Topological and Enzymatic Analysis of Human Alg2 Mannosyltransferase

2021 ◽  
Author(s):  
Meng-Hai Xiang ◽  
Xin-Xin Xu ◽  
Chun-Di Wang ◽  
Shuai Chen ◽  
Si Xu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Biosynthetic Pathway ◽  
Membrane Binding ◽  
Binding Domain ◽  
Enzymatic Analysis ◽  
Kinetic Assay ◽  
Physiological Conditions ◽  
Single Membrane ◽  
Er Membrane

Abstract N-glycosylation starts with the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum. Alg2 mannosyltransferase adds both the α1,3- and α1,6-Man onto ManGlcNAc2-pyrophosphate-dolichol (M1Gn2-PDol) in either order to generate the branched M3Gn2-PDol product. The well-studied yeast Alg2 interacts with ER membrane through four hydrophobic domains. Unexpectedly, we show that Alg2 structure has diverged significantly between yeast and humans. Human Alg2 (hAlg2) associates with the ER via a single membrane-binding domain and is markedly more stable in vitro. These properties were exploited to develop an LC-MS quantitative kinetic assay for studying purified hAlg2. Under physiological conditions, hAlg2 prefers to transfer α1,3-Man on to M1Gn2 before adding the α1,6-Man. However, this bias is altered by an excess of GDP-Man donor or an increased level of M1Gn2 substrate, both of which trigger production of the M2Gn2 (α-1,6)-PDol. These results suggest that Alg2 may regulate the LLO biosynthetic pathway by controlling accumulation of M2Gn2 (α-1,6) intermediate.

scholarly journals TheSaccharomyces cerevisiaePrenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (8) ◽  
pp. 2231-2247 ◽  
Author(s):  
Julia D. Romano ◽  
Walter K. Schmidt ◽  
Susan Michaelis
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Fractionation ◽  
Endoplasmic Reticulum Membrane ◽  
Carboxyl Methylation ◽  
C Terminus ◽  
Eukaryotic Proteins ◽  
Internal Site ◽  
Membrane Spanning ◽  
Er Membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.

scholarly journals In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum.

1995 ◽  
Vol 6 (3) ◽  
pp. 283-296 ◽  
Author(s):  
L M Hendershot ◽  
J Y Wei ◽  
J R Gaut ◽  
B Lawson ◽  
P J Freiden ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Binding ◽  
Point Mutations ◽  
Binding Domain ◽  
Dominant Negative ◽  
Atp Binding ◽  
Heavy Chains ◽  
In Vivo Expression

BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo.

scholarly journals FIT2 is a lipid phosphate phosphatase crucial for endoplasmic reticulum homeostasis

2018 ◽  
Author(s):  
Michel Becuwe ◽  
Laura M. Bond ◽  
Niklas Mejhert ◽  
Sebastian Boland ◽  
Shane D. Elliott ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Morphological Changes ◽  
Normal Structure ◽  
Membrane Lipid ◽  
Molecular Function ◽  
Key Factor ◽  
Lipid Phosphate ◽  
Lipid Abnormalities ◽  
Er Membrane

SUMMARYThe endoplasmic reticulum (ER) protein Fat-Induced Transcript 2 (FIT2) has emerged as a key factor in lipid droplet (LD) formation, although its molecular function is unknown. Highlighting its importance, FIT2 orthologs are essential in worms and mice, and FIT2 deficiency causes a deafness/dystonia syndrome in humans. Here we show that FIT2 is a lipid phosphate phosphatase (LPP) enzyme that is required for maintaining the normal structure of the ER. Recombinant FIT2 exhibits LPP activityin vitroand loss of this activity in cells leads to ER membrane morphological changes and ER stress. Defects in LD formation in FIT2 depletion appear to be secondary to membrane lipid abnormalities, possibly due to alterations in phospholipids required for coating forming LDs. Our findings uncover an enzymatic role for FIT2 in ER lipid metabolism that is crucial for ER membrane homeostasis.

scholarly journals The membrane topological analysis of 3β-hydroxysteroid-Δ24 reductase (DHCR24) on endoplasmic reticulum

2011 ◽  
Vol 48 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Xiuli Lu ◽  
Yang Li ◽  
Jianli Liu ◽  
Xiangyu Cao ◽  
Xude Wang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Topological Analysis ◽  
Binding Domain ◽  
Full Length ◽  
Membrane Topology ◽  
Membrane Targeting ◽  
Ros Scavenging ◽  
Tm Domain ◽  
Fad Binding ◽  
Er Membrane

DHCR24encodes 3β-hydroxysteroid-Δ24 reductase, catalyzing the conversion of desmosterol to cholesterol. Our previous study demonstrated that DHCR24 exerts an anti-apoptotic function as a reactive oxygen species (ROS) scavenger, for which it needs its FAD-binding domain. The membrane topology of DHCR24 on endoplasmic reticulum (ER) and the functional significance of its FAD-binding domain are not completely understood. Based on the structure predicted by bioinformatics, we studied the membrane topology of DHCR24 in murine neuroblastoma cells (N2A), using the fluorescent protease protection (FPP) technique. We showed that full-length DHCR24 is localized to the membrane of ER, whereas the predicted transmembrane (TM) domain-deleted DHCR24 mutation is localized to the cytoplasm. The change of DHCR24 localization suggests that the N-terminal TM domain is essential for the ER membrane targeting of DHCR24. The FPP assay demonstrated the membrane topology of DHCR24 with an N-terminal luminal/C-terminal cytoplasmic orientation. Measurement of intracellular ROS using H2DCFDA revealed that the ROS levels of cells infected by plasmids driving expression of full-length DHCR24 or the TM domain-deleted DHCR24 mutation after H2O2exposure were lower than those of control cells, suggesting that the ER membrane targeting of DHCR24 is not required for its enzymatic ROS scavenging activity. Confocal fluorescence microscopy revealed that the DHCR24-overexpressed cells were protected from apoptosis in response to oxidative stress, which was accompanied by a decrease in DHCR24 content on the ER and activation of caspase-3, suggesting that the anti-apoptotic function of DHCR24 is associated with its cleavage by caspase.

scholarly journals c-Fos activates and physically interacts with specific enzymes of the pathway of synthesis of polyphosphoinositides

2011 ◽  
Vol 22 (24) ◽  
pp. 4716-4725 ◽  
Author(s):  
Adolfo R. Alfonso Pecchio ◽  
Andrés M. Cardozo Gizzi ◽  
Marianne L. Renner ◽  
María Molina-Calavita ◽  
Beatriz L. Caputto
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Biosynthetic Pathway ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Physical Interaction ◽  
Quiescent Cells ◽  
No Activation ◽  
Specific Lipid

The oncoprotein c-Fos is a well-recognized AP-1 transcription factor. In addition, this protein associates with the endoplasmic reticulum and activates the synthesis of phospholipids. However, the mechanism by which c-Fos stimulates the synthesis of phospholipids in general and the specific lipid pathways activated are unknown. Here we show that induction of quiescent cells to reenter growth promotes an increase in the labeling of polyphosphoinositides that depends on the expression of c-Fos. We also investigated whether stimulation by c-Fos of the synthesis of phosphatidylinositol and its phosphorylated derivatives depends on the activation of enzymes of the phosphatidylinositolphosphate biosynthetic pathway. We found that c-Fos activates CDP-diacylglycerol synthase and phosphatidylinositol (PtdIns) 4-kinase II α in vitro, whereas no activation of phosphatidylinositol synthase or of PtdIns 4-kinase II β was observed. Both coimmunoprecipitation and fluorescence resonance energy transfer experiments consistently showed a physical interaction between the N-terminal domain of c-Fos and the enzymes it activates.

scholarly journals Analysis of the N-terminal binding domain of Goα

1997 ◽  
Vol 328 (1) ◽  
pp. 23-31 ◽  
Author(s):  
Liliana BUSCONI ◽  
M. Bradley DENKER
Keyword(s):  
Amino Acids ◽  
Cultured Cells ◽  
Membrane Binding ◽  
Receptor Activation ◽  
Binding Domain ◽  
Membrane Receptors ◽  
Membrane Interactions ◽  
Cos Cells ◽  
Vitro Analysis

Signalling from membrane receptors through heterotrimeric G-proteins (Gα and Gβγ) to intracellular effectors is a highly regulated process. Receptor activation causes exchange of GTP for GDP on Gα and dissociation of Gα from Gβγ. Both subunits remain membrane-associated and interact with a series of other molecules throughout the cycle of activation. The N-terminal binding domain of Gα subunits interacts with the membrane by several partially defined mechanisms: the anchoring of Gα to the more hydrophobic Gβγ subunits, the interaction of N-terminal lipids (palmitate and/or myristate) with the membrane, and attachment of amino acid regions to the membrane {amino acids 11-14 of Goα (D[11-14]); Busconi, Boutin and Denker (1997) Biochem. J. 323, 239-244}. We characterized N-terminal mutants of Goα with known Gβγ-binding properties for the ability to interact with phospholipid vesicles and membranes prepared from cultured cells (acceptor membranes). In vitro analysis allows membrane interactions that are important to the activated and depalmitoylated state of Gα to be characterized. Subcellular localization was also determined in transiently transfected COS cells. All of the mutant proteins are myristoylated, and differences in myristoylation do not account for changes in membrane binding. Disrupting the N-terminal α-helix of Goα with a proline point mutation at Arg-9 (R9P) does not affect interactions with Gβγ on sucrose-density gradients but significantly reduces acceptor membrane binding. Deletion of amino acids 6-15 (D[6-15]; reduced Gβγ binding) or deletion of amino acids 3-21 (D[3-21]); no detectable Gβγ binding) further reduces acceptor membrane binding. When expressed in COS cells, R9P and D[6-15] are localized in the membrane similar to wild-type Goα as a result of the contribution from palmitoylation. In contrast, D[3-21] is completely soluble in COS cells, and no palmitoylation is detected. The binding of Goα and mutants translated in vitro to liposomes indicates that Goα preferentially binds to neutral phospholipids (phosphatidylcholine). R9P and D[11-14] bind to phosphatidylcholine liposomes like Goα, but D[6-15] exhibits no detectable binding. Taken together, these studies suggest that interactions of the N-terminus of Gα subunits with the membrane may be affected by both membrane proteins and lipids. A detailed understanding of Gα-membrane interactions may reveal unique mechanisms for regulating signal transduction.

scholarly journals Mitochondria supply ATP to the ER through a mechanism antagonized by cytosolic Ca2+

2019 ◽  
Author(s):  
Jing Yong ◽  
Helmut Bischof ◽  
Marina Siirin ◽  
Anne Murphy ◽  
Roland Malli ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Warburg Effect ◽  
Transport Process ◽  
Protein Misfolding ◽  
Atp Hydrolysis ◽  
Atp Level ◽  
Physiological Conditions ◽  
Atp Supply ◽  
Atp Regeneration ◽  
Er Membrane

AbstractThe endoplasmic reticulum (ER) imports ATP and uses energy from ATP hydrolysis for protein folding and trafficking. However, little is known about this vital ATP transport process across the ER membrane. Here, using three commonly used cell lines (CHO, INS1 and HeLa), we report that ATP enters the ER lumen through a cytosolic Ca2+-antagonized mechanism, or CaATiER (Ca2+-Antagonized Transport into ER) mechanism for brevity. Significantly, we observed that a Ca2+ gradient across the ER membrane is necessary for ATP transport into the ER. Therefore Ca2+ signaling in the cytosol is inevitably coupled with ATP supply to the ER. We propose that under physiological conditions, cytosolic Ca2+ inhibits ATP import into the ER lumen to limit ER ATP consumption. Furthermore, the ATP level in the ER is readily depleted by oxidative phosphorylation (OxPhos) inhibitors, and that ER protein misfolding increases ATP trafficking from mitochondria into the ER. These findings suggest that ATP usage in the ER may increase mitochondrial OxPhos while decreasing glycolysis, i.e., an “anti-Warburg” effect.Significance StatementWe report that ATP enters the ER lumen through an AXER-dependent, cytosolic Ca2+-antagonized mechanism, or CaATiER (Ca2+-Antagonized Transport into ER) mechanism. In addition, our findings suggest that ATP usage in the ER may render an “anti-Warburg” effect by increasing ATP regeneration from mitochondrial OxPhos while decreasing the portion of ATP regeneration from glycolysis.

scholarly journals The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro

1975 ◽  
Vol 146 (3) ◽  
pp. 513-526 ◽  
Author(s):  
T K Shires ◽  
C M McLaughlin ◽  
H C Pitot
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Binding Sites ◽  
Large Subunit ◽  
Relative Concentration ◽  
Membrane Binding ◽  
Ribosomal Subunits ◽  
Derivatives Of ◽  
Membrane Binding Sites

Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.

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