Presence Of Canine Microsomes Research Articles

Endomannosidase undergoes phosphorylation in the Golgi apparatus

Glucose residues from N-linked oligosaccharides are removed by glucosidases I and II in the endoplasmic reticulum (ER) or by the alternate endomannosidase pathway in the Golgi apparatus. Our morphological analysis demonstrates that recombinant rat endomannosidase exhibited a cis- and medial-Golgi localization alike the endogenous enzyme and its ER to Golgi transport is COP II mediated. Recombinant endomannosidase undergoes a posttranslational modification, which is not related to N-or O-glycosylation. A shift in molecular mass of recombinant endomannosidase was observed upon phosphatase digestion but not for ER-retained CHO cell endomannosidase. Furthermore, immunoprecipitation of (35)S- and (33)P-labeled endomannosidase expressed in CHO-K1 cells suggests that recombinant endomannosidase undergoes phosphorylation. Substitution of the single cytoplasmic threonine residue of rat endomannosidase by either an alanine or valine residue resulted in the same posttranslational modification alike the wild-type enzyme. The subcellular localization and the in vivo activity of the mutant endomannosidase were not affected. Thus, endomannosidase phosphorylation is occurring in luminal sequences. Modification was prevented when endomannosidase was synthesized using reticulocyte lysates in the presence of canine microsomes. Treatment of cells with brefeldin A blocked the posttranslational modification of endomannosidase, suggesting that phosphorylation is occurring in the Golgi apparatus, the residence of endomannosidase.

Critical roles for p22phox in the structural maturation and subcellular targeting of Nox3

Otoconia are small biominerals in the inner ear that are indispensable for the normal perception of gravity and motion. Normal otoconia biogenesis requires Nox3, a Nox (NADPH oxidase) highly expressed in the vestibular system. In HEK-293 cells (human embryonic kidney cells) transfected with the Nox regulatory subunits NoxO1 (Nox organizer 1) and NoxA1 (Nox activator 1), functional murine Nox3 was expressed in the plasma membrane and exhibited a haem spectrum identical with that of Nox2, the electron transferase of the phagocyte Nox. In vitro Nox3 cDNA expressed an approximately 50 kDa primary translation product that underwent N-linked glycosylation in the presence of canine microsomes. RNAi (RNA interference)-mediated reduction of endogenous p22phox, a subunit essential for stabilization of Nox2 in phagocytes, decreased Nox3 activity in reconstituted HEK-293 cells. p22phox co-precipitated not only with Nox3 and NoxO1 from transfectants expressing all three proteins, but also with NoxO1 in the absence of Nox3, indicating that p22phox physically associated with both Nox3 and with NoxO1. The plasma membrane localization of Nox3 but not of NoxO1 required p22phox. Moreover, the glycosylation and maturation of Nox3 required p22phox expression, suggesting that p22phox was required for the proper biosynthesis and function of Nox3. Taken together, these studies demonstrate critical roles for p22phox at several distinct points in the maturation and assembly of a functionally competent Nox3 in the plasma membrane.

Expression, purification, and evidence for the interaction of the two nucleotide-binding folds of the sulphonylurea receptor

The ATP-sensitive potassium channel is made up of four pore forming Kir6.2 subunits, surrounded by four regulatory sulphonylurea receptor (SUR) subunits. The latter subunit contains two nucleotide-binding folds (NBFs) that confer the ability on the channel to sense changes in the metabolic status ([ATP]/[ADP]) of the cell and couple the changes to the membrane potential of the cell. In an attempt to better understand the mechanisms by which NBFs influence the activity of the channel, we have expressed the NBF domains with C-terminally added epitopes (FLAG to NBF1 and His 6 to NBF2) in Escherichia coli and the rabbit reticulocyte lysate system and examined the ability of these domains to interact with each other and with Kir6.2. Both NBFs could be expressed to high levels in E. coli and purified to homogeneity from inclusion bodies. Re-folding of the proteins proved to be unsuccessful. However, we were able to obtain small amounts of radio-labelled NBFs in a soluble state. Using co-immunoprecipitation, we demonstrate that the radio-labelled NBF1 and NBF2 interact with each other. Neither of the NBFs bound to Kir6.2 expressed in the presence of canine microsomes.

Berberine bridge enzyme, a key branch-point enzyme in benzylisoquinoline alkaloid biosynthesis, contains a vacuolar sorting determinant.

In opium poppy (Papaver somniferum L.), (S)-reticuline is the last common intermediate in sanguinarine and morphine biosynthesis. Sanguinarine accumulates in the vacuole of cultured opium poppy cells in response to treatment with fungal elicitors. The first committed step in sanguinarine biosynthesis is catalyzed by the berberine bridge enzyme (BBE), which converts (S)-reticuline to (S)-scoulerine. An N-terminal signal peptide and novel vacuolar sorting determinant were identified and characterized in BBE. In vitro translation of BBE mRNA in the presence of canine pancreatic microsomes produced a glycosylated, proteolysis-resistant protein, confirming the existence of a signal peptide. Transcripts encoding a BBE N-terminal deletion series fused to beta-glucuronidase or green fluorescent protein (GFP) were also translated in the presence of canine microsomes, and introduced into cultured opium poppy cells via microprojectile bombardment. The signal peptide was restricted to the first 25 amino acids and shown to initially target BBE to the endoplasmic reticulum. Fusion of 50 N-terminal residues from BBE to GFP resulted in the localization of the reporter to the vacuole. GFP was also sorted to the vacuole when fused to a heterologous N-terminal signal peptide followed by BBE amino acids 26-50. The BBE vacuolar sorting determinant was further localized between residues 26 and 41 by deletion analysis. The final subcellular destination of BBE is consistent with the vacuolar sequestration of sanguinarine. However, the vacuolar pH is below the functional range for BBE, suggesting that the enzyme is active only prior to its entry into the vacuole.

A topological study of the human γ-glutamyl carboxylase

γ-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K–dependent posttranslational modification of glutamate to γ-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

A topological study of the human γ-glutamyl carboxylase

Abstractγ-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K–dependent posttranslational modification of glutamate to γ-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

Expression cloning of the early activation antigen CD69, a type II integral membrane protein with a C-type lectin domain.

CD69 is a very early activation Ag of T lymphocytes. It is a cell surface glycoprotein that can only be detected after stimulation of lymphocytes. Despite extensive studies on its biologic activities, little is known about its molecular function. To investigate the latter in more detail, we have cloned a cDNA encoding CD69 on the basis of its expression in COS cells. The nucleotide sequence of clone CD69.13 is 1676 bp in length and contains a single open reading frame of 600 bp encoding a protein of 199 amino acids. The predicted molecular mass of 22,559 Da could be confirmed by in vitro translation. The protein contains a hydrophobic transmembrane region between amino acids 41 and 61 but no N-terminal signal peptide, which suggests that it is a type II membrane protein. It has one potential N-glycosylation site at amino acid 166. Two glycosylated forms of 26 to 28 kDa and 32 to 34 kDa were detected both in transfected COS cells and in in vitro translation in the presence of canine microsomes. Proteinase K degradation of the N-terminal part after in vitro protein synthesis supports the view of CD69 being a type II integral membrane protein with the N-terminal 40 amino acids in the cytoplasm, a transmembrane domain of 21 amino acids, and C-terminal 138 amino acids as the extracellular domain. Homology searches revealed sequence similarity with members of a supergene family of type II integral membrane proteins with a C-type lectin domain, indicating that CD69 is involved in signal transduction.

Cotranslational Integration of Soybean (Glycine max) Oil Body Membrane Protein Oleosin into Microsomal Membranes.

Storage triglycerides in oil seeds are sequestered in discrete organelles termed oil bodies. They are bounded by a monolayer of phospholipids in which a few distinct proteins (oleosins) are embedded. Synthesis of soybean (Glycine max) 24-kD oleosin was analyzed by in vitro transcription and translation in reticulocyte lysate in the presence of canine microsomes. Our results show that 24-kD oleosin is cotranslationally integrated into microsomal membranes. We demonstrate that oleosin is integrated into a bilayer membrane in preference to the oil body monolayer membrane, indicating that oleosin is synthesized on the endoplasmic reticulum (ER). A new model of oil body assembly involving a conformational change through initial association with the ER membrane is proposed.