ1 as a novel target gene to manipulate jasmonate biosynthesis. Our results suggest that, in addition to DAD1 and PLA-Iγ1, still unidentified enzymes with sn-1 and sn-2 hydrolase activity are involved in wound- and pathogen-induced jasmonate formation, indicating functional redundancy within the lipase family.
Reid Gilmore - One of the best experts on this subject based on the ideXlab platform.
-
DAD1, THE DEFENDER AGAINST APOPTOTIC CELL DEATH, IS A SUBUNIT OF THE MAMMALIAN OLIGOSACCHARYLTRANSFERASE
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Daniel J. Kelleher, Reid GilmoreAbstract:DAD1, the defender against apoptotic cell death, was initially identified as a negative regulator of programmed cell death in the BHK21-derived tsBN7 cell line. Of interest, the 12.5-kDa DAD1 protein is 40% identical in sequence to Ost2p, the 16-kDa subunit of the yeast oligosaccharyltransferase (OST). Although the latter observation suggests that DAD1 may be a mammalian OST subunit, biochemical evidence to support this hypothesis has not been reported. Previously, we showed that canine OST activity is associated with an oligomeric complex of ribophorin I, ribophorin II, and OST48. Here, we demonstrate that DAD1 is a tightly associated subunit of the OST both in the intact membrane and in the purified enzyme. Sedimentation velocity analyses of detergent-solubilized WI38 cells and canine rough microsomes show that DAD1 cosediments precisely with OST activity and with the ribophorins and OST48. Radioiodination of the purified OST reveals that DAD1 is present in roughly equimolar amounts relative to the other subunits. DAD1 can be crosslinked to OST48 in intact microsomes with dithiobis(succinimidylpropionate). Crosslinked ribophorin II–OST48 heterodimers, DAD1–ribophorin II–OST48 heterotrimers and DAD1–ribophorin I–ribophorin II–OST48 heterotetramers also were detected. The demonstration that DAD1 is a subunit of the OST suggests that induction of a cell death pathway upon loss of DAD1 in the tsBN7 cell line reflects the essential nature of N-linked glycosylation in eukaryotes.
Dorothea Ellinger - One of the best experts on this subject based on the ideXlab platform.
-
Involvement of DAD1-like lipases in response to salt and osmotic stress in Arabidopsis thaliana.
Plant signaling & behavior, 2010Co-Authors: Dorothea Ellinger, Ines Ingeborg KubigsteltigAbstract:Acyl hydrolases remodel biological membranes and release signaling molecules in response to a variety of biotic and abiotic stresses. After wounding or pathogen treatment lipases are necessary to release fatty acids as substrate for jasmonate biosynthesis. In osmotic stressed tissue they maintain integrity and functionality of membranes and during senescence lipases destroy and recycle membranes. Recently the role of several acyl hydrolases including DEFECTIVE IN ANTHER DEHISCENCE (DAD1) and DAD1-like lipase, e.g. DONGLE (DGL) and the phospholipase A (PLA) PLA-Iγ1 in jasmonate biosynthesis after wounding were investigated and functional redundancy within this family has been stated. Here we report necessity of diverse DAD1-like lipases in response to salt and sorbitol treatment. The lipase PLA-Iγ1 and PLA-Iβ2, which were both impaired in wound response, were also affected in response to osmotic stress in seed germination assays. Based on our observations and interpretations of transcription analyses gener...
-
dongle and defective in anther dehiscence1 lipases are not essential for wound and pathogen induced jasmonate biosynthesis redundant lipases contribute to jasmonate formation
Plant Physiology, 2010Co-Authors: Dorothea Ellinger, Nadja Stingl, Ines Ingeborg Kubigsteltig, Thomas Bals, Melanie Juenger, Stephan Pollmann, Susanne Berger, Danja Schuenemann, Martin J MuellerAbstract:Lipases are involved in the generation of jasmonates, which regulate responses to biotic and abiotic stresses. Two sn-1-specific acyl hydrolases, DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) and DONGLE (DGL), have been reported to be localized in plastids and to be essential and sufficient for jasmonate biosynthesis in Arabidopsis (Arabidopsis thaliana) leaves. Here, we show that levels of 12-oxo-phytodienoic acid (OPDA) and jasmonic acid in three different DGL RNA interference lines and the DAD1 mutant were similar to wild-type levels during the early wound response as well as after Pseudomonas infection. Due to the lack of sn-2 substrate specificity, synthesis of dinor OPDA was not expected and also not found to be affected in DGL knockdown and DGL-overexpressing lines. As reported, DAD1 participates in jasmonate formation only in the late wound response. In addition, DGL protein was found to be localized in lipid bodies and not in plastids. Furthermore, jasmonate levels in 16 additional mutants defective in the expression of lipases with predicted chloroplast localization did not show strong differences from wild-type levels after wounding, except for a phospholipase A (PLA) PLA-Iγ1 (At1g06800) mutant line that displayed diminished wound-induced dinor OPDA, OPDA, and jasmonic acid levels. A quadruple mutant defective in four DAD1-like lipases displayed similar jasmonate levels as the mutant line of PLA-Iγ1 after wounding. Hence, we identify PLA-Iγ1 as a novel target gene to manipulate jasmonate biosynthesis. Our results suggest that, in addition to DAD1 and PLA-Iγ1, still unidentified enzymes with sn-1 and sn-2 hydrolase activity are involved in wound- and pathogen-induced jasmonate formation, indicating functional redundancy within the lipase family.
