(Received for publication, August 3, 1995; and in revised form, October 12, 1995)
From the
Biochemical and morphometric approaches were combined to examine
whether constitutive secretory transport might be controlled by plasma
membrane receptors, as this possibility would have significant
physiological implications. Indeed, IgE receptor stimulation in rat
basophilic leukemia cells potently increased the rate of transport of
soluble pulse-labeled S-sulfated glycosaminoglycans from
distal Golgi compartments to the cell surface. This effect was largely
protein kinase C (PKC)-dependent. Direct activation of PKC also
stimulated constitutive transport of glycosaminoglycans, as indicated
by the use of agonistic and antagonistic PKC ligands. PKC ligands also
had potent, but different, effects on the exocytic transport from
distal Golgi compartments to the plasma membrane of a membrane-bound
protein (vesicular stomatitis virus glycoprotein), which was slightly
stimulated by activators and profoundly suppressed by inhibitors of
PKC. Morphological analysis showed impressive changes of the organelles
of the secretory pathway in response to IgE receptor stimulation and to
direct PKC activation (enhanced number of buds and vesicles originating
from the endoplasmic reticulum and Golgi and increase in surface and
volume of Golgi compartments), suggestive of an overall activation of
exocytic movements. These results show that rapid and large changes in
constitutive transport fluxes and in the morphology of the exocytic
apparatus can be induced by membrane receptors (as well as by direct
PKC stimulation).
Constitutive membrane transport is fundamental to a number of cellular functions including growth and differentiation, secretion of proteins such as immunoglobulins, proteoglycans, serum, matrix, and milk proteins, as well as generation, homeostasis, and turnover of cellular organelles. Such essential functions are likely to be precisely controlled.
Certain basic steps in membrane and protein transport such as vesicle formation, docking, and fusion have been studied extensively over the last decade, and a fundamental set of molecular mechanisms executing these events has been elucidated(1) . Superimposed on this core machinery, regulatory mechanisms must exist to fine tune membrane traffic, maintain organelle homeostasis, and mediate adaptive responses of transport to variations in extracellular conditions. In spite of their potentially great physiologic importance, however, they have received relatively little attention.
Evidence has nevertheless accumulated showing that
regulatory molecules play a role in constitutive membrane traffic.
Heterotrimeric GTP-binding proteins have been implicated in the
secretion of proteoglycans(2) , apical secretion in polarized
cells(3) , endosome-endosome fusion(4) ,
transcytosis(5, 6) , in the association of coatomer
with Golgi membranes(7) , vesicle formation from the distal
Golgi(8) , and ER ()to Golgi transport(9) .
Protein phosphorylation has been shown to be involved in the regulation
of traffic; ER to Golgi transport is suppressed by protein phosphatase
inhibitors(10) . Vesicle formation from the trans Golgi/trans
Golgi network (TG/TGN) is dependent on protein
phosphorylation(11) ; hyperphosphorylation of a yet
unidentified protein(s) leads to a block of homologous endosome fusion in vitro(12) ; the mitotic kinase Cdc2 has also been
proposed to be responsible for the endosome fusion block at
mitosis(13) .
On the basis of these considerations, we have recently investigated the possibility that signal transduction pathways might control constitutive membrane traffic and reported that protein kinase C (PKC) and PKC-coupled receptors modulate the association of ADP-ribosylation factor (ARF) to the Golgi apparatus(14) . ARF binding to Golgi membranes is a key molecular step in vesicular traffic(1) . This finding has helped bring into focus the link between constitutive traffic and signal transduction and further stimulated interest in the question as to whether segments of the membrane traffic pathways may be regulated by second messengers (15) and by which mechanisms. Most recently, a number of reports have produced evidence that, indeed, diverse transport pathways can be affected by bacterial toxins or drugs modifying the level of second messengers(16, 17, 18, 19, 20, 21) .
To assess the physiological significance of these regulatory phenomena, we have studied the effects of receptor stimulation on constitutive membrane traffic. Biochemical analysis indicates that in RBL cells, the IgE receptor can control membrane and solute exocytic fluxes from the TG/TGN to the cell surface mostly via PKC. Morphological analysis extends this observation, revealing changes of the secretory apparatus (enhanced number of buds and vesicles originating from both the ER and the Golgi complex, and increase in surface and volume of Golgi compartments), suggestive of an overall acceleration of exocytic transport. The results suggest that signal transduction systems can play a crucial role in the mechanisms controlling size and transport activity of the exocytic organelles.
Figure 1:
Cell-free formation of secretory
vesicle from the TG/TGN in PC12 cells. A postnuclear supernatant from
PC12 cells prelabeled with [S]sulfate, was
incubated at 37 °C to allow vesicle formation in vitro.
After the reaction, the suspension was centrifuged on a continuous
sucrose gradient to separate small vesicles (upper fractions)
from larger membranes (lower fractions). Fractions were
analyzed by SDS-PAGE, and radioactivity was quantified with an
InstantImager. The distribution of labeled proteoglycans between upper
and lower fractions (from fraction 1 to 12) is shown for a sample
incubated for 30 min at 37 °C and a control sample incubated for
the same time at 0 °C. The distribution of sialyltransferase
activity across the gradient is also shown for samples incubated at 0
°C (filled symbols) and at 37 °C (empty
symbols). The ratio between radioactivity in the upper versus lower fractions is taken as an index of vesicle formation from the
TG/TGN (see Table 3).
The Golgi apparatus was defined as a complex of cisternae organized in stacks and tubular structures in the Golgi exclusion zone (Fig. 2A). In these areas, there may be two non-Golgi tubular structures: endosomes and transitional elements of the ER. The latter were excluded based on continuity with rough ER, presence of ribosomes on a contour, characteristic floccular content, and more abrupt membrane outline. Tubular endosomes were excluded based on their thinner diameter (40-50 instead of 60-90 nm for Golgi-associated tubules), very uniform width, and worm-like appearance; moreover, experiments designed to visualize the endosomal compartment by horseradish peroxidase staining showed that endosomes represent a negligible fraction of the membranes in the Golgi exclusion zone (Fig. 2B). Buds were defined as spherical-cylindrical elevations (diameter 60-100 nm) protruding from the surface of the ER (Fig. 2C) or of the Golgi complex (Fig. 2D) by at least 60% of their diameter, showing a direct connection with the donor membrane (ER or Golgi complex) and covered with electron-dense coats distinguishable from clathrin coats (Fig. 2D, inset). Vesicular profiles were defined as 50-80-nm round structures with a slightly denser content than that of Golgi cisternae. Given the high prevalence of vesicular (round) over tubular or oval profiles, we used the approximation that all round profiles represent vesicles, in order to calculate their number, surface, and volume. The tubular-vesicular clusters (TVC) derived from the ER (putative transport intermediates, see ``Results'') were defined as distinct aggregates of at least three vesicles adjacent to the ER or of at least two vesicles associated with an ER bud (Fig. 2C and inset). The average number of vesicular profiles in thin sections of such clusters was six.
Figure 2: Ultrastructure of the components of the secretory pathway. A, a Golgi apparatus with membrane stacks (arrows), tubules, vesicles, and vacuoles (arrowheads). B, horseradish peroxidase labeling of early endosomes treated with 1 µM PMA for 15 min and processed for cytochemical visualization of horseradish peroxidase (see ``Experimental Procedures''). The horseradish peroxidase-labeled endosomes (arrows) were clearly distinct from the Golgi structure (arrowheads), and no horseradish peroxidase was detected in the Golgi area. C, ER-derived buds (arrows) and tubular-vesicular clusters (arrowheads and inset, asterisk) are situated near the ER (small arrows; see definitions in ``Experimental Procedures''). D, tangential section of the Golgi cisternae (G) and buds therefrom (arrows). Clathrin-coated vesicles (arrowhead and inset) are distinguishable from non-clathrin-coated buds and vesicular profiles. Bars, 0.1 µM.
To assess the volume and surface density (V and S
, respectively) of
the above defined structures, five random meshes on a grid, each
containing at least 30 cell profiles from the same ultrasection, were
used for each treatment and time point, photographed at 3000
,
and photographically enlarged 14,000 times (to analyze whole cells),
31,120 times (for the ER), or 66,700 times (for the Golgi complex and
TVC). The mean area (S
) and perimeter (P
) on thin sections of the above structures and
of whole cells (S
) was estimated with Videoplan 2,
and V
and S
were calculated
by the formulas: V
= S
/S
and S
=
2P
/S
(33) . The number
density (N
) of buds, vesicles, and TVC in cells
was estimated by the formula N
= C
N
V
,
where N
is the mean number of profiles of these
structures per cell sectional area and C is a constant related
to their shape(33) . Since these structures are rather
spherical, we used a C coefficient equal to 1. The correction
of bias due to section thickness and section compression was done
according to (33) . The correction factor for bud and vesicle
number density was 0.41, as in Fig. 4in (33) . The
correction factors for Golgi volume and surface were estimated as 0.6
and 0.64, respectively, based on Fig. 5and Fig. 6in (33) and on the reasonable assumption that two-thirds of the
Golgi complex was composed of cisternae with an average length of 1
µm and one-third of tubules of 70 nm in diameter. The absolute
values of surface (S
), volume (V
) and number (N
) of the
structures were calculated by the formulae V
= V
MCV, S
= S
MCV, and N
= N
MCV.
Figure 4:
Stimulation of constitutive glycan release
by IgE receptor stimulation and by PMA in RBL cells. A,
IgE-primed RBL cells were preincubated in the presence of xyloside,
pulse-labeled with [S]sulfate, and then chased
in calcium-free medium for 30 min in the presence (circles) or
in the absence (squares) of 10 µg/ml BFA, under basal
conditions (empty symbols) or with 100 ng/ml DNP
BSA (filled symbols). B, IgE-primed RBL cells were
treated as in A and then chased in calcium-free medium for 30
min in the absence (white bars) or in the presence of 100
ng/ml DNP
BSA (striped bars) and, where indicated,
exposed to 5 µM Ro 31-8220 or 1 µM calphostin
C. C, cells preincubated in the presence of xyloside and then
pulse-labeled for 5 min with [
S]sulfate were
chased for various times with (filled symbols) or without (empty symbols) 100 nM PMA in the absence (squares) or presence (circles) of 10 µg/ml BFA.
Release was expressed as the percentage of the total cellular content
as assayed by the cetylpiridinium chloride method. Values represent
means ± S.D. Experiments were performed at least 3 times with
similar results.
Figure 5:
Effects of specific stimulators and
inhibitors of PKC on constitutive glycan release in RBL cells. A, cells preincubated in the presence of xyloside and then
pulse-labeled with [S]sulfate were chased for 15
min in the presence of 10, 100, or 1000 nM PMA (squares), thymeleatoxin (triangles), DPP (empty
circles), or DPPA (filled circles). Release was expressed
as the percentage of the total cellular content as assayed by the
cetylpiridinium chloride method. B, cells preincubated in the
presence of xyloside and then pulse-labeled with
[
S]sulfate were chased for 15 min in the absence (white bars) or presence (striped bars) of 100 nM PMA together with 1 µM calphostin C, 3 µM Ro 31-8220, or, after 6 h of treatment, with 1 µM PMA
which down-regulates PKC isoforms
and
(71, 72) . Release was expressed as above. Values
represent means ± S.D. Experiments were performed at least 3
times with similar results.
Figure 6:
Regulation of VSV-G transport from the
TG/TGN to the cell surface in RBL cells. RBL cells, infected with VSV
and labeled with [S]methionine were blocked at
19.5 °C to accumulate material in the TG/TGN. Cells were then
chased at 37 °C in the presence or absence of 100 nM PMA
or 5 µM Ro 31-8220 (Ro). Biotinylated cell
surface proteins were precipitated with streptavidin-agarose and
quantified with the InstantImager after SDS-PAGE. Top panel shows the autoradiogram of a time-course of VSV-G transport to the
cell surface. Bottom panel shows InstantImager
quantification.
We have studied the effects of membrane receptor stimulation on constitutive transport of soluble and membrane markers from TG/TGN to plasma membrane and on the morphology of the exocytic apparatus. Most experiments were carried out in RBL cells, where we had previously described the modulatory role of membrane receptors and PKC on ARF binding(14) ; other cell lines were employed to confirm and extend the results.
Figure 3:
Characterization of constitutive glycan
release in RBL cells. Cells were pulse-labeled with
[S]sulfate with or without preincubation in
xyloside-containing medium. Total proteoglycans and GAG were separated
by SDS-PAGE. A, sample image from the Packard InstantImager
showing the separation between proteoglycans (1, high M
material) and GAG (2, low M
material), with the radioactivity profile. B, InstantImager quantification. In the graph, striped
bars represent proteoglycans, and gray bars represent
GAG. C, cells preincubated in the presence of xyloside and
then pulse-labeled with [
S]sulfate were chased
for various times in the absence (circles) or presence of 100
nM PMA (triangles). Both content and released
material were analyzed after SDS-PAGE separation by InstantImager
quantification. Filled symbols represent proteoglycans, and empty symbols represent GAG. D, cells preincubated in
xyloside and labeled as in B were chased for 6 h and then
stimulated with 100 nM PMA with or without 1 µM A23187 for 30 min. Content and released material were then
analyzed after SDS-PAGE separation by InstantImager quantification. Striped bars represent proteoglycans, and gray bars represent GAG. Values represent means ± S.D. Experiments
were performed at least 3 times with similar
results.
Of
note, proteoglycans (the high M sulfated material
in Fig. 3A) appeared to behave similarly (although not
identically) to GAG both in time course of secretion and responsiveness
to stimulants (Fig. 3C) (experiments using various
stimulants are described below). As GAG measurement involving SDS-PAGE
and imaging is expensive and time consuming, we measured the release of
sulfated material by cetylpyridinium chloride precipitation (a simpler
technique that collectively detects GAG and proteoglycans, hereafter
indicated as ``glycans''), as an index of constitutive
release. As expected, a good numerical correspondence was found between
the two methods (for total glycans and GAG; Fig. 4). The
cetylpyridinium chloride method was thus routinely used, and the
SDS-PAGE/InstantImager technique was only employed to separately
quantitate GAG and proteoglycans in selected experiments.
While the above clearly suggests the
involvement of PKC in the regulation of constitutive exocytosis, it has
been recently shown that a domain homologous to the phorbol ester
binding region of PKC is present in other
proteins(38, 39) , raising the possibility that these
proteins might be responsible for the stimolatory effects of PMA. A
series of experiments was thus carried out to determine if the effect
of PMA was due to PKC activation. Indeed, several other phorbol esters
among which DPP, DPPA, and thymeleatoxin had a stimulatory effect on
glycan release at concentrations shown to be active on purified PKC in vitro (Fig. 5A)(40) ; of notice,
DPPA, which has been shown to exclusively stimulate PKC in
vitro(40) had the least effect on glycan release, being
slightly effective only at the highest concentration (1
µM). The inactive phorbol
-PDD had no effect (not
shown). The specific PKC inhibitor calphostin C, which acts directly on
the phorbol binding site of PKC(41) , markedly reduced the
stimulatory effect of PKC (Fig. 5B). Ro 31-8220, a
highly specific PKC inhibitor acting at the ATP binding site of
PKC(42) , completely abolished the stimulatory effect of PMA (Fig. 5B). H7 and staurosporine, less selective
compounds of the latter class, had similar effects (not shown). In
addition, PMA was no longer able to stimulate glycan release after
down-regulation of PKC with 1 µM PMA treatment for 6 h (Fig. 5B). Calphostin C and Ro 31-8220 did not have
effects on basal glycan release during the first 15 min after labeling,
while an inhibition developed at later times (data not shown). The
above results strongly suggest that PKC is responsible for the
stimulatory effects of PMA on glycan release.
As noted above, RBL
cells also feature regulated exocytosis and a considerable fraction of
proteoglycans, and GAG (this report) accumulates in secretory granules.
PMA alone does not stimulate release from secretory granules in RBL
cells ( Table 2and Beaven et al.(36) ), and, in
addition, regulated secretion of serotonin was strictly dependent on
external calcium, whereas PMA-stimulated glycan release was not.
Finally, BFA did not inhibit serotonin release in clear contrast with
its ability to inhibit glycan release. Thus, the pattern of
responsiveness of the regulated secretory route in RBL cells to a
variety of stimulators and inhibitors is completely different from that
of the constitutive pathway. To further verify the separatedness of the
two pathways, we followed the time-course of entry of GAG into the
regulated compartment. After chases of increasing lengths, RBL cells
were treated with PMA or DNPBSA in the absence of extracellular
calcium (to stimulate constitutive release) or a combination of calcium
ionophore and PMA or DNP
BSA in the presence of external calcium
(to induce regulated exocytosis, which is absolutely calcium-dependent
in these cells; (36) and this report). When release was
measured immediately after labeling, PMA (or DNP
BSA in the
absence of calcium) potently stimulated it. After 1 h of chase in the
absence of stimuli, PMA moderately stimulated GAG release, whereas
PMA/A23187 induced the release of about 60% of the total GAG content
(not shown); the same was observed with IgE stimulation in the absence
or the presence of external calcium, respectively. After 6 h of chase (Fig. 3D), PMA alone or IgE stimulation in the absence
of external calcium (not shown) had no effect, whereas the vast
majority of GAG content was released by PMA/A23187 or IgE stimulation
in the presence of external calcium (not shown); the response to the
latter stimuli precisely paralleled that seen with serotonin. This is
taken to indicate that GAG had moved into a nonconstitutively releasing
compartment, functionally colocalized with the serotonin-containing
granule compartment. Thus, the kinetic data are in line with the other
above results, indicating that in RBL cells, constitutive and regulated
GAG secretion can be readily distinguished and that the former is
selectively stimulated by PMA or by IgE receptor activation in the
absence of calcium.
Figure 7:
Stimulation of glycan release by PMA in
PC12 cells and other cell lines. A, HL-60, Madin-Darby canine
kidney, and PC12 cells were preincubated in the presence of xyloside
and then pulse-labeled with [S]sulfate and
chased for 15 min with (stripedbars), or without 100
nM PMA (white bars). B, PC12 cells,
preincubated in the presence of xyloside and then pulse-labeled with
[
S]sulfate were chased for various times with (filled symbols) or without (empty symbols) 100
nM PMA in the absence (squares) or presence (circles) of 10 µg/ml BFA. Release was expressed as above.
Values represent means ± S.D., and experiments were performed at
least 3 times with similar results.
To measure
vesicle production in vitro, PC12 cells were homogenized after
a short pulse with [S]sulfate to produce a
postnuclear supernatant containing, among other membranes, the Golgi
complex. This postnuclear supernatant was incubated at 37 °C for
various lengths of time in the presence or absence of 100 nM PMA or 10 µM Ro 31-8220 and then fractionated by
velocity gradient centrifugation to separate smaller secretory vesicles
from larger TGN membranes between upper and lower fractions at 0 °C
and 37 °C, according to (26) (see Fig. 1). The
addition of PMA stimulated the transfer of labeled proteoglycans from
lower (Golgi) into upper (vesicular) fractions (Table 3). The
inclusion of Ro 31-8220 during the incubation inhibited vesicle
formation (not shown). A similar increase in vesicle formation by PMA,
albeit only in the presence of okadaic acid, was recently
reported(11) . These results indicate that PMA can increase
vesicle formation and therefore that at least part of the acceleration
of glycan release in vivo by PMA is due to a direct effect on
export from the TG/TGN.
When cells were treated with PMA, the number of buds on the ER increased by 90% within 2 min (Fig. 8A), while the distribution of the buds and the shape and size of the ER did not change appreciably. The number of ER-derived vesicular profiles and TVC rose to a somewhat lesser extent (Fig. 8A), while the number of vesicular profiles per TVC did not change. The number of total vesicular profiles was also significantly increased (Fig. 8D). Only less than half of this effect could be ascribed to ER vesicles; we did not attempt to determine the nature of the non-ER vesicular profiles, but they are likely to be mostly endocytic and Golgi-derived. A similar effect of PMA on cytoplasmic vesicles has been observed previously in human basophils(46) . Concomitantly (within 2 min), the volume and surface of the Golgi apparatus increased by 40 and 55%, respectively (Fig. 8B), and the number of Golgi buds increased to a similar extent (Fig. 8C). The rims of Golgi profiles appeared to have become larger, and the number of Golgi profiles per cell appear to be slightly increased. The latter effect, however, might be due to increased probability of section through larger stacks rather than to an actual increase in stack profile number. Endocytosis in RBL cells can be stimulated by PMA(47) ; thus, to examine whether the changes attributed to the Golgi complex might in part be due to endocytic structures located in the Golgi area, cells were incubated with a high concentration of horseradish peroxidase (15 mg/ml) for 2 h to allow extensive labeling of the endosomal system. Several structures, mostly located in the cell periphery and identifiable as endosomes, became strongly horseradish peroxidase-labeled. In contrast, no horseradish peroxidase was detectable in the Golgi area both in control cells and in cells treated with PMA (Fig. 2B). Endosomes are thus unlikely to interfere with the identification of Golgi structures in RBL cells. Of note, these data also make it unlikely that stimulation of endocytosis by PMA might affect TG/TGN to plasma membrane traffic via an increased fluid input into the TG/TGN (see ``Discussion''). At 10 min after PMA addition, the density of buds on the ER and the number of associated vesicular clusters began to decline; at 30 min of PMA treatment the number of ER buds and vesicular clusters had reached near basal levels (Fig. 8A). The Golgi complex also decreased in size, albeit with a delay, and began returning toward control values after 10-15 min of exposure to PMA (Fig. 8B). Treatments with PMA for up to 60 min had no significant effect on cell volume.
Figure 8: Ultrastructural morphometric analysis of RBL cells stimulated with PMA. RBL cells were treated with 1 µM PMA (which caused a stimulation of glycan release comparable with 100 nM PMA, see Fig. 5A) and then processed for morphometric analysis (see ``Experimental Procedures''). A significant and rapid (2-15 min) increase of ER buds and TVC and a quick normalization of these parameters were observed (A). The number of Golgi buds and vesicles (C and D, respectively) increased to a slightly lesser degree. In a parallel manner, an increase of Golgi complex volume and surface (B) was observed.
IgE receptor stimulation caused morphological effects similar to those of PMA. Indeed, receptor activation induced both an increase in number of ER buds and of associated vesicular clusters, and an enlargement of the Golgi complex (Fig. 9). These effects were slower to develop and less prominent than those of PMA but more prolonged in time, and they reached a maximal value after 60 min.
Figure 9:
Ultrastructural morphometric analysis
after IgE receptor activation in RBL cells. IgE-primed cells were
stimulated with 100 ng/ml DNPBSA for different times and then
processed for morphometry as described under ``Experimental
Procedures.'' The number of vesicles (A), ER buds (B), and the Golgi surface (C) rapidly increased
reaching a plateau in about 20 min.
The mechanism by which signals
generated at the plasma membrane by the IgE receptor translocate to the
transport apparatus is probably linked to the fact that IgE receptor
activation stimulates the activity of soluble tyrosine
kinases(48) , which can, in turn, activate cytosolic
phospholipase C1(49) ; PKC could thus be stimulated
directly on the target organelle through the translocation of activated
phospholipase C
1 and the consequent local production of DAG, the
natural activator of the kinase. Very recently, we have found that
other receptors such as growth factor receptors, can stimulate
constitutive secretion in a variety of cell lines, and that they can
exert this effect even through PKC-independent mechanisms.
Thus, different signaling pathways appear to control constitutive
traffic. To understand the physiological significance of these
responses, it will be important to determine which types of receptors
activate specific stations of the transport route and which second
messengers are involved. It is likely that receptor-induced modulation
of intracellular protein transport and sorting is involved in a variety
of physiological phenomena. For instance, the stimulation of the
membrane transport apparatus in mast cells via the IgE receptor might
be required to prepare the complex recovery process that these cells
must undergo after degranulation.
A relevant question concerning the role of PKC in constitutive release is whether the kinase can act directly on the TG/TGN to stimulate export toward the plasma membrane. In fact, in addition to the ER to Golgi traffic segment(20) , endocytosis (19, 51) and apical endosome-plasma membrane recycling in polarized cells (16) have been reported to be stimulated by PMA. It is thus possible that the stimulation of TG/TGN to plasma membrane transport might be due to enhanced membrane flow in other districts of the cell rather than a direct activation of export from the TG/TGN. For instance, the stimulation of endocytosis and ER to Golgi transport could increase the input of surface and volume into the TG/TGN, thereby stimulating a compensatory output from it. However, the demonstration, in PC12 cells, that PMA accelerates the formation of proteoglycan-containing vesicles from cell-free TG/TGN preparations (where such indirect effects are very unlikely to occur) indicates that at least part of the effect of PMA on glycan release is due to a direct action on the TG/TGN. It should be noted, at the same time, that this experiment does not exclude the possibility that other (indirect) effects of PMA may occur in vivo, especially because the effect observed in cell-free vesicle formation experiments is smaller than the overall acceleration of glycan release seen in intact cells (it is also possible, however, that this discrepancy may simply be due to a lower efficiency of the in vitro reaction). Further support for a direct action of PMA on the TG/TGN comes from a different set of experiments in intact RBL cells, showing that there is no detectable input from the endocytic pathway into the TG/TGN (see Fig. 2) and that, after a prolonged temperature block at 19.5 °C, IgE receptor and PKC activation strongly accelerate exit from the TG/TGN. In conclusion, the collective data indicate that the stimulatory action of PMA on TG/TGN to plasma membrane transport is, at least in part, due to a direct activation of membrane export from the TG/TGN.
Another question concerns the differential roles of PKC in the transport of fluid-phase and membrane markers, since the transport of GAG (a fluid-phase marker) was potently accelerated by PKC activators, whereas the transport of VSV-G (a membrane protein) was rapidly and markedly inhibited by PKC inhibitors (compare Fig. 4and Fig. 6). A difficulty in interpreting these results is that the appearance of membrane markers on the plasma membrane depends on the balance between exocytic and endocytic activities, whereas the release of soluble molecules is mainly due to exocytic transport. Nevertheless, it seems to us that the most economical explanation of the results may be based on recent reports that soluble and membrane proteins are transported to the plasma membrane in distinct vesicular carriers(43, 44) . Another alternative explanation is based on the fact that, in RBL cells, a large amount of soluble glycans are eventually accumulated in secretory granule, which would occur via intermediates generally referred to as ``immature granule.'' Constitutive exit from this compartment has clearly been shown (for a review, see (52) ), and it is thus possible that PKC and receptor activation might accelerate this constitutive-like (i.e. not calcium-dependent) transport toward the plasma membrane. Thus, if indeed there are different transport mechanisms for GAG and VSV-G, they might be differentially regulated by PKC.
What is the target of PKC in the traffic machinery? A good candidate is the mechanism of ARF activation and/or binding to Golgi membranes, since this is a key event in vesicle formation already known to be modulated by PKC(14) . The involvement of ARF in promoting the assembly of different kinds of coat onto Golgi membranes, is indicated both by direct evidence and by the use of BFA that blocks the activation of ARF (7, 57, 58) . With regard to the TG/TGN, BFA is known to block constitutive transport from the TG/TGN to the plasma membrane (59) (this report); moreover, ARF has also been found to be associated with proteoglycan-carrying post-TGN vesicles in rat hepatocytes(42) . Other targets of PKC in the traffic machinery cannot be excluded. For instance, the phospholipase D-stimulating activity of ARF (60, 61) has been recently shown to be potentiated by PMA in human neutrophils (62) .
The identity
and the localization of the PKC isoform(s) that controls membrane
traffic is presently unclear. PKC is part of a family of several
isozymes with different sensitivities to modulators and substrate
specificities(63, 64) . The ,
, and
isoforms have previously been hypothesized to be involved in the
regulation of membrane
traffic(14, 16, 19, 65) . A series
of experiments in COS 7 cells, which normally express these isoforms
and respond to PMA with an increase in glycan secretion have, however,
revealed that these kinases are not involved in regulating membrane
traffic (at least in these cells). In fact, when
- and
-PKC
were overexpressed together with the secretory mutant of human alkaline
phosphatase(66) , neither the basal secretion rate of the
secretory mutant of human alkaline phosphatase nor the potency of PMA
in eliciting the secretory response appeared to change,
as
one would have expected if the overexpressed isoforms had been involved
in the PMA stimulation of membrane traffic(67, 68) .
Interestingly when the
isoform was overexpressed, the response to
PMA was completely lost
in accordance with a recent
report(65) . More work using the co-transfection approach
employed in this study will be necessary to identify the relevant PKC
isoform(s).
If one makes the reasonable assumption that the increase in Golgi size (60% in 2 min) is mostly due to enhanced membrane input from the ER, one obtains that the observed PMA-induced Golgi increase requires an acceleration in net membrane transport from the ER to the Golgi corresponding to at least 5% of the ER surface/min. Clearly this effect, if continued, would rapidly lead to gross imbalances in the sizes of the secretory organelles. Similar considerations can be made based on functional (GAG release) data, with respect to the TG/TGN. The half-time of GAG release from the cell lines here used ranges from 15 to 30 min; these estimations, assuming that GAG are homogenous markers of the TG/TGN luminal space, would also reflect the half-time of turnover of this organelle. If so, a 100% increase in export rate from the TG/TGN such as that described here would, again, lead to substantial changes in the size of the organelle within a short time if homeostatic mechanisms were not in place.
Thus, this study introduces a novel appreciation of the rapidity and extent of the variations in membrane traffic rates that may occur under physiological conditions and supports the notion that there must be ways in which traffic movements in different organelles are precisely matched to preserve the ``homeostasis'' of cell membranes. While this idea has occasionally surfaced in the literature, the mechanisms presiding over such fine regulation have not been investigated. One possibility that may now be suggested is that DAG or other second messengers (see recent data implicating cAMP in the regulation of transcytosis and endocytosis) (17, 18, 19) may be involved not only in mediating the variations in traffic rates induced by a membrane receptor, as shown here, but also in restoring, when necessary, ``normal'' conditions in the secretory pathway. It is possible, for instance, that individual organelles may possess sensors for excessive changes in their size and shape that would generate messengers to re-establish ``correct'' functioning status. The heterotrimeric G proteins located on secretory organelles might play a role in these intracellular signaling systems functioning in and between organelles (55) . Interestingly, an example of interorganelle signaling (albeit apparently not involving G proteins) has been recently provided by the finding that events in the lumen of the ER can be sensed and signaled via a protein kinase to the nucleus, to control the transcription of ER chaperon proteins(69, 70) . A variety of transduction signals may be used as one of the mechanisms functioning to maintain the homeostasis of intracellular traffic pathways and to mediate their adaptive responses to receptor stimuli.