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Results for “protein translocation”

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At least 19 recordsLinked to original sources
PubMed1977

Evidence for contractile protein translocation in macrophage spreading, phagocytosis, and phagolysosome formation.

Macrophage pseudopodia that surround objects during phagocytosis contain a meshwork of actin filaments and exclude organelles. Between these pseudopodia at the base of developing phagosomes, the organelle exclusion ceases, and lysosomes enter the cell periphery to fuse with the phagosomes. Macrophages also extend hyaline pseudopodia on the surface of nylon wool fibers and secrete lysosomal enzymes into the extracellular medium instead of into phagosomes. To analyze biochemically these concurrent alterations in cytoplasmic architecture, we allowed rabbit lung macrophages to spread on nylon wool fibers and then subjected the adherent cells to shear. This procedure caused the selective release of beta-glucoronidase into the extracellular medium and yielded two fractions, cell bodies and isolated pseudopod blebs resembling podosomes, which are plasma-lemma-bounded sacs of cortical cytoplasm. Cytoplasmic extracts of the cell bodies eluted from nylon fibers contained two-thirds less actin-binding protein and myosin, and approximately 20 percent less actin and two-thirds of the other two proteins were accounted for in podosomes. The alterations in protein composition correlated with assays of myosin-associated EDTA-activated adenosine triphosphatase activity, and with a diminution in the capacity of extracts of nylon wool fiber-treated cell bodies to gel, a property dependent on the interaction between actin-binding protein and F-actin. However, the capacity of the remaining actin in cell bodies to polymerize did not change. We propose that actin-binding protein and myosin are concentrated in the cell cortex and particularly in pseudopodia where prominent gelation and syneresis of actin occur. Actin in the regions from which actin-binding protein and myosin are displaced disaggregates without depolymerizing, permitting lysosomes to gain access to the plasmalemma. Translocation of contractile proteins could therefore account for the concomitant differences in organelle exclusion that characterize phagocytosis.

Actins↗
PubMed1979

Subcellular particles involved in the translocation of proteins in rat brain.

Protein translocation systems which are inhibited by vinblastine, colchicine, and low calcium concentrations have been found in the cells of the brain slice. The early steps in the translocation pathways of newly synthesized protein have been studied by use of a double-label experiment in conjunction with subcellular fractionation. Certain subcellular particles have been positioned on the pathways with reference to vinblastine-sensitive translocation steps. There appears to be many subcellular organelles that are located downstream from a vinblastine-sensitive translocation step and which receive significant quantities of translocated protein within an hour of its synthesis. Some of these organelles co-enrich with the enzyme marker 5'-AMPase. Myelinated axons, Golgi derived vesicles, and smooth and rough endoplasmic reticulum all are enriched in fractions which contain a net vinblastine-sensitive importation of protein. The major particles, which lie upstream from a vinblastine-sensitive translocation step and are net exporters of protein on this system, are found in a brain capillary fraction. It is suggested that the most likely exporter present in these capillaries are the end feet of astrocyte glial cells.

Animals↗
PubMed1976

Enhanced template activity in chromatin from adrenal medulla after phosphorylation of chromosomal proteins.

Translocation of protein kinase to the nucleus had been implicated earlier in the transsynaptic control of gene expression mediated by cholinergic nerves in adrenal medulla. Phosphorylation of chromosomal proteins by adenosine 3',5'-monophosphate-dependent protein kinase and adenosine 3',5'-monophosphate enhances the template activity of chromatin from adrenal medulla. When homologous RNA polymerase II is used the relative activation is greater than that obtained with Escherichia coli RNA polymerase. The substrate for such phosphorylation does not seem to be RNA polymerase II. Phosphorylation of specific acidic protein probably mediates this enhancement of template activity.

Adenosine Triphosphate↗
PubMed1979

Translocation of proteins across membranes: the signal hypothesis and beyond.

Proteins are translocated across membranes either coupled to translation (co-translationally) or after translation (post-translationally). The information for both modes of translocation is encoded in the protein in the form of a short-lived sequence extension (signal sequence). Additional information resides in the ribosome in the case of co-translational translocation, which proceeds via a ribosome--membrane junction. Translocation is mediated by specific receptors (ribosome and/or signal receptors) which are restricted in their location to distinct cellular membranes. In most cases the signal sequence is removed by a signal peptidase operating in an endoproteolytic mode. Membranes endowed with receptors for co-translational translocation are: the rough endoplasmic reticulum (RER) including the outer nuclear envelope membrane, the inner mitochondrial membrane and the thylakoid membrane of chloroplasts, in eukaryotic cells; and the plasma membrane in prokaryotic cells. Each of these membranes presumably contains a single distinctive signal receptor, ribosome receptor and signal peptidase. Membranes endowed with one distinct receptor each for post-translational translocation are both mitochondrial membranes, the chloroplast envelope membrane and the peroxisomal membrane. A signal sequence for co-translational translocation across the RER membrane that is identical in its secondary structure is shared by secretory, lysosomal and certain bitopic integral membrane proteins. Some integral membrane proteins presumably share another common sequence--referred to as stop-transfer sequence--which serves to interrupt translocation and thereby to orient the polypeptide chain in the lipid bilayer. Furthermore, the existence of a few specific 'sorting' sequences is postulated. These would be common to many proteins and would serve to route them to their final destination following translocation across or orientation within the membrane. Thus, the topological information which determines the intracellular pathway and the final location of a great number of proteins appears to reside in a small repertoire of specific sequences which are either a transient or a permanent part of the protein.

Animals↗
PubMed1978

Cytosol protein-independent translocation of isomeric spin-labelled radioactive lipids from isolated guinea pig liver microsomal to mitochondrial membranes.

Intermembranous translocation of membrane-bound radioactive lipids covalently labelled with 5-, 12, and 16-doxyl stearic acid was studied. Guinea pig liver microsomal membranes containing known amounts of isomeric spin-labelled radioactive phosphatidic acid, phosphatidylcholine, and diglycerides were incubated with unlabelled mitochondria; reisolated mitochondria contained around 28-31% of microsomal labelled lipids above the microsomal contamination. The effect of adding crude or 'pH 5.1' 105 000 X g cytosol supernatant on the amount and composition of translocated labelled lipids was studied. While the translocation of labelled phosphatidylcholine was slightly stimulated by the addition of these cytosol supernatants, no significant increase of the amount of translocated labelled phosphatidic acic and diglycerides was observed by this addition. In view of these results, a probable mechanism for the cytosol protein-independent translocation of lipids between biological membranes is proposed.

Animals↗
PubMed1976

Activation and nuclear translocation of protein kinase during transsynaptic induction of tyrosine 3-monooxygenase.

The tyrosine-3-monooxygenase activity [L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] of rat adrenal medulla is induced 20-24 hr after the injection of reserpine (16 mumol/kg intraperitoneally). This and other inducing stimuli increase the 3': 5'-cyclic AMP (cAMP) content in the medulla for longer than 60 min and activate the cAMP-dependent protein kinase (ATP: protein phosphotransferase; EC 2.7.1.37) for several hours. Corticotropin (ACTH), dopamine, and propranolol do not induce the monooxygenase, but elicit an increase in the cAMP content of the medulla which fails to activate protein kinase and lasts less than 1 hr. A high- and low-molecular-weight protein kinase are separated by gel filtration from the 20,000 X g pellet extract of adrenal medulla homogenate. The activity of the low-molecular-weight enzyme is expressed as its ability to phosphorylate histone. The protein kinase activity of the pellet is increased between 3 and 17 hr after reserpine injection. Our evidence indicates that this increase is due to a translocation from cytosol to subcellular structures of a kinase that utilizes lysine-rich histone as phosphate acceptor. The protein kinase activity that is extracted from a purified nuclear fraction prepared from the adrenal medulla of rats injected 7 hr previously with reserpine is greater than that extracted from medulla of saline-treated rats.

Adrenal Medulla↗
PubMed1979

Trans-membrane translocation of proteins. The direct transfer model.

As a start towards a deeper understanding of the transmembrane transport of proteins, the transfer of a nascent chain through the lipophilic core of a membrane is discussed from a physico-chemical point of view. Some simple considerations of the energetics of protein structure, together with experimental data on the transfer process, form the basis for a detailed and quantifiable model, accounting for the extrusion of secreted proteins as well as for the insertion of trans-membrane proteins.

Amino Acid Sequence↗
PubMedDate not supplied

Studies on the mechanism of translocation in ribosomes. IV. The role of ribisomal proteins S12 in translocation.

It is shown that ribosomes, the 30S subparticles of which are reconstituted without protein S12, read out poly(U) and synthesize polyphenylalanine in the absence of protein elongation factors (EF-T and EF-G) and GTP, i.e. perform "non-enzymatic" translation. On the contrary, ribosomes, the 30S subparticles of which are reconstituted with protein S12, do not display "non-enzymatic" translation without its activation with parachloromercuribenzoate. This means that a complete removal of protein S12 from the ribosome, as well as its damage with para-chloromercuribenzoate, leads to the unblocking of the potential ability of ribosomes for spontaneous ("non-enzymatic") translocation. The presence of intact protein S12 in the ribosome prevents spontaneous (EF-G-GTP-independent) translocation. A suggestion is made that the intact protein S12 forms an additional contact between the ribosomal subparticles and thus participates in the ribosomal mechanism of translocation by affecting the locking-unlocking of the subparticles.

Escherichia coli↗
PubMed2026

Pairwise transmembrane domain insertion during multipass protein biogenesis.

The ∼2,500 multipass membrane proteins encoded in the human genome are constructed mostly or entirely of transmembrane domain (TMD) pairs: exceptionally common biosynthetic units comprised of two TMDs separated by a short non-cytosolic loop. It has long been thought that each TMD of a pair sequentially enters the lipid bilayer through a lateral gate in the Sec61 protein translocation channel. Here, we show that TMD pairs can access multiple insertion routes and that most are completely impervious to small-molecule blockade of Sec61's lateral gate. Obligate use of Sec61 is seen only for exceptional cases where the translocated loop exceeds ∼60 amino acids. TMD pairs with shorter loops typically use either EMC or GEL, insertase complexes of the universally conserved Oxa1 superfamily. Our results suggest that, contrary to long-held Sec61-based models, the fundamental biosynthetic unit of nearly all multipass membrane proteins uses the Oxa1 family for insertion.

Humans↗
PubMed1979

Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein.

Replication in vitro of the replicative form (RF) I DNA of bacteriophage varphiX174 requires the phage-induced cistron A (cisA) protein, the host rep protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The rep protein is a single-stranded DNA-dependent ATPase. In this paper we show that varphiX174 RF I DNA cut by the cisA protein acts as a duplex DNA cofactor for the rep protein ATPase activity, provided that DNA-binding protein is present. In this latter reaction the duplex DNA is unwound by the rep protein with concomitant hydrolysis of ATP. The extents of ATP hydrolysis, DNA unwinding, and, where appropriate, DNA synthesis are proportional to the amounts of DNA-binding protein present. Two ATP molecules are hydrolyzed per base pair unwound. We propose that the obligatory requirement for the cisA protein in the unwinding of varphiX174 RF I DNA is not simply due to its endonuclease activity but rather is due to its provision of a site for the binding of the rep protein. The rep protein in the presence of DNA-binding protein, but in the absence of cisA protein, unwinds duplex DNA when one strand extends to generate a single-stranded leader region preceding the duplex. We show that rep protein translocates along the leader single strand in a 5'-to-3' direction only and then invades the duplex DNA. The rep protein shows a directional specificity for translocation and unwinding. A model is presented to explain the mechanism of DNA unwinding catalyzed by the rep protein.

Adenosine Triphosphatases↗
PubMed1979

Resolution and reconstitution of active transport of calcium by a protein(s) from Mycobacterium phlei.

Membrane protein(s) responsible for the active transport of calcium in membrane vesicles from Mycobacterium phlei have been solubilized from membranes by sodium cholate treatment and partially purified using a hydrophobic resin. Reconstitution of calcium transport was demonstrated by reconstitution of detergent extracted membranes with the partially purified protein. The uptake of calcium in the reconstituted system was sensitive to proton-conducting uncouplers. Liposomes prepared with partially purified calcium translocating protein were capable of accumulating calcium. The uptake of calcium in this system occurred as a result of an artificial proton gradient generated by the reduction of entrapped ferricyanide with ascorbate-benzoquinone serving as a hydrogen carrier. The addition of the ionophore A23187 caused efflux of accumulated calcium in both native and proteoliposomal-reconstituted system.

Biological Transport, Active↗
PubMed1976

Purification of the carbodiimide-reactive protein component of the ATP energy-transducing system of Escherichia coli.

The ATP-energy transducing system in membranes of Escherichia coli is inhibited by dicyclohexylcarbodiimide. The protein component of this complex with which carbodiimides covalently react to inhibit function was previously identified by labeling wild type and dicyclohexylcarbodiimide-resistant mutants with dicyclohexyl[14C]carbodiimide (Fillingame, R. H. (1975) J. Bacteriol. 124, 870-883). This specific carbodiimide-reactive protein has now been purified. The protein was extracted from the membrane with chloroform:methanol and chromatographed on DEAE-cellulose and hydroxypropyl Spehadex G-50 in this sulvent mixture. The resultant 700-fold purification yielded a protein that was homogeneous on dodecyl sulfate-acrylamide gel electrophoresis and virtually free of phospholipid. It remained soluble in neutral chloroform:methanol throughout the purification procedure. The amino acid composition of the purified protein was extraordinary in that only 16% of the amino acids present could be considered polar. Histidine, serine, cysteine, and tryptophan were not found. Abnormally high contents of methionine, glycine, alanine, and leucine were present. One mole of lysine and threonine were found/mole of dicyclohexyl[14C]carbodiimide bound. The minimum molecular weight based on the amino acid composition was 8400. The specific carbodiimide-reactive protein has also been purified without prior modification by dicyclohexylcarbodiimide. The unmodified protein eluted from DEAE-cellulose at a higher salt concentration than the dicyclohexylcarbodiimide-modified form, which suggested that the reaction with the carbodiimide neutralized the negative charge. Only one-third of the total carbodiimide-reactive protein in the membrane was modified by dicyclohexylcarbodiimide under conditions which maximally inhibited adenosine triphosphatase activity. These results rais the possibility that the carbodiimide-reactive protein may be present as an oligomer in the energy-transducing complex. The purification of the unmodified carbodiimide-reactive protein should permit assessment of tis biological function, particularly its role in the protein-translocation process that is catalyzed by this energy-transducing complex.

Adenosine Triphosphate↗
PubMed1979

Translocation of cytosol protein kinase into nuclei and the induction of tyrosine hydroxylase in NBD-2 neuroblastoma cells.

Exposure of neuroblastoma cells (NBD-2) to 8-bromo-adenosine 3',5'-cyclic monophosphate (0.2-1.0 mM) (8-Br-cAMP) for 15 min caused a long term increase in the Vmax of tyrosine-3-monooxygenase activity (TH) beginning about 1 day after 8-Br-cAMP application. Cyclic AMP-dependent histone kinase was maximally activated in about 30 min and stayed activated above pretreatment levels for one hour. In cells exposed to 8-Br-cAMP for 15 min, separation of soluble and particle bound histone kinase showed that the total histone kinase activity in the soluble fraction decreased by 40%. This decrease was accompanied by an increase in protein kinase activity in the particulate fraction, suggesting enzyme translocation. After translocation, the enzyme appears to acquire a different substrate affinity because it prefers as a PO43- acceptor, acidic protein rather than histone. In NBD-2 cells this kinase appears to precede, and may be related to, the delayed increase in TH Vmax.

Cell Nucleus↗