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 The effects of low temperatures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes

1986 ◽  
Vol 237 (1) ◽  
pp. 33-39 ◽  
Author(s):  
E Fries ◽  
I Lindström
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Low Temperatures ◽  
Intracellular Transport ◽  
Rat Hepatocytes ◽  
Subcellular Fractionation ◽  
Isolated Rat Hepatocytes ◽  
Different Temperatures ◽  
Lag Period ◽  
Time Courses

Isolated rat hepatocytes were pulse-labelled with [35S]methionine at 37 degrees C and subsequently incubated (chased) for different periods of time at different temperatures (37-16 degrees C). The time courses for the secretion of [35S]methionine-labelled albumin and haptoglobin were determined by quantitative immunoprecipitation of the detergent-solubilized cells and of the chase media. Both proteins appeared in the chase medium only after a lag period, the length of which increased markedly with decreasing chase temperature: from about 10 and 20 min at 37 degrees C to about 60 and 120 min at 20 degrees C for albumin and haptoglobin respectively. The rates at which the proteins were externalized after the lag period were also strongly affected by temperature, the half-time for secretion being 20 min at 37 degrees C and 200 min at 20 degrees C for albumin; at 16 degrees C no secretion could be detected after incubation for 270 min. Analysis by subcellular fractionation showed that part of the lag occurred in the endoplasmic reticulum and that the rate of transfer to the Golgi complex was very temperature-dependent. The maximum amount of the two pulse-labelled proteins in Golgi fractions prepared from cells after different times of chase decreased with decreasing incubation temperatures, indicating that the transport from the Golgi complex to the cell surface was less affected by low temperatures than was the transport from the endoplasmic reticulum to the Golgi complex.

scholarly journals PROTEIN SYNTHESIS, STORAGE, AND DISCHARGE IN THE PANCREATIC EXOCRINE CELL

1964 ◽  
Vol 20 (3) ◽  
pp. 473-495 ◽  
Author(s):  
Lucien G. Caro ◽  
George E. Palade
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Zymogen Granules ◽  
Intravenous Injections ◽  
Pancreatic Exocrine ◽  
Exocrine Cell ◽  
Secretory Products ◽  
Electron Microscopical

The synthesis, intracellular transport, storage, and discharge of secretory proteins in and from the pancreatic exocrine cell of the guinea pig were studied by light- and electron microscopical autoradiography using DL-leucine-4,5-H3 as label. Control experiments were carried out to determine: (a) the length of the label pulse in the blood and tissue after intravenous injections of leucine-H3; (b) the amount and nature of label lost during tissue fixation, dehydration, and embedding. The results indicate that leucine-H3 can be used as a label for newly synthesized secretory proteins and as a tracer for their intracellular movements. The autoradiographic observations show that, at ∼5 minutes after injection, the label is localized mostly in cell regions occupied by rough surfaced elements of the endoplasmic reticulum; at ∼20 minutes, it appears in elements of the Golgi complex; and after 1 hour, in zymogen granules. The evidence conclusively shows that the zymogen granules are formed in the Golgi region by a progressive concentration of secretory products within large condensing vacuoles. The findings are compatible with an early transfer of label from the rough surfaced endoplasmic reticulum to the Golgi complex, and suggest the existence of two distinct steps in the transit of secretory proteins through the latter. The first is connected with small, smooth surfaced vesicles situated at the periphery of the complex, and the second with centrally located condensing vacuoles.

scholarly journals Assembly of influenza hemagglutinin trimers and its role in intracellular transport.

1986 ◽  
Vol 103 (4) ◽  
pp. 1179-1191 ◽  
Author(s):  
C S Copeland ◽  
R W Doms ◽  
E M Bolzau ◽  
R G Webster ◽  
A Helenius
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Quaternary Structure ◽  
Subunit Interactions ◽  
Oligomer Formation ◽  
Influenza Hemagglutinin ◽  
Triton X

The hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.

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.

The role of subcompartments of the Golgi complex in protein intracellular transport

1982 ◽  
Vol 300 (1099) ◽  
pp. 173-184 ◽  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Concanavalin A ◽  
Intracellular Transport ◽  
Subcellular Fractionation ◽  
Membrane Glycoproteins ◽  
Myeloma Cells ◽  
Golgi Stack ◽  
Wheatgerm Agglutinin

The functioning of the Golgi complex in protein intracellular transport is most simply understood in terms of its being composed of a sequence of functionally distinct subcompartments. For example, the influence of perturbation of cellular Na + -K + balance on the transport of secretory and membrane glycoproteins is to greatly slow their passage from relatively proximal to relatively distal subcompartments. To further the understanding of the nature of these subcompartments a rat IgM myeloma has been subjected to analytical subcellular fractionation. Fractions selectively enriched in distinct Golgi-associated activities have been prepared and their membrane proteins compared with those of rough microsomal fractions. The subfractionation is extensive and suggests the possibility of obtaining well resolved Golgi subfractions. Myeloma cells stained intracellularly with Goncanavalin A - and wheatgerm agglutinin-peroxidase conjugates show distinct labelling patterns. Concanavalin A stains the entirety of the rough endoplasmic reticulum as well as the proximal face of the Golgi stack. Wheatgerm agglutinin stains the distal face of the stack of Golgi cisternae. The staining patterns are not due to immunoglobulin as they are also observed in myeloma variants that fail to synthesize immunoglobulin.

scholarly journals The differential effects of dithiothreitol and 2-mercaptoethanol on the secretion of partially and completely assembled immunoglobulins suggest that thiol-mediated retention does not take place in or beyond the Golgi.

1994 ◽  
Vol 5 (12) ◽  
pp. 1311-1324 ◽  
Author(s):  
C Valetti ◽  
R Sitia
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Reducing Agents ◽  
Differential Effects ◽  
Golgi Transport ◽  
Disulphide Bonds ◽  
Golgi Compartment ◽  
Interchange Reactions

Dithiothreitol (DTT) blocks the endoplasmic reticulum (ER)-Golgi transport of newly synthesized immunoglobulin (Ig) molecules, whereas 2-mercaptoethanol (2ME) allows secretion of unpolymerized Igs otherwise retained intracellularly by disulphide interchange reactions. To understand this dichotomy, we have compared the effects of DTT and 2ME on the assembly, intracellular transport, and secretion of a panel of chimeric Igs that are either constitutively secreted or retained intracellularly. Our results demonstrate that DTT, but not 2ME, reduces some of the inter- and intrachain disulphide bonds and causes partial disassembly of H2L2 complexes and unfolding of individual chains in the ER. Upon DTT removal, heavy (H) and light (L) chains reform hapten-binding H2L2 molecules, which are later secreted. Reduction of the H2L2 interchain disulphide bonds can occur along the entire secretory pathway; however, in or beyond the Golgi this does not result in efficient H-L disassembly or unfolding. As a consequence, DTT does not block the exit from the Golgi. Moreover, unpolymerized Igs--normally retained in a pre-Golgi compartment--no longer require reducing agents to be secreted once they have reached the Golgi. Thus, little if any thiol-mediated retention seems to take place in or beyond the Golgi complex.

scholarly journals The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

1997 ◽  
Vol 8 (9) ◽  
pp. 1805-1814 ◽  
Author(s):  
J S Cox ◽  
R E Chapman ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Secretory Pathway ◽  
Lipid Synthesis ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

scholarly journals New Mutants of Saccharomyces cerevisiae Affected in the Transport of Proteins From the Endoplasmic Reticulum to the Golgi Complex

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 393-406 ◽  
Author(s):  
Linda J Wuestehube ◽  
Rainer Duden ◽  
Arlene Eun ◽  
Susan Hamamoto ◽  
Paul Korn ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Secretory Protein ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Membrane Structures ◽  
Α Subunit ◽  
Complementation Groups ◽  
Vacuolar Protein

Abstract We have isolated new temperature-sensitive mutations in five complementation groups, sec31-sec35, that are defective in the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex. The sec31-sec35 mutants and additional alleles of previously identified sec and vacuolar protein sorting (vps) genes were isolated in a screen based on the detection of α-factor precursor in yeast colonies replicated to and lysed on nitrocellulose filters. Secretory protein precursors accumulated in sec31-sec35 mutants at the nonpermissive temperature were core-glycosylated but lacked outer chain carbohydrate, indicating that transport was blocked after translocation into the ER but before arrival in the Golgi complex. Electron microscopy revealed that the newly identified sec mutants accumulated vesicles and membrane structures reminiscent of secretory pathway organelles. Complementation analysis revealed that sec32-1 is an allele of BOS1, a gene implicated in vesicle targeting to the Golgi complex, and sec33-1 is an allele of RET1, a gene that encodes the α subunit of coatomer.

scholarly journals Dissection of the asynchronous transport of intestinal microvillar hydrolases to the cell surface.

1988 ◽  
Vol 106 (6) ◽  
pp. 1853-1861 ◽  
Author(s):  
B Stieger ◽  
K Matter ◽  
B Baur ◽  
K Bucher ◽  
M Höchli ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Colon Adenocarcinoma ◽  
Subcellular Fractionation ◽  
Adenocarcinoma Cell Line ◽  
Dipeptidylpeptidase Iv ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Novel subcellular fractionation procedures and pulse-chase techniques were used to study the intracellular transport of the microvillar membrane hydrolases sucrase-isomaltase and dipeptidylpeptidase IV in the differentiated colon adenocarcinoma cell line Caco-2. The overall rate of transport to the cell surface was two fold faster for dipeptidylpeptidase IV than for sucrase-isomaltase, while no significant differences were observed in transport rates from the site of complex glycosylation to the brush border. The delayed arrival of sucrase-isomaltase in the compartment where complex glycosylation occurs was only in part due to exit from the endoplasmic reticulum. A major slow-down could be ascribed to maturation in and transit of this enzyme through the Golgi apparatus. These results suggest that the observed asynchronism is due to more than one rate-limiting step along the rough endoplasmic reticulum to trans-Golgi pathway.

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