scholarly journals Deletion of the propeptide of apolipoprotein A-I impairs exit of nascent apolipoprotein A-I from the endoplasmic reticulum

1994 ◽  
Vol 302 (3) ◽  
pp. 641-648 ◽  
Author(s):  
R S McLeod ◽  
C Robbins ◽  
A Burns ◽  
Z Yao ◽  
P H Pritchard
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Wild Type ◽  
Mature Form ◽  
Human Apolipoprotein ◽  
Apolipoprotein A

Human apolipoprotein (apo) A-I is secreted as a proprotein of 249 amino acids and is processed extracellularly to the mature form (243 amino acids) by removal of a six-residue propeptide segment. We have examined the role of the apoA-I propeptide in intracellular transport and secretion using transfected baby hamster kidney cells that secreted either proapoA-I (from the wild-type cDNA, A-Iwt) or mature-form apoA-I (from A-I delta pro, a cDNA in which the propeptide sequence was deleted). Deletion of the propeptide from the apoA-I sequence did not affect the rate of apoA-I synthesis, nor did it affect the fidelity of proteolytic removal of the prepeptide. However, the propeptide deletion caused mature-form apoA-I to accumulate within the cells as determined by pulse-chase experiments; the intracellular retention times for the mature-form apoA-I in which the propeptide was prematurely removed was three times longer than that of proapoA-I (t1/2 > 3 h compared with approximately 50 min). There was no detectable degradation of either form of newly synthesized apoA-I. Immunofluorescence microscopy revealed that, whereas the proapoA-I was located predominantly in the Golgi apparatus, large quantities of the mature-form apoA-I were detected in the endoplasmic reticulum and very little was in the Golgi apparatus of A-I delta pro-transfected cells. These findings suggest that the propeptide sequence may be involved in the intracellular transport of apoA-I from the endoplasmic reticulum to the Golgi apparatus. We propose that the function of the propeptide sequence is to facilitate efficient transport of apoA-I through the secretory pathway.

scholarly journals The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells.

1989 ◽  
Vol 108 (5) ◽  
pp. 1647-1655 ◽  
Author(s):  
T J Stoller ◽  
D Shields
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Signal Peptide ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Gh3 Cells ◽  
Alpha Globin ◽  
Regulated Secretory Pathway

We have investigated the role of the somatostatin propeptide in mediating intracellular transport and sorting to the regulated secretory pathway. Using a retroviral expression vector, two fusion proteins were expressed in rat pituitary (GH3) cells: a control protein consisting of the beta-lactamase signal peptide fused to chimpanzee alpha-globin (142 amino acids); and a chimera of the somatostatin signal peptide and proregion (82 amino acids) fused to alpha-globin. Control globin was translocated into the endoplasmic reticulum as determined by accurate cleavage of its signal peptide; however, alpha-globin was not secreted but was rapidly and quantitatively degraded intracellularly with a t 1/2 of 4-5 min. Globin degradation was insensitive to chloroquine, a drug which inhibits lysosomal proteases, but was inhibited at 16 degrees C suggesting proteolysis occurred during transport to the cis-Golgi apparatus. In contrast to the control globin, approximately 30% of the somatostatin propeptide-globin fusion protein was transported to the distal elements of the Golgi apparatus where it was endoproteolytically processed. Processing of the chimera occurred in an acidic intracellular compartment since cleavage was inhibited by 25 microM chloroquine. 60% of the transported chimera was cleaved at the Arg-Lys processing site in native prosomatostatin yielding "mature" alpha-globin. Most significantly, approximately 50% of processed alpha-globin was sorted to the regulated pathway and secreted in response to 8-Br-cAMP. We conclude that the somatostatin propeptide mediated transport of alpha-globin from the endoplasmic reticulum to the trans-Golgi network by protecting molecules from degradation and in addition, facilitated packaging of alpha-globin into vesicles whose secretion was stimulated by cAMP.

Immunolocalization of the intracellular sites of accumulation of mutant influenza hemagglutinins by Electron Microscopy

1989 ◽  
Vol 47 ◽  
pp. 968-969
Author(s):  
K. McCammon ◽  
M. Segal ◽  
J. Sambrook ◽  
M. J. Gething ◽  
A. McDowall
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Cysteine Residue ◽  
Homologous Sequence ◽  
Golgi Compartment ◽  
Golgi Network

The hemagglutinin (HA) of influenza virus has been used as a model system to study the biosynthesis and intracellular transport of integral membrane proteins in mammalian cells. To investigate the role of protein structure in facilitating transport along the secretory pathway, we have examined the expression in monkey CV-1 cells of a large number of mutant HA molecules. The majority of the HA mutants do not progress along the secretory pathway and accumulate in the endoplasmic reticulum (ER), and we have shown that assembly of newly-synthesized HA monomers into correctly folded trimeric structures is required for transport of the protein to the Golgi apparatus. By contrast, only one HA mutant has beegn characterized whose transport is blocked at a post-Golgi stage of the pathway and thus little is known about the factors involved in the sorting of the HA molecule from the Golgi apparatus to the plasma membrane (PM). In this study we are using electron microscopy to precisely define the intracellular site of accumulation of two mutant HAs whose transport is blocked at different stages of the secretory pathway. In mutant HAJS67, a cysteine residue (cys67) involved in a key disulfide bond has been substituted by a serine residue. In mutant HA164, the 10 amino acid cytoplasmic tail of the wild-type HA has been replaced by a non-homologous sequence of 16 amino acids. Biochemical and immunof1uoresence analyses have indicated that HAJS67 molecules remain in the ER compartment while HA164 is largely confined to a post-Golgi compartment, possibly the trans Golgi network (TGN).

Potassium depletion inhibits the intracellular transport of secretory proteins between the endoplasmic reticulum and the Golgi complex

1989 ◽  
Vol 92 (2) ◽  
pp. 173-185
Author(s):  
J.D. Judah ◽  
K.E. Howell ◽  
J.A. Taylor ◽  
P.S. Quinn
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Subcellular Fractionation ◽  
Secretory Proteins ◽  
Mature Form ◽  
Processing Enzymes ◽  
Microscopic Studies ◽  
K Depletion

In this paper we show that hepatocytes that have been depleted of K+ secrete albumin, alpha-1-anti-trypsin and transferrin at a slower rate than cells to which K+ has been returned. K+ depletion has no effect on the intracellular nucleotide pools, and we provide evidence that the inhibitions of secretion caused by depletion of K+ and depletion of ATP are independent. Studies of the processing of alpha-1-anti-trypsin show that K+ depletion inhibits the formation of the mature form of the protein, but that immature forms are never secreted. In cells to which K+ was returned, secretion of the mature form was restored. This implies that transport is blocked at a point before the proteins reach the processing enzymes. Proteins delayed by K+ depletion are not removed from the secretory pathway, but are free to mix with protein synthesized subsequently. These data are supported by subcellular fractionation experiments, which show that the secretory proteins are delayed before reaching the Golgi complex, and by immunoelectron microscopic studies. These show that in K+-deficient cells the morphology of both the endoplasmic reticulum and the Golgi complex is normal. The secretory proteins are trapped in smooth vesicles that contain reaction product when incubated for glucose-6-phosphatase, a marker for the endoplasmic reticulum.

scholarly journals Biosynthesis of high density lipoprotein by chicken liver: intracellular transport and proteolytic processing of nascent apolipoprotein A-1.

1985 ◽  
Vol 101 (4) ◽  
pp. 1219-1226 ◽  
Author(s):  
D Banerjee ◽  
T K Mukherjee ◽  
C M Redman
Keyword(s):  
Amino Acids ◽  
Sequence Analysis ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Proteolytic Processing ◽  
Chicken Liver ◽  
Golgi Cell ◽  
Apolipoprotein A ◽  
Cell Fractions

To study the in vivo processing and secretion of Apolipoprotein A-I (Apo A-I), young chickens were administered individual L-[3H]amino acids intravenously and the time of intracellular transport of nascent Apo A-I from rough endoplasmic reticulum (RER) to the Golgi apparatus was measured. Within 3 to 9 min there was maximal incorporation of radioactivity into Apo A-I in both the RER and the Golgi cell fractions. By contrast, the majority of radioactive albumin was also present in the RER by 3 to 9 min, but did not reach peak amounts in the Golgi fraction until 9 to 25 min. Both radioactive Apo A-I and albumin appeared in the blood at about the same time (between 20 and 30 min). NH2-terminal amino acid sequence analysis of nascent intracellular Apo A-I showed that it contains a pro-hexapeptide extension identical to that of human Apo A-I. After 30 min of administration of radioactive amino acids radioactive Apo A-I was isolated by immunoprecipitation from the liver and serum. NH2-terminal sequence analysis of 20 amino acids indicated that chicken liver contained an equal mixture of nascent pro-Apo A-I and fully processed Apo A-I, whereas the serum only contained processed Apo A-I. Further studies showed that the RER only contained pro-Apo A-I, whereas a mixture of pro-Apo A-I and processed Apo A-I was found in the Golgi complex. These results indicate that, in chicken hepatocytes, there is a more rapid transport of Apo A-I than of albumin from the RER to the Golgi cell fractions, and that Apo A-I remains in the Golgi apparatus for a longer period of time before it is secreted into the blood. In addition these studies show that the in vivo proteolytic processing of chicken pro-Apo A-I to Apo A-I occurs in the Golgi cell fractions.

scholarly journals Dynein Supports Motility of Endoplasmic Reticulum in the FungusUstilago maydis

2002 ◽  
Vol 13 (3) ◽  
pp. 965-977 ◽  
Author(s):  
Roland Wedlich-Söldner ◽  
Irene Schulz ◽  
Anne Straube ◽  
Gero Steinberg
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Cytoplasmic Dynein ◽  
Organelle Transport ◽  
Minor Importance ◽  
Dependent Transport

The endoplasmic reticulum (ER) of most vertebrate cells is spread out by kinesin-dependent transport along microtubules, whereas studies in Saccharomyces cerevisiae indicated that motility of fungal ER is an actin-based process. However, microtubules are of minor importance for organelle transport in yeast, but they are crucial for intracellular transport within numerous other fungi. Herein, we set out to elucidate the role of the tubulin cytoskeleton in ER organization and dynamics in the fungal pathogen Ustilago maydis. An ER-resident green fluorescent protein (GFP)-fusion protein localized to a peripheral network and the nuclear envelope. Tubules and patches within the network exhibited rapid dynein-driven motion along microtubules, whereas conventional kinesin did not participate in ER motility. Cortical ER organization was independent of microtubules or F-actin, but reformation of the network after experimental disruption was mediated by microtubules and dynein. In addition, a polar gradient of motile ER-GFP stained dots was detected that accumulated around the apical Golgi apparatus. Both the gradient and the Golgi apparatus were sensitive to brefeldin A or benomyl treatment, suggesting that the gradient represents microtubule-dependent vesicle trafficking between ER and Golgi. Our results demonstrate a role of cytoplasmic dynein and microtubules in motility, but not peripheral localization of the ER inU. maydis.

scholarly journals A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast.

1988 ◽  
Vol 107 (3) ◽  
pp. 851-863 ◽  
Author(s):  
A Nakano ◽  
D Brada ◽  
R Schekman
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Membrane Fraction ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Genomic Library ◽  
Membrane Glycoprotein ◽  
Mobility Shift ◽  
Cloned Gene

SEC12, a gene that is required for secretory, membrane, and vacuolar proteins to be transported from the endoplasmic reticulum to the Golgi apparatus, has been cloned from a genomic library by complementation of a sec12 ts mutation. Genetic analysis has shown that the cloned gene integrates at the SEC12 locus and that a null mutation at the locus is lethal. The DNA sequence predicts a protein of 471 amino acids containing a hydrophobic stretch of 19 amino acids near the COOH terminus. To characterize the gene product (Sec12p) in detail, a lacZ-SEC12 gene fusion has been constructed and a polyclonal antibody raised against the hybrid protein. The antibody recognizes Sec12p as a approximately 70-kD protein that sediments in a mixed membrane fraction that includes endoplasmic reticulum. Sec12p is not removed from the membrane fraction by treatment at high pH and high salt and is not degraded by exogenous protease unless detergent is present. Glycosylation of Sec12p during biogenesis is indicated by an electrophoretic mobility shift of the protein that is influenced by tunicamycin and by imposition of an independent secretory pathway block. We suggest that Sec12p is an integral membrane glycoprotein with a prominent domain that faces the cytoplasm where it functions to promote protein transport to the Golgi apparatus. In the process of transport, Sec12p itself may migrate to the Golgi apparatus and function in subsequent transport events.

scholarly journals Role of N-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation

2000 ◽  
Vol 348 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Armando J. PARODI
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Cell Recognition ◽  
Glucosidase Ii ◽  
Membrane Bound ◽  
Quaternary Structures ◽  
Lectin Recognition

The endoplasmic reticulum (ER) is the subcellular site where proteins following the secretory pathway acquire their proper tertiary and, in certain cases, quaternary structures. Species that are not yet properly folded are prevented from exit to the Golgi apparatus and, if permanently misfolded, are transported to the cytosol, where they are degraded in the proteasomes. This review deals with a mechanism, applicable to proteins that are N-glycosylated in the ER, by which the quality control of folding is performed. Protein-linked monoglucosylated glycans, formed by glucosidase I- and glucosidase II-dependent partial deglucosylation of the oligosaccharides transferred from dolichol diphosphate derivatives in N-glycosylation (Glc3Man9GlcNAc2), mediate glycoprotein recognition by two ER-resident lectins, membrane-bound calnexin (CNX) and its soluble homologue, calreticulin (CRT). A still not yet fully confirmed interaction between the lectins and the protein moieties of folding glycoproteins may occur after lectin recognition of monoglucosylated structures. Further deglucosylation of glycans by glucosidase II, and perhaps also by a change in CNX/CRT and/or in the substrate glycoprotein conformation, liberates the glycoproteins from their CNX/CRT anchors. Glycans may be then reglucosylated by the UDP-Glc:glycoprotein glucosyltransferase (GT), and thus be recognized again by CNX/CRT, but only when linked to not yet properly folded protein moieties, as this enzyme behaves as a sensor of glycoprotein conformation. Deglucosylation/reglucosylation cycles catalysed by the opposing activities of glucosidase II and GT only stop when proper folding is achieved. The interaction between CNX/CRT and a monoglucosylated glycan is one of the alternative mechanisms by which cells retain not yet properly folded glycoproteins in the ER; in addition, it enhances folding efficiency by preventing protein aggregation and thus allowing intervention of classical chaperones and other folding-assisting proteins. There is evidence suggesting that both glycoprotein glucosylation and mannose removal, respectively mediated by GT and ER mannosidase I, might be involved in cell recognition of permanently misfolded glycoproteins bound for proteasome degradation.

scholarly journals Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

2021 ◽  
Author(s):  
Amit Ketkar ◽  
Lane Smith ◽  
Callie Johnson ◽  
Alyssa Richey ◽  
Makayla Berry ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mutation Frequency ◽  
Control Sequence ◽  
Wild Type ◽  
Binding Properties ◽  
High Affinity ◽  
G4 Dna ◽  
G Quadruplex ◽  
Forward Mutagenesis

Abstract We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

Sign in / Sign up

Export Citation Format

Share Document