scholarly journals INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS IN THE PANCREATIC EXOCRINE CELL

1967 ◽  
Vol 34 (2) ◽  
pp. 577-596 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Guinea Pig ◽  
Golgi Complex ◽  
Structural Damage ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Cell Fractionation ◽  
Secretory Cycle ◽  
Pancreatic Exocrine ◽  
Rough Er

It has been established by electron microscopic radioautography of guinea pig pancreatic exocrine cells (Caro and Palade, 1964) that secretory proteins are transported from the elements of the rough-surfaced endoplasmic reticulum (ER) to condensing vacuoles of the Golgi complex possibly via small vesicles located in the periphery of the complex. To define more clearly the role of these vesicles in the intracellular transport of secretory proteins, we have investigated the secretory cycle of the guinea pig pancreas by cell fractionation procedures applied to pancreatic slices incubated in vitro. Such slices remain viable for 3 hr and incur minimal structural damage in this time. Their secretory proteins can be labeled with radioactive amino acids in short, well defined pulses which, followed by cell fractionation, makes possible a kinetic analysis of transport. To determine the kinetics of transport, we pulse-labeled sets of slices for 3 min with leucine-14C and incubated them for further +7, +17, and +57 min in chase medium. At each time, smooth microsomes ( = peripheral elements of the Golgi complex) and rough microsomes ( = elements of the rough ER) were isolated from the slices by density gradient centrifugation of the total microsomal fraction. Labeled proteins appeared initially (end of pulse) in the rough microsomes and were subsequently transferred during incubation in chase medium to the smooth microsomes, reaching a maximal concentration in this fraction after +7 min chase incubation. Later, labeled proteins left the smooth microsomes to appear in the zymogen granule fraction. These data provide direct evidence that secretory proteins are transported from the cisternae of the rough ER to condensing vacuoles via the small vesicles of the Golgi complex.

scholarly journals INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS IN THE PANCREATIC EXOCRINE CELL

1968 ◽  
Vol 39 (3) ◽  
pp. 589-603 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Oxidative Phosphorylation ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Antimycin A ◽  
Cell Fractionation ◽  
Pancreatic Exocrine ◽  
Exocrine Cell ◽  
Cell Synthesis

Since in the pancreatic exocrine cell synthesis and intracellular transport of secretory proteins can be uncoupled (1), it is possible to examine separately the metabolic requirements of the latter process. To this intent, guinea pig pancreatic slices were pulse labeled with leucine-3H for 3 min and incubated post-pulse for 37 min in chase medium containing 5 x 10-4 M cycloheximide and inhibitors of glycolysis, respiration, or oxidative phosphorylation. In each case, the effect on transport was assessed by measuring the amount of labeled secretory proteins found in zymogen granule fractions isolated from the corresponding slices. This assay is actually a measure of the efficiency of transport of secretory proteins from the cisternae of the rough endoplasmic reticulum (RER) to the condensing vacuoles of the Golgi complex which are recovered in the zymogen granule fraction (16). The results indicate that transport is insensitive to glycolytic inhibitors (fluoride, iodoacetate) but is blocked by respiratory inhibitors (N2, cyanide, Antimycin A) and by inhibitors of oxidative phosphorylation (dinitrophenol, oligomycin). Except for Antimycin A, the effect is reversible. Parallel radioautographic studies and cell fractionation procedures applied to microsomal subfractions have indicated that the energy-dependent step is located between the transitional elements of the RER and the small, smooth-surfaced vesicles at the periphery of the Golgi complex. Radiorespirometric data indicate that the substrates oxidized to support transport are endogenous long-chain fatty acids.

scholarly journals SYNTHESIS, INTRACELLULAR TRANSPORT, AND DISCHARGE OF SECRETORY PROTEINS IN STIMULATED PANCREATIC EXOCRINE CELLS

1971 ◽  
Vol 50 (1) ◽  
pp. 135-158 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Protein Synthesis ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Cell Fractionation ◽  
Secretory Product ◽  
Zymogen Granules ◽  
Pancreatic Exocrine ◽  
Storage Granules

Our previous observations on the synthesis and transport of secretory proteins in the pancreatic exocrine cell were made on pancreatic slices from starved guinea pigs and accordingly apply to the resting, unstimulated cell. Normally, however, the gland functions in cycles during which zymogen granules accumulate in the cell and are subsequently discharged from it in response to secretogogues. The present experiments were undertaken to determine if secretory stimuli applied in vitro result in adjustments in the rates of protein synthesis and/or of intracellular transport. To this intent pancreatic slices from starved animals were stimulated in vitro for 3 hr with 0.01 mM carbamylcholine. During the first hour of treatment the acinar lumen profile is markedly enlarged due to insertion of zymogen granule membranes into the apical plasmalemma accompanying exocytosis of the granule content. Between 2 and 3 hr of stimulation the luminal profile reverts to unstimulated dimensions while depletion of the granule population nears completion. The acinar cells in 3-hr stimulated slices are characterized by the virtual complete absence of typical condensing vacuoles and zymogen granules, contain a markedly enlarged Golgi complex consisting of numerous stacked cisternae and electron-opaque vesicles, and possess many small pleomorphic storage granules. Slices in this condition were pulse labeled with leucine-3H and the route and timetable of intracellular transport assessed during chase incubation by cell fractionation, electron microscope radioautography, and a discharge assay covering the entire secretory pathway. The results showed that the rate of protein synthesis, the rate of drainage of the rough-surfaced endoplasmic reticulum (RER) compartment, and the over-all transit time of secretory proteins through the cells was not accelerated by the secretogogue. Secretory stimulation did not lead to a rerouting of secretory proteins through the cell sap. In the resting cell, the secretory product is concentrated in condensing vacuoles and stored as a relatively homogeneous population of spherical zymogen granules. By contrast, in the stimulated cell, secretory proteins are initially concentrated in the flattened saccules of the enlarged Golgi complex and subsequently stored in numerous small storage granules before release. The results suggest that secretory stimuli applied in vitro primarily affect the discharge of secretory proteins and do not, directly or indirectly, influence their rates of synthesis and intracellular transport.

scholarly journals INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS IN THE PANCREATIC EXOCRINE CELL

1968 ◽  
Vol 39 (3) ◽  
pp. 580-588 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Protein Synthesis ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Cell Fractionation ◽  
Maximum Inhibition ◽  
Granule Fraction ◽  
Pancreatic Exocrine ◽  
Exocrine Cell ◽  
Acinar Lumen

Experiments have been carried out to determine whether intracellular transport of pancreatic secretory proteins is obligatorily coupled to protein synthesis or whether it is a separable process which can be independently regulated. To this intent, guinea pig pancreatic slices were pulse labeled with leucine-3H for 3 min and incubated post-pulse for 37 min in chase medium containing cycloheximide up to concentrations sufficient to inhibit protein synthesis by 98%. In controls, newly synthesized secretory proteins are transported over this interval to condensing vacuoles of the Golgi complex. Since the latter are recovered in the zymogen granule fraction upon cell fractionation, intracellular transport was assayed by measuring the amount of protein radioactivity found in the zymogen granule fraction after a (3 + 37) min incubation. The results indicated that at maximum inhibition of protein synthesis (5 x 10-4 M cycloheximide), transport proceeded with an efficiency ∼80% of control. Parallel radioautographic studies on intact slices confirmed these data and further indicated that all the steps of intracellular transport, including discharge to the acinar lumen, were independent of protein synthesis. We conclude that: (1) transport and protein synthesis are separable processes; (2) intracellular transport is not the result of a continuous delivery of secretory proteins from attached polysomes to the cisternae of the rough endoplasmic reticulum; and (3) transport is not dependent on the synthesis of "specific" nonsecretory proteins within the time limits tested.

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 COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG

1971 ◽  
Vol 49 (1) ◽  
pp. 109-129 ◽  
Author(s):  
J. Meldolesi ◽  
J. D. Jamieson ◽  
G. E. Palade
Keyword(s):  
Guinea Pig ◽  
Gradient Centrifugation ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Marker Enzymes ◽  
Zymogen Granules ◽  
Terminal Web ◽  
Granule Fraction ◽  
Pancreatic Exocrine ◽  
Exocrine Cell

The subcellular components involved in the synthesis, transport, and discharge of secretory proteins in the guinea pig pancreatic exocrine cell have been isolated from gland homogenates by differential and gradient centrifugation. They include rough and smooth microsomes derived respectively from the rough endoplasmic reticulum and Golgi periphery, a zymogen granule fraction consisting mainly of mature zymogen granules and a smaller population of condensing vacuoles, and a plasmalemmal fraction. Membrane subfractions were obtained from the particulate components by treatment with mild (pH 7.8) alkaline buffers which extract the majority (>95%) of the content of secretory proteins, allowing the membranes to be recovered from the extracting fluid by centrifugation. The purity of the fractions was assessed by electron microscopy and by assaying marker enzymes for cross-contaminants. The rough and smooth microsomes were essentially free of mitochondrial contamination; the smooth microsomes contained <15% rough contaminants. The zymogen granule fraction and its derived membranes were free of rough microsomes and contained <3% contaminant mitochondria. The plasmalemmal fraction was heterogeneous as to origin (deriving from basal, lateral, and apical poles of the cell) and contained varying amounts of adherent fibrillar material arising from the basement membrane and terminal web. The lipid and enzymatic composition of the membrane fractions are described in the following reports.

scholarly journals DYNAMICS OF CYTOPLASMIC MEMBRANES IN GUINEA PIG PANCREATIC ACINAR CELLS

1974 ◽  
Vol 61 (1) ◽  
pp. 1-13 ◽  
Author(s):  
J. Meldolesi
Keyword(s):  
Molecular Weight ◽  
Membrane Proteins ◽  
Guinea Pig ◽  
Intracellular Transport ◽  
Sodium Dodecyl ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Double Labeling

The rate of synthesis and the turnover of cytoplasmic membrane proteins were determined in the acinar cells of guinea pig pancreas with the aim of investigating the mechanisms by which the intracellular transport of secretion products occurs. These cells are highly specialized toward protein secretion. By means of in vitro pulse-chase experiments and in vivo double-labeling experiments, using radioactive L-leucine as the tracer, it was found that the turnover of secretory proteins is much faster than that of all membranes involved in their transport (rough and smooth microsome and zymogen granule membranes). Sodium dodecyl sulfate-polyacrylamide disk gel electrophoresis of membrane proteins revealed that in each of these membranes there is a marked heterogeneity of turnover; generally the high molecular weight polypeptides have a shorter half-life than the low molecular weight polypeptides. These data indicate that the membranes participating in the intracellular transport of secretory proteins are not synthesized concomitantly with the latter. Rather, they are probably reutilized in several successive secretory cycles. The possible relevance of these findings to other secretory systems is discussed.

scholarly journals INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS IN THE PANCREATIC EXOCRINE CELL

1967 ◽  
Vol 34 (2) ◽  
pp. 597-615 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Intracellular Transport ◽  
Gradient Centrifugation ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Electron Microscopic ◽  
Intact Cells ◽  
Secretory Cycle ◽  
Granule Fraction ◽  
Electron Microscopic Radioautography

In the previous paper we described an in vitro system of guinea pig pancreatic slices whose secretory proteins can be pulse-labeled with radioactive amino acids. From kinetic experiments performed on smooth and rough microsomes isolated by gradient centrifugation from such slices, we obtained direct evidence that secretory proteins are transported from the cisternae of the rough endoplasmic reticulum to condensing vacuoles of the Golgi complex via small vesicles located in the periphery of the complex. Since condensing vacuoles ultimately become zymogen granules, it was of interest to study this phase of the secretory cycle in pulse-labeled slices. To this intent, a zymogen granule fraction was isolated by differential centrifugation from slices at the end of a 3-min pulse with leucine-14C and after varying times of incubation in chase medium. At the end of the pulse, few radioactive proteins were found in this fraction; after +17 min in chaser, its proteins were half maximally labeled; they became maximally labeled between +37 and +57 min. Parallel electron microscopic radioautography of intact cells in slices pulse labeled with leucine-3H showed, however, that zymogen granules become labeled, at the earliest, +57 min post-pulse. We assumed that the discrepancy between the two sets of results was due to the presence of rapidly labeled condensing vacuoles in the zymogen granule fraction. To test this assumption, electron microscopic radioautography was performed on sections of zymogen granule pellets isolated from slices pulse labeled with leucine-3H and subsequently incubated in chaser. The results showed that the early labeling of the zymogen granule fractions was, indeed, due to the presence of highly labeled condensing vacuoles among the components of these fractions.

scholarly journals COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG

1972 ◽  
Vol 55 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Jacopo Meldolesi ◽  
Dario Cova
Keyword(s):  
Guinea Pig ◽  
Intracellular Transport ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Composition ◽  
Zymogen Granule ◽  
Exocrine Cells ◽  
Acid Method ◽  
Two Systems ◽  
Pancreatic Exocrine

Two methods of polyacrylamide gel electrophoresis (the acid method of Eytan and Ohad and the Na dodecylsulfate (SDS) disc method of Maizel) have been used for analyzing the proteins of gel fractions isolated from the guinea pig pancreatic exocrine cells and in particular the proteins bound to the membranes involved in the synthesis, intracellular transport, and discharge of secretory enzymes: rough (RM) and smooth microsome (SM) membranes, zymogen granule (ZG) membranes, and plasma membranes (PM). Since in the two systems the electrophoretic mobility of proteins depends on different factors (size, shape, and net charge of molecules in the acid system; size only in the SDS system) a deeper insight into the protein composition of the fractions could be obtained. The gel patterns of RM, SM, and ZG membranes turned out to be accounted for mainly by segregated secretory enzymes (in rough microsomes also by ribosome proteins) and thus were found to share most of the bands. In contrast, with highly purified membrane fractions different patterns were obtained: RM and SM membrane proteins turn out to contain a large number of different proteins with molecular weights varying between ∼150,000 and 15,000 daltons. The pattern of ZG membranes was greatly different in the two systems: only two bands were separated by the acid method and as many as 23 by the SDS method. PM gave a rather complex pattern in either system. Both ZG membranes and PM were found to contain a large proportion of low molecular weight proteins. Nothing appears in common between the proteins of SM membranes (primarily of Golgi origin) and those of ZG membranes, while the latter and PM exhibit a certain degree of similarity. By amino acid analysis we found only slight differences: relative to the other fractions: RM membranes were higher in basic amino acids and ZG membranes contained a larger amount of methionine. Taken together with recent data on lipid composition and enzyme activities of the same fractions, these results indicate that the membranes of the pancreatic exocrine cells are chemically and functionally distinct, and hence do not mix randomly with one another during the transport of secretory products.

scholarly journals COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG

1971 ◽  
Vol 49 (1) ◽  
pp. 150-158 ◽  
Author(s):  
J. Meldolesi ◽  
J. D. Jamieson ◽  
G. E. Palade
Keyword(s):  
Electron Transport ◽  
Guinea Pig ◽  
Enzyme Activities ◽  
Transport Systems ◽  
Zymogen Granule ◽  
Electron Transport Chains ◽  
Microsomal Membranes ◽  
Pancreatic Exocrine ◽  
Exocrine Cell ◽  
Secretory Products

A comparative study of the enzymic activities of membrane fractions derived from guinea pig pancreatic homogenates has yielded the following results: Rough microsomal membranes (derived from the rough ER) have the reductase activities of the two microsomal electron transport systems but lack enzyme activities of Golgi-type (TPPase) and plasmalemmal-type (5'-nucleotidase, ß-leucyl naphthylamidase, Mg-ATPase). Smooth microsomal membranes (derived primarily from the Golgi complex), zymogen granule membranes, and plasmalemmal fractions possess overlapping enzyme activities of plasmalemmal type, in different relative concentrations for each fraction. In addition, the smooth microsomal membranes exhibit TPPase and ADPase activity and share with rough microsomes the reductase activities of the two electron transport chains. Taken together with recent data on the lipid composition of the same fractions (2), these results indicate that the membranes of the pancreatic exocrine cell are chemically and functionally distinct, and hence do not mix with one another during the transport of secretory products.

scholarly journals Studies on the synthesis and intracellular transport of lipoprotein particles in rat liver.

1975 ◽  
Vol 64 (2) ◽  
pp. 356-377 ◽  
Author(s):  
H Glaumann ◽  
A Bergstrand ◽  
J L Ericsson
Keyword(s):  
Chemical Composition ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
High Rate ◽  
Very Low Density Lipoproteins ◽  
Lipoprotein Particles ◽  
Parallel Change ◽  
Rough Er

Lipoprotein particles (d less than 1.03 g/ml) were isolated from rough and smooth microsomes and from the Golgi apparatus of rat liver, and were characterized chemically and morphologically. The rough endoplasmic reticulum (ER) particles were rich in protein (50%) and contained phospholipids (PLP) and triglycerides (TG) in smaller amounts, whereas the lipoprotein particles emanating from the smooth ER, and especially the Golgi apparatus, were rich in TG and PLP, resembling very low density lipoproteins (VLDL) of serum. The difference in chemical composition among the particles was associated with change in size both in situ and in isolated lipoprotein fractions. The rough ER particles were 200-800 A in diameter (mean similar to 420 A); the smooth er particles 200-900 A (mean similar to 520 A); the Golgi particles 350-950 A (mean similar to 580A); and serum VLDL 300-800 A (mean similar to 450 A). Generally, lipoprotein particles were rare in the rough ER, frequent but diffusely dispersed in smooth ER, and occurring mainly in clusters in "secretory vesicles" of the Golgi complex. They were seldom observed in the cisternal compartments of the Golgi complex. At short intervals (less than 15 min), intravenously injected radioactive glycerol was preferentially channelled into TG, whereas at later time points the majority of the isotope was recovered in the PLP. Three TG pools were distinguished: (a) a cytoplasmic pool with a slow turnover rate; (b) a membrane-associated TG pool; and (c) a pool corresponding to the TG moiety of lipoprotein particles, which showed the highest initial rate of labeling and fastest turnover. When, after pulse labeling, the appearance of incorporation of radioactive glycerol into TG or PLP of isolated lipoproteins was followed from one subcellular fraction to the other, a sequence of labeling was noted. During the first interval, TG from both rough and smooth microsomal lipoproteins displayed a high rate of labeling with peak value at 6 min, followed by a quick fall-off, while the Golgi lipoproteins reached maximal level at 10-20 min after administration. There was an interval of 10-15 min before the appearance of labeled VLDL in serum. It is concluded that the assembly of the apoproteins and lipid moieties into lipoprotein particles-presumed to be precursors of liver VLDL-begins in the rough ER and continues in the smooth ER. Also, there is a parallel change in chemical composition and size of the lipoprotein particles as they make their way through the ER and the Golgi apparatus. Some remodeling of the particles may take place in the Golgi apparatus before discharge into the circulation.

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