scholarly journals Active translocon complexes labeled with GFP–Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum

2002 ◽  
Vol 158 (3) ◽  
pp. 497-506 ◽  
Author(s):  
Andrei V. Nikonov ◽  
Erik Snapp ◽  
Jennifer Lippincott-Schwartz ◽  
Gert Kreibich
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Membrane Proteins ◽  
Effective Diffusion ◽  
Diffusion Constant ◽  
Temperature Sensitive ◽  
Lateral Mobility ◽  
Nascent Polypeptide ◽  
Membrane Bound ◽  
Polypeptide Chains

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.

scholarly journals Craniofacial Diseases Caused by Defects in Intracellular Trafficking

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 726
Author(s):  
Chung-Ling Lu ◽  
Jinoh Kim
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Intracellular Trafficking ◽  
Critical Role ◽  
Treatment Strategies ◽  
Soluble Proteins ◽  
Genes Encoding ◽  
Membrane Bound ◽  
Signaling Receptors ◽  
Craniofacial Morphogenesis

Cells use membrane-bound carriers to transport cargo molecules like membrane proteins and soluble proteins, to their destinations. Many signaling receptors and ligands are synthesized in the endoplasmic reticulum and are transported to their destinations through intracellular trafficking pathways. Some of the signaling molecules play a critical role in craniofacial morphogenesis. Not surprisingly, variants in the genes encoding intracellular trafficking machinery can cause craniofacial diseases. Despite the fundamental importance of the trafficking pathways in craniofacial morphogenesis, relatively less emphasis is placed on this topic, thus far. Here, we describe craniofacial diseases caused by lesions in the intracellular trafficking machinery and possible treatment strategies for such diseases.

Studies on the site of addition of sialic acid and glucosamine to rat α1-acid glycoprotein

1977 ◽  
Vol 55 (4) ◽  
pp. 408-414 ◽  
Author(s):  
J. C. Jamieson
Keyword(s):  
Endoplasmic Reticulum ◽  
Sialic Acid ◽  
Rat Liver ◽  
Golgi Complex ◽  
Smooth Endoplasmic Reticulum ◽  
Main Site ◽  
Acid Glycoprotein ◽  
Membrane Bound ◽  
Polypeptide Chains

Ultrasonic extracts of rough and smooth endoplasmic reticulum fractions and Golgi fractions from rat liver were examined by immunoelectrophoresis using antiserum to α1-acid glycoprotein. Rough endoplasmic reticulum fractions contained only sialic acid free α1-acid glycoprotein, whereas smooth endoplasmic reticulum and Golgi fractions also contained sialic acid containing α1-acid glycoprotein. Determination of the sialic acid contents of immune precipitates isolated from the extracts suggested that the Golgi complex was the main site of addition of sialic acid to α1-acid glycoprotein. Immunological studies on puromycin extracts of polyribosomes showed that polypeptide chains of α1-acid glycoprotein and albumin were assembled mainly on membrane-bound polyribosomes. Evidence is presented from incorporation studies with labelled leucine and glucosamine that initial glycosylation of α1-acid glycoprotein occurs mainly or entirely after release of nascent polypeptide from the ribosomal site.

scholarly journals Mutants of the Yeast Yarrowia lipolyticaDefective in Protein Exit from the Endoplasmic Reticulum Are Also Defective in Peroxisome Biogenesis

1998 ◽  
Vol 18 (5) ◽  
pp. 2789-2803 ◽  
Author(s):  
Vladimir I. Titorenko ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Oleic Acid ◽  
Membrane Proteins ◽  
Matrix Proteins ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Mutant Cells

ABSTRACT Mutations in the SEC238 and SRP54 genes of the yeast Yarrowia lipolytica not only cause temperature-sensitive defects in the exit of the precursor form of alkaline extracellular protease and of other secretory proteins from the endoplasmic reticulum and in protein secretion but also lead to temperature-sensitive growth in oleic acid-containing medium, the metabolism of which requires the assembly of functionally intact peroxisomes. The sec238A andsrp54KO mutations at the restrictive temperature significantly reduce the size and number of peroxisomes, affect the import of peroxisomal matrix and membrane proteins into the organelle, and significantly delay, but do not prevent, the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX1 and PEX6 genes, which encode members of the AAA family of N-ethylmaleimide-sensitive fusion protein-like ATPases, not only affect the exit of precursor forms of secretory proteins from the endoplasmic reticulum but also prevent the exit of the peroxisomal membrane proteins Pex2p and Pex16p from the endoplasmic reticulum and cause the accumulation of an extensive network of endoplasmic reticulum membranes. None of the peroxisomal matrix proteins tested associated with the endoplasmic reticulum in sec238A,srp54KO, pex1-1, and pex6KO mutant cells. Our data provide evidence that the endoplasmic reticulum is required for peroxisome biogenesis and suggest that inY. lipolytica, the trafficking of some membrane proteins, but not matrix proteins, to the peroxisome occurs via the endoplasmic reticulum, results in their glycosylation within the lumen of the endoplasmic reticulum, does not involve transport through the Golgi, and requires the products encoded by the SEC238, SRP54,PEX1, and PEX6 genes.

scholarly journals The ADP Ribosylation Factor-Nucleotide Exchange Factors Gea1p and Gea2p Have Overlapping, but Not Redundant Functions in Retrograde Transport from the Golgi to the Endoplasmic Reticulum

2001 ◽  
Vol 12 (4) ◽  
pp. 1035-1045 ◽  
Author(s):  
Anne Spang ◽  
Johannes M. Herrmann ◽  
Susan Hamamoto ◽  
Randy Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Transport ◽  
Retrograde Transport ◽  
Guanine Nucleotide Exchange Factors ◽  
Temperature Sensitive ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Membrane Bound

The activation of the small ras-like GTPase Arf1p requires the action of guanine nucleotide exchange factors. Four Arf1p guanine nucleotide exchange factors have been identified in yeast: Sec7p, Syt1p, Gea1p, and its homologue Gea2p. We identifiedGEA2 as a multicopy suppressor of asec21-3 temperature-sensitive mutant.SEC21 encodes the γ-subunit of coatomer, a heptameric protein complex that together with Arf1p forms the COPI coat.GEA1 and GEA2 have at least partially overlapping functions, because deletion of either gene results in no obvious phenotype, whereas the double null mutant is inviable. Conditional mutants defective in both GEA1 andGEA2 accumulate endoplasmic reticulum and Golgi membranes under restrictive conditions. The two genes do not serve completely overlapping functions because a Δgea1Δarf1 mutant is not more sickly than a Δarf1 strain, whereas Δgea2Δarf1 is inviable. Biochemical experiments revealed similar distributions and activities for the two proteins. Gea1p and Gea2p exist both in membrane-bound and in soluble forms. The membrane-bound forms, at least one of which, Gea2p, can be visualized on Golgi structures, are both required for vesicle budding and protein transport from the Golgi to the endoplasmic reticulum. In contrast, Sec7p, which is required for protein transport within the Golgi, is not required for retrograde protein trafficking.

scholarly journals Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Benjamin D Hobson ◽  
Linghao Kong ◽  
Erik W Hartwick ◽  
Ruben L Gonzalez ◽  
Peter A Sims
Keyword(s):  
Protein Synthesis ◽  
Messenger Rna ◽  
Mammalian Cells ◽  
Acyl Transfer ◽  
Proximity Ligation Assay ◽  
Nascent Polypeptide ◽  
Rna Translation ◽  
Biochemical Assays ◽  
Messenger Rna Translation ◽  
Polypeptide Chains

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.

scholarly journals Elongation Inhibitors do not Prevent the Release of Puromycylated Nascent Polypeptide Chains from Ribosomes

2020 ◽  
Author(s):  
Benjamin D. Hobson ◽  
Linghao Kong ◽  
Erik W. Hartwick ◽  
Ruben L. Gonzalez ◽  
Peter A. Sims
Keyword(s):  
Protein Synthesis ◽  
Messenger Rna ◽  
Acyl Transfer ◽  
Proximity Ligation Assay ◽  
Nascent Polypeptide ◽  
Rna Translation ◽  
Proximity Ligation ◽  
Biochemical Assays ◽  
Messenger Rna Translation ◽  
Polypeptide Chains

ABSTRACTPuromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.

scholarly journals PRODUCTION OF MEMBRANE WHORLS IN RAT LIVER BY SOME INHIBITORS OF PROTEIN SYNTHESIS

1974 ◽  
Vol 62 (1) ◽  
pp. 20-31 ◽  
Author(s):  
Kou M. Hwang ◽  
Linda C. Yang ◽  
Christine K. Carrico ◽  
Rose A. Schulz ◽  
John B. Schenkman ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Cytochrome C ◽  
Peptide Synthesis ◽  
Reductase Activity ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Nascent Peptide ◽  
Membrane Bound ◽  
Nadph Cytochrome C Reductase

Inhibitors of protein synthesis capable of differential effects on nascent peptide synthesis on membrane-bound and free polyribosomes were employed to investigate the structure and function of cellular membranes of liver. The formation of membranous whorls in the cytoplasm and distension of nuclear membranes were induced by inhibitors of protein synthesis (i.e., cycloheximide and emetine) which predominantly interfere with nascent peptide synthesis on membrane-bound polyribosomes in situ. Other inhibitors of protein synthesis such as puromycin and fusidic acid, which inhibit nascent peptide synthesis on both free and membrane-bound polyribosomes, and chloramphenicol, which inhibits mitochondrial protein synthesis, did not induce these alterations. Cycloheximide, puromycin, and chloramphenicol produce some common cellular lesions as reflected by similar alterations in morphology, such as swelling of mitochondria, degranulation of rough endoplasmic reticulum, and aggregation of free ribosomes. The process of whorl formation in the cytoplasm, the incorporation of [3H]leucine and of [3H]choline into endoplasmic reticulum and the total NADPH-cytochrome c reductase activity of the endoplasmic reticulum were determined. During maximum formation of membranous whorls, [3H]leucine incorporation into cytoplasmic membranes was inhibited, while [3H]choline incorporation into these structures was increased; maximum inhibition of protein synthesis and stimulation of choline incorporation into endoplasmic reticulum, however, preceded whorl formation. Cycloheximide decreased the activity of NADPH-cytochrome c reductase of rough endoplasmic reticulum, but increased NADPH-cytochrome c reductase activity of smooth endoplasmic reticulum. In addition, cycloheximide decreased the content of hemoprotein in both the microsomal and mitochondrial fractions of rat liver, and the activities of mixed function oxidase and of oxidative phosphorylation were impaired to different degrees. Succinate-stimulated microsomal oxidation was also inhibited. The possible mechanisms involved in the formation of membranous whorls, as well as their functions, are discussed.

Thermolability of protein synthesis in a cell-free system from the obligately psychrophilic yeast Candida gelida

1969 ◽  
Vol 15 (4) ◽  
pp. 339-343 ◽  
Author(s):  
C. H. Nash ◽  
D. W. Grant ◽  
N. A. Sinclair
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Thermal Inactivation ◽  
Kinetic Studies ◽  
Temperature Sensitive ◽  
Free System ◽  
Psychrophilic Yeast ◽  
A Cell ◽  
Polypeptide Chains ◽  
Soluble Enzymes

A subcellular amino-acid-incorporating system from the obligately psychrophilic yeast, Candida gelida, was completely inhibited after incubation at 35 C for 30 minutes. The thermal inactivation of protein synthesis was due, in part, to the presence of unusually temperature-sensitive aminoacyl-sRNA synthetases in C. gelida extracts. Of the 13 specific synthetases examined, 7 retained less than 50% of their activity after being held at 35 C for 30 minutes. Kinetic studies of thermal inactivation of leucyl-sRNA synthetase demonstrated that this enzyme is 50% inactivated after only 7 minutes at 35 C. None of the 10 sRNA species tested was temperature sensitive. In addition to temperature-sensitive synthetases, C. gelida possesses thermolabile soluble enzymes involved in the formation of ribosomal-bound polypeptide chains.

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