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The Golgi complex is a series of membrane compartments through which proteins destined for the plasma membrane, secretory vesicles, and lysosomes move sequentially. A model is proposed whereby these three different classes of proteins are sorted into different vesicles in the last Golgi compartment, the trans Golgi network. This compartment corresponds to a tubular reticulum on the trans side of the Golgi stack, previously called Golgi endoplasmic reticulum lysosomes (GERL).
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The trans Golgi Network: Sorting at the Exit Site of the Golgi Complex
Author(s): Gareth Griffiths and Kai Simons
Source:
Science,
New Series, Vol. 234, No. 4775 (Oct. 24, 1986), pp. 438-443
Published by: American Association for the Advancement of Science
Stable URL: http://www.jstor.org/stable/1697339
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The
trans
Golgi
Network:
Sorting
at
the
Exit
Site
of
the
Golgi
Complex
GARETH GRIFFITHS AND KAI SIMONS
The Golgi complex is a series of membrane
compartments
through which proteins destined for the plasma mem-
brane, secretory
vesicles, and lysosomes move sequential-
ly. A model is proposed whereby these three different
classes of proteins are sorted into different vesicles in the
last Golgi compartment, the tramns
Golgi network. This
compartment
corresponds to a tubular reticulum on the
trans side of the Golgi stack, previously called Golgi
endoplasmic
reticulum lysosomes (GERL).
P ROTEINS THAT ARE INSERTED INTO, OR TRANSLOCATED
across, the membrane of the rough endoplasmic reticulum
(ER) must contain information that determines their final
destination (1). This destination may be intra- or extracellular.
For
some membrane proteins the target organelle is the ER itself, and
they are not transported further (2). All other proteins are carried to
the Golgi complex, which is a series of membrane compartments
through which proteins in transit pass sequentially (3-7).
Most proteins that are synthesized in the ER are glycoproteins.
The elaborate series of covalent modifications that glycoproteins
undergo in the ER and subsequently in the Golgi complex are now
well established (8). The structure of the oligosaccharide moiety at
any one time after synthesis indicates which enzymes of the process-
ing pathway the protein has been exposed to. In many cases this
information can be used to indicate the compartment reached
during transport. However, this characterization is not absolute,
because our knowledge of the molecular composition of the com-
partments in the pathway is still rudimentary.
Newly synthesized proteins that pass through the Golgi complex
can be classified into three groups characterized by their exit
pathway from the Golgi. The first group consists of plasma mem-
brane proteins and proteins that are constitutively secreted. The
second group consists of secretory proteins that are packaged into
secretory granules that are, in turn, released in response to an
external signal. Lysosomal enzymes constitute the third group.
These three classes of proteins seem to be transferred
through an
identical pathway until the last compartment of the Golgi complex,
because (i) proteins of all three classes are found in all Golgi
cisternae by immunocytochemistry (9) and (ii) the oligosaccharide
moieties of glycoproteins in all three classes (with the exceptions of
some oligosaccharides of lysosomal enzymes) are covalently modi-
fied by ER and Golgi enzymes in a similar fashion (8). Therefore,
the sorting of proteins into separate pathways occurs either in the
last Golgi compartment or after exit from the Golgi. We now discuss
the structure and function of the intracellular site where these
sorting events are proposed to occur.
The authors are at the European Molecular Biology Laboratory, Postfach 10.2209,
6900 Heidelberg, Federal Republic of Germany.
438
Current Models of the Golgi Complex
In the simplest possible model, the Golgi stack consists of three
discrete cisternae, or groups of cisternae, which can be distinguished
on the basis of distinct cytochemical reactivities (Fig. 1). Although
this interpretation may be an oversimplification (10), these cyto-
chemically defined zones have greatly influenced most current
models of Golgi functions (3-7) (Fig. 1). In present models, the
different
cytochemical reactions in the Golgi cisternae
are
thought to
reflect the presence of three different functional compartments
through which proteins pass vectorially. Various functions of the
Golgi have been proposed to occur in each of these three compart-
ments, although most of these assignments are still tentative.
Another feature of these models is that selective transfer
of lipid
and proteins from one compartment to the next occurs through
vesicular transport (7, 11). Vesicles are thought to enter the system
by budding off specialized regions of the ER, termed the transitional
elements, and then to fuse with the cis compartment of the Golgi
stack. After a succession of sequential budding and fusion events
that transport the proteins from one cisterna to the next, the
proteins exit by budding off the trans cisterna or cisternae (Fig. 1).
The vesicles that have been proposed to mediate transport between
the subcompartments have proven elusive. There is still no direct
evidence for their existence.
The current models of the Golgi complex usually do not incorpo-
rate the Golgi endoplasmic reticulum lysosome (GERL) structure
(12, 13). Its role has remained controversial despite documentation
of the existence of this Golgi-associated structure in many morpho-
logical studies of different cell types.
The GERL Concept
The recognition of the importance of the GERL structure began
in the 1960's with Novikoffs pioneering morphological studies of
lysosomes, in which acid phosphatase was used as the cytochemical
marker (12). Novikoff saw reaction product not only in lysosomes,
but also in a reticular structure in the perinuclear region of spinal
neurons. The location of this structure in the light microscope
corresponded precisely with the cytochemical reaction for thiamine
pyrophosphatase, which was already an accepted marker for the
Golgi region in light microscopic studies. Subsequent electron
microscopic observations led him to propose that this acid phospha-
tase-reactive structure was a specialized region of smooth ER that
was directly continuous with the rough ER, as well as with dense
bodies (lysosomes). He believed that this structure was specialized
for the biogenesis of lysosomes, and he introduced the acronym
GERL to indicate that this structure is "intimately related to the
Golgi saccule (G), that it is part of the ER, and that it forms
lysosomes (L)" (12, p. 358). He then suggested that after lysosomal
enzymes were synthesized by the ER, they bypassed the Golgi stack
via the GERL. The same claim was later made for secretory proteins
SCIENCE, VOL. 234
m
Fig. 1. Prevailing
model of the Gol-
gi complex.
The major
feature
is the
presence of three compartments
through which transit proteins
move
sequentially.
The
cytochemical
activities
and the specific
funmctions
ascribed to the compartments
are
shown (6, 7). The model proposes
that
proteins
move
from
one station
to the next by vesicular
transport.
Abbreviations:
AGT, N-acetylglu-
cosamine-transferase
I; GT, galacto-
syl
transferase;
ST, sialyl
transferase;
OS04, reduced
osmium tetroxide;
NADPase,
nicotinamide
adenine di-
nucleotide phosphatase; TPPase,
thiamine
pyrophosphatase.
^^ ? . .. .*
0
CN9ADPase Medial 9
0
CTPPase trans GT ST7K
when it became clear that immature secretory granules were bud-
ding off the GERL (14).
The morphological and cytochemical differences between GERL
and the cisternae that made up the Golgi stack are well documented
(15). In most cell types, the GERL structure that stained for acid
phosphatase had morphological features distinct from the Golgi
cisternae.
However, part of the GERL often appeared
to be cisternal
and, in the absence of a cytochemical reaction, was difficult to
distinguish from the other cisternae on the trans side of the Golgi
stack. The membranes forming the GERL were usually more
tubular, were thicker in cross section, and had luminal contents that
often reacted differently with electron-opaque strains from Golgi
cisternae. The presence of budding, coated vesicles and extensive
coated membrane regions further distinguished GERL from other
Golgi cisternae (12, 13, 16). Electron microscopy showed that it was
the trans Golgi cisternae adjacent
to GERL that contained thiamine
pyrophosphatase and that, in most cases, the GERL structure itself
did not (14, 17).
GERL was always a strictly morphological concept, and, al-
though biochemists often referred loosely to the "Golgi-GERL
region" of the cell, the GERL itself was not generally considered
part of the exocytic pathway. Morphologists were divided between
those who accepted Novikoffs theories and those who believed that
GERL was the last (3), or most "mature,"
cisterna
of the Golgi stack
(18). In fact, little evidence corroborated its continuity with the ER.
Several lines of evidence argue strongly against such continuities
(19). For this reason the acronym GERL no longer seems to be
appropriate for this structure, which has been given many other
names, including the "boulevard peripherique" (20), the trans
tubular network (21), and the trans Golgi reticulum (22). To avoid
confusion with the ER or with the original GERL concept, we
propose to call this structure the trans Golgi network (TGN). This is
a modification of the terminology used by Rambourg et al. (21) and
emphasizes that the TGN belongs to the Golgi complex.
The TGN-the Exit Compartment of the
Golgi Complex?
In the model we are proposing, the TGN is a specialized organelle
on the trans side of the Golgi stack that is responsible for routing
proteins to lysosomes, secretory vesicles, and the plasma membrane
from the Golgi complex (Fig. 2).
Novikoff used acid phosphatase as a marker for the TGN. This
cytochemical reaction is probably produced by phosphatases in
transit to the lysosomes. Recently, other markers
labeling the TGN
have been identified. Within the Golgi complex, the TGN appears
to be the main, and perhaps exclusive, site of clathrin localization
24 OCTOBER
1986
(23, 24). Although coated, presumably budding structures on the
rim of the cisternae in the Golgi stack are frequently observed; these
could not be labeled with antibodies against clathrin (23, 24), which
suggests that a different protein forms these structures in the Golgi
stack. Sialyl- and galactosyltransferase
have also been localized in the
TGN in hepatocytes (25, 26). These enzymes, which have well-
defined functions at the terminal stages of nitrogen-linked glycosyla-
tion, may be present in the trans cisternae of the Golgi stack (27).
The distinction between the trans cisterna and the TGN in Fig. 2 is a
tentative one, based only on the nonoverlapping localizations, in
many cell types, of TPPase (thiamine pyrophosphatase) reactivity
(trans) and acid phosphatase (TGN) by cytochemistry (15).
Exit of Plasma Membrane Proteins from the
Golgi Complex
A significant step in defining the TGN as a sorting organelle for
outgoing membrane traffic was the observation that, at 20?C, the
transport of newly synthesized viral glycoproteins from the ER to
the cell surface was blocked (28). The proteins were terminally
glycosylated and sialylated (29), but remained inside the cell. On
warming the cells, these viral proteins were transported rapidly to
the cell surface, which indicated that the block was fully reversible.
Vesicular stomatitis virus (VSV) G protein accumulated in the
TGN, which was apparent
on one side of the Golgi stack as a tubular
network that reacted for acid phosphatase (24). The large amount of
G protein that accumulated in this structure caused the protein to be
packed into regular arrays.
This facilitated the identification of the
compartment, even in the absence of additional markers.
During the
low-temperature block, the TGN had many budding structures
with
cytoplasmic coats. Although many of these coated structures reacted
immunocytochemically with antibodies to clathrin, others unexpect-
edly failed to do so. Both types of coated, budding structures failed
to react with antibodies to the G protein, in agreement with earlier
studies (30). Thus, clathrin-coated vesicles do not seem to transport
newly synthesized plasma membrane proteins from the Golgi to the
cell surface, in contrast to the proposal by Rothman et al. (31).
Recent studies in yeast have also provided compelling evidence
against a role for clathrin in exocytosis. Deletion of the clathrin
heavy-chain gene had little influence on the rate of intracellular
transport and secretion of newly synthesized invertase (32).
Nonpolarized cells such as fibroblasts and macrophages are
thought to exocytose plasma membrane proteins uniformly over
their surfaces, although, when they are carefully
examined, preferen-
c cis D
C Medial D
( trans
~~~~~~~~~~~~~~~~~.,
_
I l
:onstitutive Regulated
pathway pathway
I i
Plasma membrane
Fig. 2. Model for sorting in the
TGN. The distinction
between
the
compartment
labeled
"trans"
and the
TGN is a tentative
one. It is based
only
on the consistent
colocalization
in many
cell
types
of TPPase
reactiv-
ity (trans) and acid phosphatase
(TGN) by cytochemistry
(15). A
part of the TGN may appear
as a
cisterna
and be difficult
to distin-
guish from other Golgi cisternae
[see,
for
example,
figure
4b in (24)].
The mechanism
of transport be-
tween the Golgi compartments
has
not been considered
in this scheme
(Fig. 1). In epithelial
cells,
it appears
likely
that
two types
of vesicles
bud
off the TGN and carry
apical
and
basolateral
plasma
membrane
pro-
teins, respectively,
to their surface
domains
(35).
ARTICLES 439
tial exocytosis has been observed at the leading, motile edge of some
cells (33). Polarized epithelial cells, which make up the bulk of the
cell types in tissues, contain two different plasma membrane do-
mains, apical and basolateral, which are separated from each other
by tight junctions (34, 35). In the Madin-Darby canine kidney
(MDCK) cell line, which is polarized in culture, membrane proteins
targeted to the apical domain are sorted from those destined for the
basolateral domain at the Golgi complex (36). The site of sorting is
probably the TGN because the viral membrane proteins that are
differentially targeted to the two domains can be colocalized by
immunocytochemistry throughout the Golgi stack (37), and they
remain in contact with each other when their intracellular
transport
is blocked at 20?C (29).
Those secretory proteins that are constitutively secreted by eu-
karyotic cells are presumably transported by a mechanism similar, if
not identical, to that responsible for the transport of plasma
membrane proteins. The two groups of proteins may be carried to
the cell surface in the same vesicles (38). Although this mechanism is
likely, the only direct data in its support are the colocalization of
VSV membrane glycoprotein with albumin and transferrin in
putative transport vesicles (39).
Exit of Secretory Proteins from the
Golgi Complex
Regulated secretory cells concentrate proteins destined for export
into storage granules. In the early stages of formation these are
clearly continuous with the TGN (14). Secretory cells can carry
out
both constitutive and regulated secretion simultaneously, using
separate carrier vesicles (38).
Proteins that have moved through the Golgi complex and are
destined for the regulated secretory pathway seem to condense in
the lumen of the TGN before they are sorted into a developing
secretory granule (38, 40). These developing secretory granules all
contain patches of clathrin (41). Because the patches do not cover
the entire cytoplasmic surface of the budding granule, it is unlikely
that clathrin is necessary for the budding of the secretory granules.
The clathrin remains bound even after the secretory granule has
been released from the TGN, but dissociates as the granule matures
and becomes more electron-opaque. This bound clathrin may
recycle excess membrane back to the TGN (38). The proteolytic
cleavage of prohormones such as proinsulin occurs in these coated
granules (42). Whether this proteolytic processing starts while the
granule is still attached to the TGN membrane is not known.
Another posttranslational modification of secretory proteins, sulfa-
tion of tyrosine residues, probably occurs in the TGN (43), al-
though this has yet to be confirmed by immunocytochemical
localization of the sulfotransferase responsible for this reaction.
Exit of Lysosomal Proteins from the
Golgi Complex
The biochemical changes that lysosomal enzymes undergo during
their transport from ER to lysosomes are now being elucidated (8,
44, 45). After the enzymes are transferred from the ER to the Golgi
complex, a specific subset of high-mannose oligosaccharide chains
are phosphorylated at the 6-carbon position of mannose by the
concerted action of two early Golgi enzymes. This alteration serves
as an address tag that allows these proteins to bind to the 215-kD
mannose-6-phosphate (M6P) receptor. Further biochemical events
in the pathway are a low pH-mediated dissociation of the ligands
from the receptor and two other, relatively
late changes, a proteolyt-
440
ic cleavage and a dephosphorylation. The latter changes probably
occur in the lysosome itself. Phosphorylation of lysosomal enzymes
is thought to occur in the cis Golgi compartment (46), although
conclusive evidence is lacking. Exactly where the lysosomal enzymes
bind to the receptor is not yet clear. In many cases, the oligosaccha-
ride chains of lysosomal enzymes that are not phosphorylated
undergo the same modifications seen in other glycoproteins, includ-
ing the trimming of mannose residues and the addition of the
terminal sugars, N-acetylglucosamine, galactose, fucose, and sialic
acid. Thus, those lysosomal enzymes which become sialylated must
move through the whole Golgi stack into the TGN. This conclusion
is supported by cytochemical data (19, 47) and by recent immuno-
cytochemical studies (26, 48), both of which reveal low levels of the
enzymes throughout the Golgi stack and a higher concentration in
the TGN.
There is still some dispute as to whether the lysosomal enzymes in
the TGN are bound to the M6P receptor and whether this receptor
is at all present in this compartment. The receptor may be concen-
trated in TGN and in coated membrane buds of this structure in
Chinese hamster ovary (CHO) cells (49). In this study, however, no
markers were used to distinguish the TGN from endocytic struc-
tures (Fig. 3). In human hepatoma cells (Hep G 2) (26), albumin
was used as a specific marker to distinguish the exocytic pathway
from the endocytic one. In these cells, double-labeling experiments
with frozen-section immunocytochemistry showed that the M6P
receptor as well as lysosomal enzymes were concentrated in TGN
and included in coated buds. Whether these label with antibodies to
clathrin is not yet known.
Other studies, based on immunoperoxidase localization, have
suggested that the M6P receptor is concentrated in the cis compart-
ment of the Golgi complex and that it is absent from the trans side
(50). We believe that these differences are technical in nature (50)
and that the most reasonable
interpretation of all the available data is
that the M6P receptor and lysosomal enzymes are present in low
concentrations throughout the Golgi stack, but are concentrated in
the TGN before they exit from the Golgi complex (Fig. 2). The
vesicles transporting the receptor-bound lysosomal enzymes from
the Golgi complex have not yet been characterized. Clathrin-coated
vesicles leaving from the TGN are the most attractive candidates at
present (51).
There is also an alternative mechanism for lysosomal enzyme
sorting by the M6P signal that depends on another receptor, a 46-
kD protein (52). Pathways even exist in which nonphosphorylated
lysosomal enzymes seem to be correctly targeted to lysosomes (45).
To what extent these alternative pathways involve the TGN is not
known. It is also not known how lysosomal membrane proteins
reach the lysosomes. The few that have been characterized are
heavily sialylated (53), which indicates that they have passed
through the TGN. However, these proteins are not phosphorylated,
suggesting that the mechanism of targeting does not depend on an
M6P recognition system (53).
The Size andpH of the TGN
The TGN seems to be able to vary in size in response to the cell's
requirements. A "hypertrophy" of TGN in secretory cells, for
example, occurs after cells are stimulated to secrete (54). Stereologi-
cal studies of the TGN in baby hamster kidney (BHK) cells indicate
that this compartment almost doubles in size after VSV infection at
20?C; within minutes of reversing the temperature block, it returns
to its normal size (55). Moreover, in a number of pathological
conditions that require more lysosomal enzyme production, there is
also a hypertrophy of the TGN (15).
SCIENCE, VOL. 234
Fig. 3. Endosomes labeled
by endocytosed
peroxidase.
Thick section (0.5
uim)
through the Golgi region of a BHK cell that had endocytosed
horseradish
peroxidase
for
2 hours
at 20?C.
Stained
endosome structures are
very
close to the Golgi stack
(arrow),
and the two sets of structures would
not be distinguished
from each
other in the absence
of a marker.
The two
compartments
are, however, functionally
distinct (24) [from the study
described
in (63)]. Abbreviation:
N, nucleus.
x42,120.
Recent studies indicate that the TGN may be mildy acidic. This
was shown with the lysosomotropic agent 3-(2,4-dinitroanilino)-3'-
amino-N-methyl-dipropylamine (DAMP), which accumulates
selec-
tively in acidic organelles (56). In the protonated form, DAMP can
be visualized in the electron microscope either by indirect immuno-
peroxidase staining or by the use of antibodies to DAMP marked by
colloidal gold. In fibroblasts this reagent accumulates in membrane
structures on the trans side of the Golgi stack. Although not
unequivocally identified in these studies, we suggest that this labeled
structure is the TGN. An acidic pH in this organelle could explain
the effects of lysosomotropic agents on exocytic sorting (57).
If the TGN is acidic, its pH would not be expected to fall below 6
for several reasons. Because many of the lysosomal enzymes undergo
a proteolytic cleavage at a late stage of transport and probably in the
lysosome itself, these enzymes are likely to be in the precursor,
uncleaved form in the TGN. Biochemical and cytochemical evidence
suggests, however, that these precursors would be active at low pH
(58). It may, therefore, be important for the pH in the TGN, where
lysosomal enzymes are mixed with other transit proteins, to be
above the pH optimum for lysosomal enzyme activity, that is, above
pH 6.0. Further, because the M6P receptor and lysosomal enzymes
seem to be bound to each other in the TGN, the pH must be above
6. At lower pH's the ligands would dissociate from the receptor (8,
44, 45). Finally, a number of viral spike proteins function as
fusogens atpH's around 6 or below (59). Even brief exposure to low
pH causes irreversible loss of the fusogenic activity essential for
infectivity. Thus, these proteins are probably not exposed to a pH
significantly below 6 during their transport to the cell surface (60).
Target Organelles for Proteins Exiting
from the TGN
The vesicles that carry newly synthesized plasma membrane
proteins from the TGN have not yet been characterized, although
preliminary immunocytochemical data suggest potential candidates
(24, 28, 61). Those vesicles containing newly synthesized VSV G
proteins seem to fuse directly with the plasma membrane rather than
with an intermediate organelle, such as an endosome compartment
(62). During the low temperature-dependent accumulation of VSV
24 OCTOBER 1986
G protein in TGN (24), exogenously added horseradish
peroxidase was simultaneously endocytosed by the same cells. At
20?C, this marker selectively fills endocytic compartments without
being transported to the lysosomes (63). In a double-labeling
procedure, both G protein and peroxidase were visualized in the
same sections and endosome structures and the TGN, although
often adjacent, were nevertheless distinct (Fig. 3). No G protein
could be detected in endosome structures at any time during
exocytosis. The same conclusion has been recently obtained in a
similar double-labeling procedure with exocytosing G protein for
the outward pathway and recycling transferrin for the inward one
(64).
Of the three classes of proteins that exit from the TGN, the
lysosomal proteins are those whose sorting is best understood at the
molecular level. The key processes in this sorting are a neutral pH
binding of phosphorylated lysosomal enzymes to the M6P receptor
shortly after leaving the ER and a low-pH dissociation of ligand and
receptor at a later step in transport, which allows the receptor to
recycle for further rounds of sorting. The precise cellular pathway
that the lysosomal enzymes follow from the ER to lysosomes is still
not clear.
Both biochemical (8, 44, 45) and immunocytochemical (26, 48,
49) data suggest that the M6P receptor is in the TGN, in the
endosomes, and on the plasma membrane, where it seems to be
concentrated in coated pits. Both approaches have failed to detect
the receptor in lysosomes. The receptor on the cell surface can bind
exogenous lysosomal enzymes with high affinity and target them to
lysosomes. However, this pathway is likely to be a relatively minor
one because an excess of M6P in the culture medium (which
effectively removes any bound ligand from the receptor) failed to
alter the correct targeting of newly synthesized enzymes (65). The
most likely target of TGN-derived vesicles carrying lysosomal
enzymes seems to be an endosome compartment. Since there are
compelling arguments against the presence of significant acid hydro-
lase activity in the total endosome population (62), lysosomal
enzymes bound to the M6P receptor may be directed to a specific
subset of endosomes where their dissociation occurs. A likely
candidate is a class of multivesicular bodies, referred to as a
multivesicular endosome (66), commonly found in the Golgi region
of cells. The fusion of the TGN-derived vesicles with a multivesicu-
lar endosome may be the event that converts a "late" endosome
compartment into a lysosome.
How does the presence of the M6P receptor on the cell surface fit
into the above scheme? It may be a subset of M6P receptor that has
been missorted from the TGN. Another, perhaps more likely,
possibility is that unoccupied receptor or a fraction of it, after
releasing its ligand in a late endosome compartment, may recycle to
the Golgi complex via the plasma membrane. This model has, in
part, already been proposed (44). In both cases exogenous ligand,
when present, could be targeted correctly to lysosomes by receptor-
mediated endocytosis.
M6P receptors in their different cellular locations are in dynamic
equilibrium with each other. When antibodies to the M6P receptor
were added to the culture medium of fibroblasts, the bulk of the
cellular receptors became bound to antibodies and were inactivated
within 2 hours (44, 67). This treatment led to an aberrant secretion
of lysosomal enzymes, presumably because fimctional receptors
became depleted from the Golgi complex.
TGN and Membrane Recycling
If the TGN is sorting and segregating proteins into separate
carrier vesicles for delivery to different post-Golgi destinations, the
ARTICLES 441
loss of TGN membrane in these sorting processes must be compen-
sated for by membrane recycling. The pathway for the return of
membrane components to the Golgi complex is not yet clear.
Various electron-opaque tracers or exogenous ligands that bind to
cell-surface
receptors have often been shown to be endocytosed into
tubular elements on the trans side of the Golgi stack. In most of
these studies, however, it has been difficult to conclude whether
uptake had occurred into endosome structures in the vicinity of the
Golgi complex or whether the tracers had actually moved into the
TGN or other Golgi compartments (Fig. 3). Double-label electron
microscopy can distinguish between these alternatives, but such
studies have been rare. Endocytosed ligands may be localized, at
least transiently, in structures with the cytochemical properties of
TGN (68). Cell-surface markers have been shown in a few cases to
enter into the cisternae of the Golgi stack and even into the newly
formed secretory granules (6, 69). It has also been shown that in
CHO cells, endocytosed transferrin, initially found in a pH 5.5
endosomal compartment, later moved into tubular structures in the
vicinity of the Golgi stack that had a pH of 6.4 (70). Whether the
latter is the TGN or a late endosome compartment is not yet clear
[see also (64)].
Both transferrin and the transferrin receptor, if desialylated in
vitro, seem to be resialylated during membrane recycling (71). This
resialylation presumably occurs in the TGN. The half-time for
resialylation-2 to 3 hours in the case of the receptor-is much
slower than that needed for the movement of receptors from the
plasma membrane to the endosomes and back, a process requiring
only minutes. The transferrin receptor might have to make many
endocytic rounds on average, before it is diverted into the TGN.
This could explain why it has often been difficult to observe
membrane recycling into the TGN.
More work is necessary to elucidate how the exocytic and the
endocytic pathways are linked. The phenotype of some yeast
mutants suggests that endocytosis may be coupled obligatorily to
the last steps of protein secretion (72). Furthermore, mutants of
CHO cells that are defective in acidification of endosomes had
abnormal terminal glycosylation of some viral spike glycoproteins
and of several endogenous secretory glycoproteins (73). Although
the molecular nature of the defects in these mutants is unknown, one
possibility is that endosomes and the Golgi complex share gene
products necessary for the function of both organelles (57).
Conclusions
We propose that the sorting of three distinct classes of proteins
into separate pathways is accomplished by the trans Golgi network.
This structure is defined as the last station along the Golgi pathway
involved in the final processing steps ofN-linked glycosylation in the
cell. The TGN must now be further characterized in different cell
types to establish its precise role in sorting of plasma membrane,
lysosomal, and secretory proteins.
The pathways that connect the TGN with endosomes, lysosomes,
and plasma membrane must be studied in well-defined systems. This
task will best be accomplished by combining immunocytochemical
and biochemical approaches under conditions in which a defined
transit protein is blocked in a specific and identified site, then
released and followed kinetically in a quantitative manner. The
elucidation of molecular mechanisms of membrane sorting will have
to await a reconstitution of TGN functions in the test tube, an
approach already in use for studying transport between Golgi
compartments
(7).
REFERENCES AND NOTES
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10. Although
much
insight
into the three-dimensional structure
of the Golgi
stack
has
been obtained
from an excellent
serial-section
reconstruction
study (21), the
number of discrete
compartments
in the Golgi complex
is not known. This
approach
is limited
by the diameters
of the cisternac
and many
of the tubular
connections between adjacent
structures,
which are near the thickness
of the
section.
Small connections can therefore be missed. A complete
three-dimensional
model
of the Golgi stack
would be possible only if each
compartment
could be
selectively
and completely
filled
with different
electron-opaque
markers
prior
to
serial
thin-section reconstruction.
This has not yet occurred. Current models
assume that the
different
cytochemical
reactions
do not
overlap,
although
no two
of
the three
reactions have ever
been localized
simultaneously
in the same
Golgi
stack.
In fact,
there is evidence
in some
systems
that the different
cytochemical
reactions
may
overlap
(17, 19).
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17. Although
the distinction
between
these two structures
is usually
clear
cytochemi-
cally,
the acid
phosphatase
and
thiamine
pyrophosphatase
reactions
overlap
or mix
in a few cases. This is true in some tissue
culture cells
[G. Griffiths,
P. Quinn,
G.
Warren,J.
Cell
Biol.
96, 835 (1983)] and
in cells
during particular developmental
stages
or in response
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physiological
or pathological
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[C.
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Cvytochem.
31, 1041 (1983); A. I. Doine,
C.
Oliver,
A. R. Hand,
ibid.
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18. During
this period
the "maturation"
model of the Golgi
stack had
support
[D. J.
Morr6,
J. Kartenbeck,
W. W. Franke,
Biochim.
Biophys.
Acta
559, 71 (1979)]. This
theory proposed
that
the cisternae
themselves
"moved"
across the stack,
maturing
in the process.
For arguments
against
this model,
see (7).
19. S. Goldfischer,J.
Histochem.
Cytochem.
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20. D. J. Morr6 and L. Ovtracht,
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61 (1977).
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Y. Clermont,
L. Hermo,
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G. Warren,
I. Stuhlfauth,
B. M. Jockusch,
Eur.
J. Cell
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26, 52
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M. Amherdt,
D. Louvard,
A. Perrelet,
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J. W. Slot, G. J. A. M. Strous,
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101, 2253 (1985).
27. Galactosyl
transferase
is colocalized
with TPPase
in HeLa cells [J. Roth and E.
Berger,
ibid.
93, 223 (1985)]. The TGN was not identified
in this study,
and
thiamine
pyrophosphatase
might
react in HeLa
cells
with both the trans
compart-
ment and the TGN, as it does in other undifferentiated
cells (17). Galactosyl
transferase
is present
in the TGN of hepatoma
cells
and liver
hepatocytes
(26).
H. J.
Geuze
(personal
communication)
has
also
consistently
found
low concentrations
of
this enzyme
in one or two cisternae
on the trans
side of the Golgi stack. These
results
suggest
that
galactosyl
transferase
may
be present
in the preceding
Golgi
compartment
(trans
in Fig. 2) as well as in the TGN. Further
data are needed
to
clarify
this point
both for this enzyme
and for sialyl
transferase.
28. K. Matlin
and K. Simons,
Cell
34, 233 (1983); J. Saraste
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E. Kuismanen,
ibid.
38, 535 (1984).
29. S. D. Fuller,
R. Bravo,
K. Simons,
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4, 297 (1985).
30. J. Wehland,
M. C. Willingham,
M. G. Gallo,
I. Pastan,
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28, 831 (1982).
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H. G. Pettegrew,
R. E. Fine,J. CellBiol.
86, 162 (1980).
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Spring
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1101 (1984).
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99, 2131 (1984); D. E. Misek,
E. Bard,
E.
Rodriguez-Boulan,
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S. D. Fuller,
K. Simons,J.
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SCIENCE, VOL. 234
442
37. M. J. Rindler,
I. E. Ivanov,
H. Plesken,
E. Rodriguez-Boulan,
D. D. Sabatini,J.
Cell
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98, 1304 (1984).
38. R. B. Kelly,
Science
230, 25 (1985).
39. G. J. A. M. Strous
et al.,J. Cell Biol.
97, 1815 (1983).
40. J. Tooze and
S. Tooze, ibid.,
in press.
41. L. Orci et al., Cell
39, 39 (1984).
42. L. Orci et al., ibid.
42, 671 (1985).
43. W. B. Huttner,
Nature (London) 299, 273 (1985); R. W. H. Lee and W. B.
Huttner,
Proc.
Natl. Acad.
Sci.
U.S.A.
82, 6143 (1985); D. B. Baeuerle
and W. B.
Huttner,
in preparation.
44. G. G. Sahagian,
Biol.
Cell.
51, 207 (1984).
45. G. E. Creek and W. S. Sly,
in
Lysosomes
in Biology
and
Pathology,
J. T. Dingle,
R. T.
Dean,
W. S. Sly, Eds. (North-Holland,
Amsterdam,
1984).
46. D. Goldberg
and S. Kornfeld,J.
Biol.
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258, 3159 (1983).
47. D. F. Bainton,
and M. G. Farquhar,J.
Cell
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45, 54 (1970).
48. H. J. Geuze et al., ibid.
98, 2047 (1984).
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I. H. Pastan,
G. G. Sahagian,
J. Histochem.
Cytochem.
31, 1
(1983).
50. Using detergent
permeabilization
and immunoperoxidase,
W. Brown
and M. G.
Farquhar
[Cell
36, 295 (1984)] have concluded
that,
within
the Golgi complex,
the M6P receptor
was observed
mainly
in the cis
compartment.
These different
results could be due to differences in cell
type
or to different
immunocytochemical
techniques
(6). With the peroxidase
technique,
a quantitatively
minor
amount of
antigen may,
however,
in some
cases,
be amplified by the enzymatic
reaction.
51. E. Schulze-Lohoff,
A. Hasilik,
K. von Figura,J.
Cell
Biol.
101, 824 (1985); C. A.
Campbell
and L. H. Rome,J.
Biol.
Chem.
258, 13347 (1983); G. C. Sahagian
and
C. J. Steer,
ibid.
260, 9838 (1985).
52. B. Hoflack and S. Kornfeld,
Proc.
Natl. Acad. Sci.
U.S.A. 82, 4428 (1985).
53. V. Lewis et al.,J. Cell
Biol.
100, 1839 (1985); J. W. Chen,
T. L. Murphy,
M. C.
Willingham,
I. Pastan,
J. T. August,
ibid.
101, 85 (1985); J. Lippincott-Schwartz
and D. M. Fambrough,
ibid. 102, 1593 (1986); I. Mellman
and A. Helenius,
personal
communication.
54. A. R. Hand and
C. Oliver,
J. Histochem.
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32, 403 (1984).
55. G. Griffiths
et al., in preparation.
56. R. G. W. Anderson
and R. K. Pathak,
Cell
40, 635 (1985).
57. I. Mellman,
R. Fuchs,
A. Helenius,
Annu. Rev. Biochem.
55, 663 (1986).
58. A. Frisch and E. F. Neufeld,
J. Biol. Chem. 256, 8242 (1981). That the
cytochemical
reactions
for
acid
phosphatase
and
aryl
sulfatase occur
atpH 5 but not
at neutral
pH's also
suggests
that
the enzymes
are
potentially
active.
37. M. J. Rindler,
I. E. Ivanov,
H. Plesken,
E. Rodriguez-Boulan,
D. D. Sabatini,J.
Cell
Biol.
98, 1304 (1984).
38. R. B. Kelly,
Science
230, 25 (1985).
39. G. J. A. M. Strous
et al.,J. Cell Biol.
97, 1815 (1983).
40. J. Tooze and
S. Tooze, ibid.,
in press.
41. L. Orci et al., Cell
39, 39 (1984).
42. L. Orci et al., ibid.
42, 671 (1985).
43. W. B. Huttner,
Nature (London) 299, 273 (1985); R. W. H. Lee and W. B.
Huttner,
Proc.
Natl. Acad.
Sci.
U.S.A.
82, 6143 (1985); D. B. Baeuerle
and W. B.
Huttner,
in preparation.
44. G. G. Sahagian,
Biol.
Cell.
51, 207 (1984).
45. G. E. Creek and W. S. Sly,
in
Lysosomes
in Biology
and
Pathology,
J. T. Dingle,
R. T.
Dean,
W. S. Sly, Eds. (North-Holland,
Amsterdam,
1984).
46. D. Goldberg
and S. Kornfeld,J.
Biol.
Chem.
258, 3159 (1983).
47. D. F. Bainton,
and M. G. Farquhar,J.
Cell
Biol.
45, 54 (1970).
48. H. J. Geuze et al., ibid.
98, 2047 (1984).
49. M. C. Willingham,
I. H. Pastan,
G. G. Sahagian,
J. Histochem.
Cytochem.
31, 1
(1983).
50. Using detergent
permeabilization
and immunoperoxidase,
W. Brown
and M. G.
Farquhar
[Cell
36, 295 (1984)] have concluded
that,
within
the Golgi complex,
the M6P receptor
was observed
mainly
in the cis
compartment.
These different
results could be due to differences in cell
type
or to different
immunocytochemical
techniques
(6). With the peroxidase
technique,
a quantitatively
minor
amount of
antigen may,
however,
in some
cases,
be amplified by the enzymatic
reaction.
51. E. Schulze-Lohoff,
A. Hasilik,
K. von Figura,J.
Cell
Biol.
101, 824 (1985); C. A.
Campbell
and L. H. Rome,J.
Biol.
Chem.
258, 13347 (1983); G. C. Sahagian
and
C. J. Steer,
ibid.
260, 9838 (1985).
52. B. Hoflack and S. Kornfeld,
Proc.
Natl. Acad. Sci.
U.S.A. 82, 4428 (1985).
53. V. Lewis et al.,J. Cell
Biol.
100, 1839 (1985); J. W. Chen,
T. L. Murphy,
M. C.
Willingham,
I. Pastan,
J. T. August,
ibid.
101, 85 (1985); J. Lippincott-Schwartz
and D. M. Fambrough,
ibid. 102, 1593 (1986); I. Mellman
and A. Helenius,
personal
communication.
54. A. R. Hand and
C. Oliver,
J. Histochem.
Cytochem.
32, 403 (1984).
55. G. Griffiths
et al., in preparation.
56. R. G. W. Anderson
and R. K. Pathak,
Cell
40, 635 (1985).
57. I. Mellman,
R. Fuchs,
A. Helenius,
Annu. Rev. Biochem.
55, 663 (1986).
58. A. Frisch and E. F. Neufeld,
J. Biol. Chem. 256, 8242 (1981). That the
cytochemical
reactions
for
acid
phosphatase
and
aryl
sulfatase occur
atpH 5 but not
at neutral
pH's also
suggests
that
the enzymes
are
potentially
active.
59. J. White,
M. Kielian,
A. Helenius,
Q. Rev.
Biophys.
16, 151 (1983).
60. To become active,
many of these spikes,
such as the hemagglutinin
(HA) of
influenza
virus,
require
a proteolytic
cleavage
late in their
transport
to the plasma
membrane.
The precise
location
of this
cleavage
is not clear,
but the TGN is a likely
candidate.
Even the uncleaved
precursors
of HA undergo
an irreversible conforma-
tional
change
at
pH's about
6.0 (R. Doms and A. Helenius,
personal
communica-
tion).
61. H. Arnheiter,
M. Dubois-Dalcq,
R. A. Lazzarini,
Cell
39, 99 (1984).
62. Endosomes are defined
here as the total population
of endocytic
prelvsosomal
acidic
organelles.
This term actually comprises
a heterogeneous
set of structures
that have
an
uncharacterized
relation to each
other
[D. A. Wall and
A. L. Hubbard,
J. Cell Biol.
90, 687 (1981); A. Helenius,
I. Mellman,
D. Wall,
A. Hubbard,
Trends
Biochem. Sci.
8, 245 (1983)].
63. In this,
as well
as in the previous study
(24), endosomes
were
defined
operationally
as those structures that accumulated horseradish
peroxidase
at 20?C for 2 hours
[M.
Marsh,
G. Griffiths,
G. E. Dean,
I. Mellman,
A. Helenius,
Proc.
Natl.
Acad.
Sci.
U.S.A. 83, 2899 (1986)]. These conditions
probably
do not resolve
different
subcompartments
of endosomes
(62).
64. K. Hedman,
K. L. Goldenthal,
A. V. Rutherford,
I. Pastan,
M. C. Willingham,
J.
Cell Biol.
101, 422a (1985).
65. W. S. Sly and
H. D. Fischer,J.
Cell.
Biochem.
18, 67 (1982).
66. C. Harding,
J. Heuser,
P. Stahl,
J. Cell Biol.
97, 329 (1983).
67. K. von Figura,
V. Gieselmann,
A. Hasilik,
EMBOJ.
3, 1281 (1984); C. Gartang,
T. Braulke,
A. Hasilik,
K. von Figura,
ibid.
4, 1725 (1985).
68. N. K. Gonatas,
S. U. Kim,
A. Stieber,
S. Avrameas,J.
CellBiol.
73, 1 (1977); E.
Essner and H. B. Haimes,
ibid.
75, 381 (1977); H. B. Haimes,
R. J. Stockert,
A.
G. Morell,
A. B. Novikoff,
Proc.
Natl. Acad.
Sci. U.S.A. 78, 6936 (1981); R. D.
Broadwell
and
C. Oliver,
J. Histochem.
Cytochem.
31, 325 (1983).
69. A. Patzak and
H. Winkler,J.
Cell Biol.
102, 510 (1986).
70. D. Y. Yamashiro,
B. Tycko,
S. F. Fluss,
F. R. Maxfield,
Cell
37, 789 (1984).
71. E. Regoeczi,
P. A. Chindemi,
M. T. Debanne,
P. A. Charlwood,
Proc. Natl.
Acad.
Sci. U.S.A.
79, 2226 (1982); M. D. Snider
and 0. C. Rogers,J.
Cell Biol.
100, 826
(1985).
72. H. Riezman,
Cell
40, 1001 (1985); M. Makarow,
EMBO
J. 4, 1861 (1985).
73. A. R. Robbins et al.,J. Cell Biol.
99, 1296 (1984).
74. We thank the following
for their
help with this reyiew:
B. Burke,
S. Fuller,
H.
Geuze, A. Helenius, K. Howell, A. Hubbard,
E. Hughson, W. Huttner, S.
Kornfeld,
D. Louvard,
M. Marsh,
K. Matlin,
I. Mellman,
L. Orci,
P. Quinn,
L.
Roman,
J. Slot, J. Tooze, and
G. Warren.
59. J. White,
M. Kielian,
A. Helenius,
Q. Rev.
Biophys.
16, 151 (1983).
60. To become active,
many of these spikes,
such as the hemagglutinin
(HA) of
influenza
virus,
require
a proteolytic
cleavage
late in their
transport
to the plasma
membrane.
The precise
location
of this
cleavage
is not clear,
but the TGN is a likely
candidate.
Even the uncleaved
precursors
of HA undergo
an irreversible conforma-
tional
change
at
pH's about
6.0 (R. Doms and A. Helenius,
personal
communica-
tion).
61. H. Arnheiter,
M. Dubois-Dalcq,
R. A. Lazzarini,
Cell
39, 99 (1984).
62. Endosomes are defined
here as the total population
of endocytic
prelvsosomal
acidic
organelles.
This term actually comprises
a heterogeneous
set of structures
that have
an
uncharacterized
relation to each
other
[D. A. Wall and
A. L. Hubbard,
J. Cell Biol.
90, 687 (1981); A. Helenius,
I. Mellman,
D. Wall,
A. Hubbard,
Trends
Biochem. Sci.
8, 245 (1983)].
63. In this,
as well
as in the previous study
(24), endosomes
were
defined
operationally
as those structures that accumulated horseradish
peroxidase
at 20?C for 2 hours
[M.
Marsh,
G. Griffiths,
G. E. Dean,
I. Mellman,
A. Helenius,
Proc.
Natl.
Acad.
Sci.
U.S.A. 83, 2899 (1986)]. These conditions
probably
do not resolve
different
subcompartments
of endosomes
(62).
64. K. Hedman,
K. L. Goldenthal,
A. V. Rutherford,
I. Pastan,
M. C. Willingham,
J.
Cell Biol.
101, 422a (1985).
65. W. S. Sly and
H. D. Fischer,J.
Cell.
Biochem.
18, 67 (1982).
66. C. Harding,
J. Heuser,
P. Stahl,
J. Cell Biol.
97, 329 (1983).
67. K. von Figura,
V. Gieselmann,
A. Hasilik,
EMBOJ.
3, 1281 (1984); C. Gartang,
T. Braulke,
A. Hasilik,
K. von Figura,
ibid.
4, 1725 (1985).
68. N. K. Gonatas,
S. U. Kim,
A. Stieber,
S. Avrameas,J.
CellBiol.
73, 1 (1977); E.
Essner and H. B. Haimes,
ibid.
75, 381 (1977); H. B. Haimes,
R. J. Stockert,
A.
G. Morell,
A. B. Novikoff,
Proc.
Natl. Acad.
Sci. U.S.A. 78, 6936 (1981); R. D.
Broadwell
and
C. Oliver,
J. Histochem.
Cytochem.
31, 325 (1983).
69. A. Patzak and
H. Winkler,J.
Cell Biol.
102, 510 (1986).
70. D. Y. Yamashiro,
B. Tycko,
S. F. Fluss,
F. R. Maxfield,
Cell
37, 789 (1984).
71. E. Regoeczi,
P. A. Chindemi,
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for their
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B. Burke,
S. Fuller,
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Geuze, A. Helenius, K. Howell, A. Hubbard,
E. Hughson, W. Huttner, S.
Kornfeld,
D. Louvard,
M. Marsh,
K. Matlin,
I. Mellman,
L. Orci,
P. Quinn,
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Roman,
J. Slot, J. Tooze, and
G. Warren.
What
Has
Happened
to
Productivity
Growth?
MARTIN
NEIL BAILY
What
Has
Happened
to
Productivity
Growth?
MARTIN
NEIL BAILY
The collapse of U.S. productivity growth is the most
severe and persistent of recent economic problems. Unless
there is an increase in growth, American living standards
will remain stagnant and problems such as the budget
deficit will plaque policy-makers. Why has this happened?
Among the important reasons are a failure to innovate,
changing demographics, and disruptions to the economy,
including oil price increases and inflation.
The collapse of U.S. productivity growth is the most
severe and persistent of recent economic problems. Unless
there is an increase in growth, American living standards
will remain stagnant and problems such as the budget
deficit will plaque policy-makers. Why has this happened?
Among the important reasons are a failure to innovate,
changing demographics, and disruptions to the economy,
including oil price increases and inflation.
A PERSISTENT
DECLINE IN PRODUCTIVITY
GROWTH IN THE
U.S. economy has prevailed since the late 1960's, and it
intensified after 1973. Multifactor productivity in nonfarm
business grew at 1.75% per year from 1953 to 1968. This rate
dropped after 1968 and fell to only 0.32% a year by 1973 to 1979
(Fig. 1). The cumulative effect of this decline on the output of the
economy was substantial. Had the pre-1968 growth rate continued,
output in 1979 would have been 12% higher than it actually was,
with no additional capital or labor used in production. This amount
of additional output is much larger than that needed to solve many
of today's economic problems, notably the budget deficit.
24 OCTOBER
I986
A PERSISTENT
DECLINE IN PRODUCTIVITY
GROWTH IN THE
U.S. economy has prevailed since the late 1960's, and it
intensified after 1973. Multifactor productivity in nonfarm
business grew at 1.75% per year from 1953 to 1968. This rate
dropped after 1968 and fell to only 0.32% a year by 1973 to 1979
(Fig. 1). The cumulative effect of this decline on the output of the
economy was substantial. Had the pre-1968 growth rate continued,
output in 1979 would have been 12% higher than it actually was,
with no additional capital or labor used in production. This amount
of additional output is much larger than that needed to solve many
of today's economic problems, notably the budget deficit.
24 OCTOBER
I986
The nonfarm business sector of the U.S. economy includes
everything except government operations, agriculture, and nonprof-
it organizations. Figure 1 also shows productivity growth in
manufacturing, which differs from that of the aggregate economy
(1). Productivity growth in manufacturing actually accelerated from
1968 to 1973, before slumping from 1973 to 1979.
Although the difference between manufacturing and the aggre-
gate economy is important, a disaggregated view of the slowdown
shows the pervasiveness of this decline. Productivity growth has
decreased in almost all of the major sectors of the economy. Within
the major sectors, the Bureau of Labor Statistics (BLS) looked at
specific industry-level performance and found that three-quarters
of
the industries in their sample had declines in productivity growth
(2).
The Dimensions of the Slowdown
Multifactor productivity growth is the concept favored by most
economists, and now by BLS also. To calculate this measure of
The nonfarm business sector of the U.S. economy includes
everything except government operations, agriculture, and nonprof-
it organizations. Figure 1 also shows productivity growth in
manufacturing, which differs from that of the aggregate economy
(1). Productivity growth in manufacturing actually accelerated from
1968 to 1973, before slumping from 1973 to 1979.
Although the difference between manufacturing and the aggre-
gate economy is important, a disaggregated view of the slowdown
shows the pervasiveness of this decline. Productivity growth has
decreased in almost all of the major sectors of the economy. Within
the major sectors, the Bureau of Labor Statistics (BLS) looked at
specific industry-level performance and found that three-quarters
of
the industries in their sample had declines in productivity growth
(2).
The Dimensions of the Slowdown
Multifactor productivity growth is the concept favored by most
economists, and now by BLS also. To calculate this measure of
The author is a senior fellow in the Economic Studies Program, Brookings Institution,
Washington, DC 20036.
The author is a senior fellow in the Economic Studies Program, Brookings Institution,
Washington, DC 20036.
ARTICLES 443
ARTICLES 443
I I
... It receives a variety of proteins from the ER and recycles some of them back to the ER (Nilsson et al., 1989;Pelham and Munro, 1993;Letourneur et al., 1994;Sato et al., 1995Sato et al., , 1997Sato et al., 1996). There are a variety of destinations for cargo transiting the Golgi, involving many different carriers to the plasma membrane and other organelles (Griffiths and Simons, 1986;Weiss and Nilsson, 2000;Rodriguez-Boulan and Müsch, 2005;Bonifacino, 2014;Dell'Angelica and Bonifacino, 2019). Recent studies have indicated that these sorting functions should be mostly ascribed to specialized regions proximal to and distal to the main body of the Golgi. ...
... Recent studies have indicated that these sorting functions should be mostly ascribed to specialized regions proximal to and distal to the main body of the Golgi. Names given to these compartments are IC (intermediate compartment) or ERGIC (ER-Golgi intermediate compartment) on the cis-side Saraste and Svensson, 1991;Plutner et al., 1992;Klumperman et al., 1998) and TGN (trans-Golgi network) on the trans-side (Roth et al., 1985;Griffiths and Simons, 1986;Taatjes and Roth, 1986;Geuze and Morré, 1991). The purpose of this review is to provide insights into the common features of these neighboring compartments, from a comparative view of yeast, plant, and animal cells. ...
... The name trans-Golgi network was given to the morphologically prominent reticular structure found at the trans side of the Golgi stack (Roth et al., 1985;Griffiths and Simons, 1986;Taatjes and Roth, 1986;Geuze and Morré, 1991), which was regarded as a part of the Golgi for some time. A variety of cargo carriers including clathrin-coated vesicles were found in this area, and the concept that this compartment is important for sorting of cargo for different destinations was established (Griffiths et al., 1989;Keller and Simons, 1997;Hinners and Tooze, 2003;Bard and Malhotra, 2006;De Matteis and Luini, 2008;Gonzalez and Rodriguez-Boulan, 2009;Chen et al., 2017). ...
Article
Full-text available
The Golgi apparatus represents a central compartment of membrane traffic. Its apparent architecture, however, differs considerably among species, from unstacked and scattered cisternae in the budding yeast Saccharomyces cerevisiae to beautiful ministacks in plants and further to gigantic ribbon structures typically seen in mammals. Considering the well-conserved functions of the Golgi, its fundamental structure must have been optimized despite seemingly different architectures. In addition to the core layers of cisternae, the Golgi is usually accompanied by next-door compartments on its cis and trans sides. The trans-Golgi network (TGN) can be now considered as a compartment independent from the Golgi stack. On the cis side, the intermediate compartment between the ER and the Golgi (ERGIC) has been known in mammalian cells, and its functional equivalent is now suggested for yeast and plant cells. High-resolution live imaging is extremely powerful for elucidating the dynamics of these compartments and has revealed amazing similarities in their behaviors, indicating common mechanisms conserved along the long course of evolution. From these new findings, I would like to propose reconsideration of compartments and suggest a new concept to describe their roles comprehensively around the Golgi and in the post-Golgi trafficking.
... The TGN is generally recognized as a membranous structure at the trans-side of the Golgi apparatus (Griffiths and Simons, 1986;Ito and Boutté, 2020), which partially corresponds to the compartment previously proposed as the Golgi-associated structure that is a part of the ER and forms Lysosomes (GERL) in the 1970s or termed the partially coated reticulum (PCR) in 1980s (Marty, 1978;Harris and Oparka, 1983;Pesacreta and Lucas, 1984;Staehelin et al., 1990). Morphologically distinct types of vesicles or multiple coat proteins have been found in the TGN, indicating that it is an important site for sorting cargo proteins with different destinations (Griffiths and Simons, 1986;Singh and Jürgens, 2018). ...
... The TGN is generally recognized as a membranous structure at the trans-side of the Golgi apparatus (Griffiths and Simons, 1986;Ito and Boutté, 2020), which partially corresponds to the compartment previously proposed as the Golgi-associated structure that is a part of the ER and forms Lysosomes (GERL) in the 1970s or termed the partially coated reticulum (PCR) in 1980s (Marty, 1978;Harris and Oparka, 1983;Pesacreta and Lucas, 1984;Staehelin et al., 1990). Morphologically distinct types of vesicles or multiple coat proteins have been found in the TGN, indicating that it is an important site for sorting cargo proteins with different destinations (Griffiths and Simons, 1986;Singh and Jürgens, 2018). Cargo proteins are thought to be transported from the Golgi to the TGN by the process of "cisternal maturation, " which has been well-studied in budding yeast (Glick and Nakano, 2009). ...
Article
Full-text available
Membrane trafficking contributes to distinct protein compositions of organelles and is essential for proper organellar maintenance and functions. The trans-Golgi network (TGN) acts as a sorting station where various cargo proteins are sorted and directed to post-Golgi compartments, such as the multivesicular body or pre-vacuolar compartment, vacuoles, and plasma membrane. The spatial and temporal segregation of cargo proteins within the TGN, which is mediated with different sets of regulators including small GTPases and cargo adaptors, is a fundamental process in the sorting machinery. Recent studies with powerful imaging technologies have suggested that the TGN possesses spatially distinct subdomains or zones for different trafficking pathways. In this review, we will summarize the spatially and dynamically characteristic features of the plant TGN and their relation to cargo protein trafficking.
... The cis-and trans faces are important sites for the sorting of proteins and lipids for delivery to specific subcellular destinations. Griffiths and Simons (1986) propose that the TGN is a specialised organelle on the trans side of the Golgi stack that is responsible for routing proteins to lysosomes, secretory vesicles, and the plasma membrane from the Golgi complex. The distinction between the trans cistema and the TGN is often determined by the nonoverlapping localisations of thiamine pyrophosphatase reactivity {trans) and acid phosphatase (TGN) as demonstrated by cytochemistry (Novikoff and Novikoff 1977). ...
... Budding structures have been seen on the rim of the cistemae in the Golgi stack but as these cannot be labelled by antibodies against clathrin, it is believed that different proteins must form these structures (Griffiths et al, 1985). It has also been suggested that a link between the exocytic and endocytic pathways exists at the TGN (Griffiths and Simons 1986). The major feature is the presence of three main compartments. ...
Thesis
p>In this study, the enhanced cytotoxic effect of the anti-CD7 IT HB2-Saporin and the anti-CD38 IT OKT10-Saporin used in combination when compared to their use individually was demonstrated on the human T-ALL cell line HSB-2 both in vivo and in vitro. In an in vitro cell proliferation assay and an in vivo SCID mouse model, OKT10-Saporin was shown to be more effective than HB2-Saporin, but neither individual IT was as potent as their combined use. In contrast, HB2-Saporin performed best in the short term protein synthesis assay (PSI) and the combination of two ITs demonstrated an intermediate potency between that of the two individual ITs. Three explanations were proposed for the improved efficacy of using two ITs simultaneously: 1) co-ligation of CD7 and CD38 might alter the individual internationalisation characteristics; 2) targeting against two molecules overcomes the heterogeneity of antigen expression on tumour cells or 3) using two immunotoxins increases the amount of toxin delivered to the target cell. It is possible that all three explanations are valid. Flow cytometry and confocal microscopy were used to determine the internalisation and intracellular routing characteristics of CD7 and CD38 on the T-ALL cell line HSB-2, when ligated by antibody individually or in combination. These studies indicate that CD7 and CD38 have very different internalisation kinetics. CD7 clears very rapidly from the cell surface following ligation by antibody, whereas only 50% of CD38 molecules internalise over a 24 hr duration. When CD7 and CD38 were ligated by antibody simultaneously, no change to the internalisation characteristics or intracellular routing of either antibody appeared altered. In these studies it has become clear that there is no direct correlation between internationalisation, intracellular routing and cytotoxic potency. Brefeldin A was used as a tool to investigate what intracellular routes these ITs may follow within the cell. These were preliminary studies and further work is required. These studies have revealed that no single factor determines IT potency. Internalisation rate is only important if degradation is avoided and trafficking occurs to an appropriate intracellular compartment from which the toxin component can translocate.</p
... The trans-Golgi Network (TGN), is the trans-most distal cisternae site of the Golgi apparatus and has a tubular network shape. TGN structure and size vary from one cell type to another, and incoming and outgoing trafficking to the TGN dynamically regulates its size and morphology [47][48][49][50]. In the classical point of view, the TGN is depicted as the ultimate sorting hub for cargo proteins and lipids, where they are sorted to their final destinations [47,51]. ...
... TGN structure and size vary from one cell type to another, and incoming and outgoing trafficking to the TGN dynamically regulates its size and morphology [47][48][49][50]. In the classical point of view, the TGN is depicted as the ultimate sorting hub for cargo proteins and lipids, where they are sorted to their final destinations [47,51]. This view has been challenged with results from polarized Madin-Darby canine kidney (MDCK) cells implicated in biosynthetic cargo trafficking. ...
Article
Full-text available
Lysosomes are key regulators of many fundamental cellular processes such as metabolism, autophagy, immune response, cell signalling and plasma membrane repair. These highly dynamic organelles are composed of various membrane and soluble proteins, which are essential for their proper functioning. The soluble proteins include numerous proteases, glycosidases and other hydrolases, along with activators, required for catabolism. The correct sorting of soluble lysosomal proteins is crucial to ensure the proper functioning of lysosomes, and is achieved through the coordinated effort of many sorting receptors, resident ER and Golgi proteins, and several cytosolic components. Mutations in a number of proteins involved in sorting soluble proteins to lysosomes result in human disease. These can range from rare diseases such as lysosome storage disorders, to more prevalent ones, such as Alzheimer’s disease, Parkinson’s disease and others, including rare neurodegenerative diseases that affect children. In this review, we discuss the mechanisms that regulate the sorting of soluble proteins to lysosomes, and highlight the effects of mutations in this pathway that cause human disease. More precisely, we will review the route taken by soluble lysosomal proteins from their translation into the ER, their maturation along the Golgi apparatus, and sorting at the trans-Golgi network. We will also highlight the effects of mutations in this pathway that cause human disease.
... As proteins move along the secretory pathway, there is a pH gradient from an almost neutral pH (7.3) in the ER lumen that progressively becomes acidic from the cis-to the trans-Golgi, with a TGN pH of around 6.1 (Griffiths and Simons, 1986;Mellman et al., 1986;Anderson and Orci, 1988;Tooze and Tooze, 1986). Our results demonstrate that mildly acidic pH (6.1), resembling the TGN milieu, is a critical driver of LLPS, whereas calcium is not necessary; this contrasts with previous studies that have indicated calcium is required for the aggregation of CGs. ...
Article
Full-text available
Insulin is synthesized by pancreatic β-cells and stored into secretory granules (SGs). SGs fuse with the plasma membrane in response to a stimulus and deliver insulin to the bloodstream. The mechanism of how proinsulin and its processing enzymes are sorted and targeted from the trans-Golgi network (TGN) to SGs remains mysterious. No cargo receptor for proinsulin has been identified. Here, we show that chromogranin (CG) proteins undergo liquid–liquid phase separation (LLPS) at a mildly acidic pH in the lumen of the TGN, and recruit clients like proinsulin to the condensates. Client selectivity is sequence-independent but based on the concentration of the client molecules in the TGN. We propose that the TGN provides the milieu for converting CGs into a “cargo sponge” leading to partitioning of client molecules, thus facilitating receptor-independent client sorting. These findings provide a new receptor-independent sorting model in β-cells and many other cell types and therefore represent an innovation in the field of membrane trafficking.
... We also found that overexpression of TopBP1 leads to Golgi dispersal, a known apical marker in MCF10A 3D cultures. Golgi dispersal is observed in various cancer cell lines, which may be the reason for altered polarity as Golgi plays a major role in maintaining polarized trafficking of proteins (Griffiths and Simons, 1986;Keller et al., 2001). ...
Preprint
Full-text available
DNA topoisomerase IIβ - binding protein 1 (TopBP1) is a mediator protein that regulates the cell cycle checkpoint signaling pathway. A plethora of studies suggests high TopBP1 levels are positively associated with various cancers. Although TopBP1 transcript, as well as protein expression levels, are high in breast cancers, its role in breast tumorigenesis is not yet explored. In our studies, we observed that TopBP1 levels are high in premalignant and malignant cells of the MCF10A cancer progression series compared to the non-tumorigenic MCF10A cells. In order to establish the role of TopBP1 in tumorigenesis, TopBP1 overexpression in non-tumorigenic MCF10A, and stable knock-down in malignant MCF10CA1a cells were performed and grown in Matrigel ™ as breast spheroids. Overexpression of TopBP1 in MCF10A spheroids induced hyperproliferation, disruption of polarity and cell-cell junctions. Moreover, TopBP1 overexpressing 3D dissociated cells exhibited EMT-like phenotype and tumorigenic properties such as increased cell migration, invasion, colony formation capabilitiy and anchorage-independent growth, indicating acquisition of cellular transformation. Finally, we demonstrated TopBP1 overexpressing cells to form tumors in athymic mice thereby confirming their tumorigenic potential. We also confirmed that overexpression of TopBP1 led to a mutation in TP53 and other genomic insults. To summarise, we observed that ectopic expression of TopBP1 transforms MCF10A breast epithelial cells. These transformed cells harbour phenotypic and genotypic characteristics similar to that of malignant cells.
... Since Furin post-translationally processes its substrates, it is not surprising that Furin is primarily localized in the trans-Golgi network (TGN) in which newly synthesized proteins are passing en route to their final destinations [18,19]. From the TGN, Furin can also traffic to the cell surface and cycle between these two compartments via endosomes depending on several sorting motifs in the cytoplasmic domain [20]. ...
Article
Furin is the first discovered proprotein convertase member and is present in almost all mammalian cells. Therefore, by regulating the maturation of a wide range of proproteins, Furin expression and/or activity is involved in various physiological and pathophysiological processes ranging from embryonic development to carcinogenesis. Since many of these protein precursors are involved in initiating and maintaining the hallmarks of cancer, Furin has been proposed as a potential target for treating several human cancers. In contrast, other studies have revealed that some types of cancer do not benefit from Furin inhibition. Therefore, understanding the heterogeneous functions of Furin in cancer will provide important insights into the design of effective strategies targeting Furin in cancer treatment. Here, we present recent advances in understanding how Furin expression and activity are regulated in cancer cells and their influences on the activity of Furin substrates in carcinogenesis. Furthermore, we discuss how Furin represses tumorigenic properties of several cancer cells and why Furin inhibition leads to aggressive phenotypes in other tumors. Finally, we summarize the clinical applications of Furin inhibition in treating human cancers.
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The maintenance of epithelial architecture necessitates tight regulation of cell size and shape. However, mechanisms underlying epithelial cell size regulation remain poorly understood. We show that the interaction of Myosin Vb with Rab11 prevents the accumulation of apically derived endosomes to maintain cell-size, whereas that with Rab10 regulates vesicular transport from the trans-Golgi. These interactions are required for the fine-tuning of the epithelial cell morphology during zebrafish development. Furthermore, the compensatory cell growth upon cell-proliferation inhibition involves a preferential expansion of the apical domain, leading to flatter epithelial cells, an efficient strategy to cover the surface with fewer cells. This apical domain growth requires post-trans-Golgi transport mediated by the Rab10-interacting Myosin Vb isoform, downstream of the mTOR-Fatty Acid Synthase (FASN) axis. Changes in trans-Golgi morphology indicate that the Golgi synchronizes mTOR-FASN-regulated biosynthetic input and Myosin Vb-Rab10 dependent output. Our study unravels the mechanism of polarized growth in epithelial cells and delineates functions of Myosin Vb isoforms in cell size regulation during development.
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Since the first fluorescent proteins (FPs) were identified and isolated over fifty years ago, FPs have become commonplace yet indispensable tools for studying the constitutive secretory pathway in live cells. At the same time, genetically encoded chemical tags have provided a new use for much older fluorescent dyes. Innovation has also produced several specialized methods to allow synchronous release of cargo proteins from the endoplasmic reticulum (ER), enabling precise characterization of sequential trafficking steps in the secretory pathway. Without the constant innovation of the researchers who design these tools to control, image, and quantitate protein secretion, major discoveries about ER-to-Golgi transport and later stages of the constitutive secretory pathway would not have been possible. We review many of the tools and tricks, some 25 years old and others brand new, that have been successfully implemented to study ER-to-Golgi transport in intact and living cells.
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Proteins in the body are produced by the cytoplasmic ribosomes and the rough endoplasmic reticulum (RER). The cytoplasmic ribosomes produce proteins necessary for cytoplasmic, mitochondrial, and peroxisomal function. The RER produces the proteins required for endoplasmic, Golgi, lysosomal function. These proteins need to be localized appropriately to carry out their tasks both intracellularly and extracellularly. The process of directing proteins to their appropriate location is termed 'protein targeting.' Protein targeting may use vesicles depending on the source of the protein. Proteins from cytoplasmic ribosomes do not use vesicles, whereas proteins from the RER use vesicles to target proteins. In protein targeting, many proteins are favorably modified by enzymes and helper proteins to improve the delivery. In the event of genetic mutations, proteins may localize inappropriately, leading to abnormal cellular function. Alterations in this process can result in fatal metabolic diseases such as inclusion-cell disease (ICD).
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Alveolar macrophages of the beige mouse mutant have a system of smooth-surfaced elements with the hallmarks of GERL. GERL also appears to produce residual bodies, and both organelles show cytochemically demonstrable acid phosphatase activity. When cells are exposed to colloidal silver, the tracer is endocytosed via pinocytic vacuoles to GERL.
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Affinity-purified, monospecific rabbit antibodies against rat pancreatic alpha-amylase and bovine pancreatic alpha-chymotrypsinogen were used for immunoferritin observations of ultrathin frozen sections of mildly fixed exocrine pancreatic tissue from secretion-stimulated (pilocarpine) rats and from overnight-fasted rats and guinea pigs. The labeling patterns for both antibodies were qualitatively alike: Labeling occurred in (a) the cisternae of the rough endoplasmic reticulum (RER) including the perinuclear cisterna, in (b) the peripheral area between the RER and cis-Golgi face, and (c) all Golgi cisternae, condensing vacuoles, and secretory granules. Labeling of cytoplasmic matrix was negligible. Structures that appeared to correspond to rigid lamellae were unlabeled. Differences in labeling intensities indicated that concentration of the zymogens starts at the boundary of the RER and cis-side of the Golgi complex. These data support the view that the Golgi cisternae are involved in protein processing in both stimulated and unstimulated cells and that Golgi cisternae and condensing vacuoles constitute a functional unit.
Article
Camillo Golgi first stained a ubiquitous cytoplasmic structure in 1898. This so-called ‘Golgi Complex’ is now known to be a dynamic crossroads for vesicular traffic; however, a systematic description of its composition and understanding of its mechanism of function are not available. Present data indicate that it is composed of a sequence of interacting subcompartments.
Article
Glutaraldehyde-fixed testes were stained “en bloc” with the Ur-Pb-Cu technique of Thiéry and Rambourg ('76) or post-fixed and stained with the osmium tetroxide-potassium ferrocyanide method of Karnovsky ('71). Thin or thick (up to 3 μm) sections were examined with the Philips (301 or 400) EM or the high voltage EM. Stereopairs were prepared with photographs of tilted specimens (± 7°). At low magnification, in thick sections (0.5–3 μm) stained with Ur-Pb-Cu, the whole Golgi apparatus formed a single network of interconnected wavy ribbon or platelike structures extending from the juxtanuclear region toward the apex of the cell. At higher magnifications, with the two staining techniques, this Golgi network showed two distinct types of regions: the “saccular region” corresponding to the conventional stack of saccules and the “intersaccular connecting region” made up of anastomotic tubules which bridge adjacent stacks. In the saccular regions, there was, on the cis-face of the stack, a tight polygonal meshwork of anastomotic tubules (osmiophilic element). Underlying it there were three to seven closely apposed saccules perforated with pores of various diameters, and finally, on the trans-face, a network of tubules was usually connected to the last saccule of the stack, which seemed to peel off from the pile, The intersaccular connecting regions showed proximal and distal zones with regard to the associated stacks. The proximal zone was made up of superimposed and parallel polygonal networks of membranous tubules which were continuous with corresponding saccules of the stack. In the distal zone they interdigitated, intertwined, anastomosed and bridged adjacent saccular regions; others turned at right angles and established connections with tubular extensions arising at various levels of the same stack. While cisternae of endoplasmic reticulum were contiguous with tubules or saccules located on the transface of the Golgi apparatus, a close association between the ER cisternae and the cis-face of the stacks was not usually observed.
Article
Enzyme cytochemistry has been used, at the light and electron microscope levels, to "mark" cytoplasmic organelles of mammalian cells. Catalase cytochemistry permitted identification of microperoxisomes, apparently ubiquitous organelles that are attached by numerous slender connections to the endoplasmic reticulum. Thiamine pyrophosphatase and acid phosphatase cytochemistry can be used to distinguish between the Golgi apparatus and a specialized acid-phosphatase-rich region of smooth endoplasmic reticulum (ER) that appears to be involved in: (a) the formation of lysosomes and melanin granules: (b) the processing and packaging of secretory materials in endocrine and exocrine cells; and (c) the metabolism of lipid. The acronym GERL has been given to this region of smooth ER because it is located at the inner or "trans" aspect of the Golgi apparatus and because it appears to produce various types of Lysosomes.
Article
Recent studies from our laboratory are described which deal with endocrine cells (insulinoma, beta-cells of the pancreas, thyroid epithelial cells), pancreatic exocrine cells, and hepatocytes. These emphasize the importance of the hydrolase-rich specialized region of endoplasmic reticulum, known as GERL, in secretory cells. Also reviewed in this paper are the varied molecular transformations which apparently occur in GERL in different cell types, as reported from other laboratories as well as our own. Evidence of the continuity of GERL with rough endoplasmic reticulum is presented. Two hydrolytic enzyme activities in GERL, in addition to acid phosphatase activity, are recorded. Finally, the use of cytochemical staining procedures in the study of microperoxisomes is briefly described.
Article
The bovine exocrine pancreatic cell produces a variety of enzymes and proenzymes for export. Biochemical studies by Greene L.J., C.H. Hirs, and G.E. Palade (J. Biol. Chem. 1963. 238:2054) have shown that the mass proportions of several of these proteins in resting pancreatic juice and zymogen granule fractions are identical. In this study we have used immunocytochemical techniques at the electron microscope level to determine whether regional differences exist in the bovine gland with regard to production of individual secretory proteins and whether specialization of product handling occurs at the subcellular level. The technique used is a modification of one previously reported (McLean, J.D., and S.J. Singer. 1970. Proc. Natl. Acad. Sci U.S.A. 69:1771) in which immunocytochemical reagents are applied to thin sections of bovine serum albumin-imbedded tissue and zymogen granule fractions. A double antibody technique was used in which the first step consisted of rabbit F(ab')2 antibovine secretory protein and the detection step consisted of sheep (F(ab')2 antirabbit F(ab')2 conjugated to ferritin. The results showed that all exocrine cells in the gland, and all zymogen granules and Golgi cisternae in each cell, were qualitatively alike with regard to their content of secretory proteins examined (trypsinogen, chymotrypsinogen A, carboxypeptidase A, RNase, and DNase). The data suggest that these secretory proteins are transported through the cisternae of the Golgi complex where they are intermixed before copackaging in zymogen granules; passage through the Golgi complex is apparently obligatory for these (and likely all) secretory proteins, and is independent of extent of glycosylation, e.g., trypsinogen, a nonglycoprotein vs. DNase, a glycoprotein.
Article
Conjugates of ricin agglutinin and phytohemagglutinin with horseradish peroxidase (HRP) were used for a cytochemical study of internalization of their plasma membrane "receptors" in cultured isolated mouse dorsal root ganglion neurons. Labeling of cells with lectin-HRP was done at 4 degrees C, and internalization was performed at 37 degrees C in a culture medium free of lectin-HRP. 15-20 min after incubation at 37 degrees C, lectin-HRP receptor complexes were seen in vesicles or tubules located near the plasma membrane. After 1-3 h at 37 degrees C, lectin-HRP-receptor complexes accumulated in vesicles and tubules corresponding to acid phosphatase-rich vesicles and tubules (GERL) at the trans aspect of the Golgi apparatus. A few coated vesicles and probably some dense bodies contained HRP after 3-6 h of incubation at 37 degrees C. Soluble HRP was not endocytosed under the conditions of this experiment or when it was present in the incubation medium at 37 degrees C. Internalization of lectin-HRP-receptor conjugates was decreased or inhibited by mitochondrial respiration inhibitors but not by cytochalasin B or colchicine. These studies indicate that lectin-labeled plasma membrane moieties of neurons are endocytosed primarily in elements of GERL.
Article
Phosphatase cytochemistry was used to distinguish between the Golgi apparatus and GERL (considered as a specialized region of endoplasmic reticulum [ER] at the inner [trans] aspect of the Golgi stack) in pancreatic exocrine cells of guinea pig, rat, rabbit, and hamster. The trans element of the Golgi stack exhibits thiamine pyrophosphatase (TPPase) but no acid phosphatase (AcPase) activity. In contrast, GERL shows AcPase but no TPPase activity. The nascent secretory granules, or condensing vacuoles, are expanded cisternal portions of GERL. Continuities of condensing vacuoles with rough ER are suggested, and it is proposed that some secretory components may have direct access to the condensing vacuoles from ER. Connections of Golgi apparatus with GERL were not seen.