Background

In addition to synthesizing proteins to carry out cellular

functions, many cells must also produce and secrete addi-
tional proteins that perform their duties outside of the cell.
The mechanism of protein secretion fascinated many cell
biologists, including Palade. In early research on secretion,
cells were disrupted and the various organelles separated
by centrifugation. These cell fractionation studies had
shown that secreted proteins are present in membrane-
bound vesicles associated with the endoplasmic reticulum
(ER), where they are synthesized, and with the plasma
membrane, where they are eventually released from the
cell. Unfortunately, results from these studies were hard to
interpret due to difficulties in obtaining clean separations
between different organelles. To further clarify the path-
way, Palade turned to a newly developed technique, high-
resolution autoradiography, that allowed him to follow
radioactively labeled proteins as they moved within the
cell. His work led to the seminal finding that secreted pro-
teins travel within vesicles from the ER to the Golgi com-
plex, and then to the plasma membrane.
The Experiment
Palade wanted to identify which cell structures and
organelles participate in protein secretion. To study such a
complex process, he carefully chose an appropriate model
system for his studies, the pancreatic exocrine cell, which
is responsible for producing and secreting large amounts
of digestive enzymes.
Palade first examined the protein secretion pathway in
vivo by injecting guinea pigs with [
3
H]leucine, which was
incorporated into newly made proteins, thereby radioac-
tively labeling them. At time points from 4 minutes to 15
hours, the animals were sacrificed, and the pancreatic tis-
sue was fixed. By subjecting the specimens to autoradiog-
raphy and viewing them in an electron microscope, Palade
could trace where the labeled proteins were in cells at var-
ious times. As expected, the radioactivity localized in vesi-
cles at the ER at time points immediately following the
[
3
H]leucine injection, and at the plasma membrane at the
later time points. The surprise came in the middle time
points. Rather than traveling straight from the ER to the
plasma membrane, the radioactively labeled proteins
Classic Experiment 17.1
Following a Protein Out
of the Cell
T
he advent of electron microscopy allowed researchers to see the cell and
its structures at an unprecedented level of detail. George Palade utilized
this tool not only to look at the fine details of the cell, but also to analyze
the process of secretion. By combining electron microscopy with pulse-chase
experiments, Palade uncovered the path proteins follow to leave the cell.
appeared to stop off at the Golgi complex in the middle of
their journey. In addition, there never was a time point
where the radioactively labeled proteins were not confined
to vesicles.
The observation that the Golgi complex was involved
in protein secretion was both surprising and intriguing. To
thoroughly address the role of this organelle in protein
secretion, Palade turned to in vitro pulse-chase experi-
ments, which permitted more precise monitoring of the
fate of labeled proteins. In this labeling technique, cells
are exposed to a radiolabeled precursor, in this case
[
3
H]leucine, for a short period of time known as the
pulse. The radioactive precursor is then replaced with its
nonlabeled form for a subsequent chase period. Proteins
synthesized during the pulse period will be labeled and
detected by autoradiography, while those synthesized during
the chase period, being nonlabeled, will not be detected.
Palade began by cutting the guinea pig pancreas into thick
slices, which were then incubated for 3 minutes in media
containing [
3
H]leucine. At the end of the pulse, he added
excess unlabeled leucine. The tissue slices were then either
fixed for autoradiography or used for cell fractionation.
To assure that his results were an accurate reflection of
protein secretion in vivo, Palade meticulously character-
ized the system. Once convinced that his in vitro system
accurately mimicked protein secretion in vivo, he pro-
(c)
(a) (b)
(d)
Figure 17.1
The synthesis and movement of guinea-pig pancreatic secretory proteins as revealed by electron microscope autoradiography. (a) At the
end of a 3-minute labeling period with [
3
H]leucine, the tissue is fixed, sectioned for electron microscopy, and subjected to auto-
radiography. M ost of the labeled proteins (the autoradiographic grains) are over the rough ER. (b) Following a 7-minute chase period with
unlabeled leucine, most of the labeled proteins have moved to the Golgi vesicles. (c) After a 37-minute chase, most of the proteins are
over immature secretory vesicles. (d) After a 117-minute chase, the majority of the proteins are over mature zymogen granules.
[Courtesy of J. Jamieson and G. Palade.]
ceeded to the critical experiment. He pulse-labeled tissue
slices with [
3
H]leucine for 3 minutes, then chased the label
for 7, 17, 37, 57, and 117 minutes with unlabeled leucine.
Radioactivity, again confined in vesicles, began at the ER,
then traveled in vesicles to the Golgi complex, and
remained in the vesicles as they passed through the Golgi
and onto the plasma membrane (see Figure17.1). As the
vesicles traveled farther along the pathway, they became
more densely packed with radioactive protein. From his
remarkable series of autoradiograms at different chase
times, Palade concluded that secreted proteins travel in
vesicles from the ER to the Golgi and onto the plasma
membrane, and that throughout this process they remain
in vesicles and do not mix with the rest of the cell.
Discussion
Palades experiments gave biologists the first clear look at
proteins traveling through the secretory pathway. His
studies on the pancreatic exocrine cells yielded two semi-
nal observations. First, that secreted proteins pass through
the Golgi complex on their way out of the cell. This was
the first function assigned to the Golgi complex. Second,
secreted proteins never mix with other cellular proteins;
they are segregated into vesicles throughout the pathway.
These findings were predicated on two important aspects
of the experimental design. Palades careful use of electron
microscopy and autoradiography allowed him to look at
the fine details of the pathway. Of equal importance was
the choice of a cell type devoted to secretion, pancreatic
exocrine cell, as a model system. In a different cell type,
significant amounts of nonsecreted proteins might have
also been produced during the pulse, possibly leading to
ambiguous results.
Palades work set the stage for more detailed studies.
Once the secretory pathway was clearly described, entire
fields of research were opened up to investigation, in the
synthesis and movement of both secreted and membrane
proteins. For this ground-breaking work, Palade was
awarded the Nobel Prize for Physiology and Medicine in
1974.