(Received for publication, November 28, 1995; and in revised form, January 23, 1996)
From the
Phosphatidylinositol (PI) 3`-kinases are a family of lipid kinases implicated in the regulation of cell growth by oncogene products and tyrosine kinase growth factor receptors. The catalytic subunit of the p85/p110 PI 3`-kinase is homologous to VPS-34, a phosphatidylinositol-specific lipid kinase involved in the sorting of newly synthesized hydrolases to the yeast vacuole. This suggests that PI 3`-kinases may play analogous roles in mammalian cells. We have measured a number of secretory and endocytic trafficking events in Chinese hamster ovary cells in the presence of wortmannin, a potent inhibitor of PI 3`-kinase. Wortmannin caused a 40-50% down-regulation of surface transferrin receptors, with a dose dependence identical to that required for maximal inhibition of the p85/p110 PI 3`-kinase in intact cells. The redistribution of transferrin receptors reflected a 60% increase in the internalization rate and a 35% decrease in the recycling rate. Experiments with fluorescent transferrin showed that entry of transferrin receptors into the recycling compartment and efflux of receptors out of the compartment were slowed by wortmannin. Wortmannin altered the morphology of the recycling compartment, which was more vesiculated than in untreated cells. Using Semliki Forest virus as a probe, we also found that delivery of the endocytosed virus to its lysosomal site of degradation was slowed by wortmannin, whereas endosomal acidification was unaffected. In contrast to these effects on endocytosis and recycling, wortmannin did not affect intracellular processing of newly synthesized viral spike proteins. Wortmannin did induce missorting of the lysosomal enzyme cathepsin D to the secretory pathway, but only at a dose 20-fold greater than that required to inhibit p85/p110 PI 3`-kinase activity or to redistribute transferrin receptors. Our data demonstrate the presence of wortmannin-sensitive enzymes at three distinct steps of the endocytic cycle in Chinese hamster ovary cells: internalization, transit from early endosomes to the recycling and degradative compartments, and transit from the recycling compartment back to the cell surface. The wortmannin-sensitive enzymes critical for endocytosis and recycling are distinct from those involved in sorting newly synthesized lysosomal enzymes.
Phosphatidylinositol (PI) ()3`-kinases are a family
of lipid kinases that contain homologous catalytic domains, but
distinct regulatory elements. The first known PI 3`-kinase was a
heterodimeric lipid kinase composed of an 85-kDa regulatory subunit and
a 110-kDa catalytic
subunit(1, 2, 3, 4, 5) .
This enzyme was discovered in transformed cells, but is also stimulated
by mitogens including platelet-derived growth factor and
insulin(6) . The p85/p110 PI 3`-kinase is activated when the
two SH2 domains in p85 bind to phosphorylated YXXM motifs in
tyrosine kinase receptors or their substrates(7, 8) ;
activation by interactions with p21
, Rho, and
the SH3 domains of Src family kinases has also been
described(9, 10, 11) . Activation of the
p85/p110 PI 3`-kinase increases the intracellular concentrations of
PI(3,4)P
and PI(3,4,5)P
(12) . Although
these lipid products activate calcium-independent protein kinase C
isoforms in vitro, their function in intact cells is not
known(13, 14) . p110 also contains a serine/threonine
protein kinase activity with an apparently limited range of
substrates(15) . In addition to multiple isoforms of both the
85- and 110-kDa subunits, the recent description of several novel PI
3`-kinase isoforms with varying substrate specificities and regulatory
mechanisms has added to the complexity of 3-phosphoinositide
signaling(16, 17, 18) .
The p110 catalytic
subunit is 55% homologous to the Saccharomyces cerevisiae protein VPS-34(2, 19) . Disruption of the VPS-34 gene causes missorting of vacuolar hydrolases to the
secretory pathway and also abolishes PI 3`-kinase activity in
yeast(20) . Although VPS-34 is a PI 3`-kinase, its substrate
specificity is limited to phosphatidylinositol, unlike the p85/p110
mammalian enzyme that also utilizes PI(4)P and PI(4,5)P(21) . Moreover, mammalian p110 is lethal when expressed
in yeast(22) . However, a mammalian homologue to VPS-34 has
been cloned, which, like VPS-34, is a lipid kinase activity specific
for phosphatidylinositol (16) . Several experiments suggest
that PI 3`-kinases play a role in vesicular trafficking in mammalian
cells. Mutagenesis of the two PI 3`-kinase-binding sites in the
platelet-derived growth factor receptor disrupts the post-endocytic
sorting of this receptor(23) . However, these same sites bind
to SH2-containing molecules other than PI 3`-kinase(24) , which
complicates the interpretation of this finding. PI kinase inhibitors
such as wortmannin and LY294002 inhibit histamine secretion in RBL-2H3
cells, exocytosis in neutrophils stimulated with chemotactic agents,
and translocation of the GLUT4 glucose transporter in
insulin-stimulated adipocytes and skeletal
muscle(25, 26, 27, 28, 29, 30, 31) .
These data have been interpreted as demonstrating a role for the
p85/p110 PI 3`-kinase in these processes. However, a number of PI
kinase isoforms are sensitive to wortmannin, including both PI
3-kinases and PI 4-kinases(16, 24, 32) .
Despite questions as to the specificity of these findings, there is a
growing body of evidence suggesting a role for 3-phosphoinositides in
intracellular trafficking.
In this study, we examined a number of endocytic and biosynthetic trafficking pathways in CHO cells using wortmannin, which irreversibly inhibits several PI kinase isoforms. Treatment of cells with 100 nM wortmannin causes a 50% reduction of cell-surface transferrin receptors due to an increase in the internalization rate and a decrease in the recycling rate. Consistent with these changes, the morphology of the recycling compartment is altered by wortmannin, and movement of transferrin into and out of the recycling compartment is slowed. We also find that wortmannin slows the delivery of endocytosed Semliki Forest virus to its lysosomal site of degradation. However, we were unable to detect any changes in the transit of newly synthesized viral glycoproteins through the Golgi apparatus and to the plasma membrane, and we detected alterations in the sorting of newly synthesized lysosomal enzymes only at micromolar concentrations of wortmannin. Our data demonstrate the presence of wortmannin-sensitive enzymes that regulate receptor-mediated endocytosis and recycling. These enzymes are pharmacologically distinct from those involved in the sorting of newly synthesized lysosomal proteases.
To
measure recycling, TRVb-1 cells were incubated with I-labeled transferrin for 2 h at 37 °C, followed by
an additional 45 min with
I-labeled transferrin in the
absence or presence of 100 nM wortmannin. The cells were
washed with 50 mM MES (pH 5.0) and 150 mM NaCl to
remove surface-bound transferrin and incubated in 600 µg/ml
unlabeled transferrin and 100 µg/ml desferoxamine in the continued
absence or presence of wortmannin. At various times, the medium and one
wash were collected, and the cells were solubilized to determine
recycled and cell-associated radioactivity.
Figure 1: Inhibition of PI 3`-kinase by wortmannin. CHO cells were incubated in the presence of 0.1% dimethyl sulfoxide or dimethyl sulfoxide containing the indicated concentrations of wortmannin. The cells were lysed, and PI 3`-kinase activity in anti-p85 immunoprecipitates was assayed. Data are the means ± S.E. of triplicates and are representative of three separate experiments.
Figure 2:
Transferrin endocytosis and recycling. A, cells were treated with the indicated concentrations of
wortmannin for 45 min at 37 °C. The cells were chilled, and surface I-labeled transferrin binding was determined. Data are
the means ± S.E. of triplicates and are representative of two
separate experiments. B, TRVb-1 cells were incubated in the
absence or presence of 100 nM wortmannin for 45 min at 37
°C. The cells were incubated for various times with
I-labeled transferrin (3 µg/ml), rapidly chilled, and
washed with either neutral or acidic buffer for determination of
cell-associated or internalized radioactivity, respectively.
Surface-bound radioactivity was calculated as the difference between
cell-associated and internalized transferrin. Data are the means
± S.E. of triplicates and are representative of five separate
experiments. C, shown is an In/Sur plot of
I-labeled transferrin uptake in control and
wortmannin-treated cells. Data are the means ± S.E. of
triplicates and are representative of nine separate experiments. D, cells were incubated in the presence of
I-labeled transferrin for 2 h at 37 °C, followed by
an additional 45 min in the presence of 100 nM wortmannin or
carrier. After a mild acid wash to remove surface-bound ligand, the
cells were incubated in medium containing 100 µg/ml desferoxamine
and 600 µg/ml unlabeled transferrin in the continued absence or
presence of wortmannin. Release of
I-labeled transferrin
into the medium was calculated as a percentage of total intracellular
transferrin at 0 min. Data are the means ± S.E. of triplicates
and are representative of seven separate
experiments.
We then
measured the uptake of I-labeled transferrin during a
10-min incubation at 37 °C in TRVb-1 cells. Pretreatment with
wortmannin for 30 min decreased the accumulation of surface-bound and
intracellular transferrin (Fig. 2B). The change in
cell-surface transferrin binding reflected a redistribution of
cell-surface transferrin receptors into an intracellular compartment,
as total cellular transferrin binding was unaffected by wortmannin
(data not shown). To determine the mechanism for this redistribution,
we measured the rates of internalization and recycling in
wortmannin-treated cells. The internalization rate constant for
I-labeled transferrin was calculated by the In/Sur
method(36) ; a representative experiment is shown in Fig. 2C. In data pooled from nine experiments, the rate
constant for transferrin receptor endocytosis was increased 55% by
wortmannin (Table 1). Similarly, the rate of
Fe
uptake was increased 60% by treatment of cells with wortmannin (Table 1). Although the calculated rate constants differed using
the two methods, in both cases, treatment of cells with 100 nM wortmannin caused a similar increase in the internalization rate (Table 1). Although there was a decrease in the absolute amount
of internalized
I-labeled transferrin in
wortmannin-treated cells (Fig. 2B), the internalization
rate constants are calculated on a per receptor basis and are in fact
increased by wortmannin.
In addition to the effects on
internalization, the rate of transferrin recycling was reduced by
treatment of cells with wortmannin. Cells were preloaded with I-labeled transferrin in the absence of wortmannin (to
prevent any differences in the concentration of intracellular ligand)
and then incubated in 100 µM desferoxamine in the absence
or presence of wortmannin (a representative experiment is shown in Fig. 2D). In data pooled from seven experiments, we
observed a 33% decrease in the rate of
I-labeled
transferrin efflux from the wortmannin-treated cells (Table 1).
The steady-state distribution of transferrin receptors between the
interior and surface of cells is determined by both the rates of
endocytosis (k) and recycling (k
). Changes in either parameter will be reflected
in a new steady-state distribution. The increase in k
and the decrease in k
observed in
wortmannin-treated cells predict a redistribution of transferrin
receptors from the cell surface to the cell interior, resulting in a
decrease in surface binding. The magnitude of the predicted change in
surface binding, based on the ratio of the changes in k
and k
, was similar to the observed decrease
in cell-surface
I-labeled transferrin binding (42% as
opposed to 45%) (Table 1).
We also measured the
internalization of I-labeled insulin in CHO/IR cells,
which overexpress the human insulin receptor. Unlike transferrin
receptors, which internalize constitutively, insulin receptors enter
coated pits in a ligand-stimulated manner(46, 47) .
Moreover, changes in the insulin receptor membrane-spanning domain that
increase the rate of receptor lateral diffusion on the cell surface
increase the rate of receptor endocytosis(48) , suggesting that
events prior to entry into coated pits are rate-limiting for insulin
receptor endocytosis. We found that the rate constant for
I-labeled insulin uptake in CHO/IR cells was unaffected
by wortmannin (k
= 0.116 ± 0.006 versus 0.125 ± 0.01 in the absence and presence of
wortmannin) (data not shown). Thus, the ligand-stimulated events that
precede entry of insulin receptors into coated pits appear to be
independent of wortmannin-sensitive enzymes.
Figure 3: Uptake of Cy3-transferrin in wortmannin-treated cells. TRVb-1 cells were incubated for 30 min in the presence of 100 nM wortmannin or carrier. After the addition of Cy3-transferrin (10 µg/ml) for 3 min, the cells were washed and chased for various times in the continued absence (A and B) or presence (C and D) of wortmannin. A and C, 2 min of chase; B and D, 12 min of chase. Serial images were collected at 0.6-µm steps over the depth of the cells and were projected using the maximum pixel method.
In contrast, pretreatment of cells with wortmannin delayed the sorting of internalized transferrin to the recycling compartment. When wortmannin-treated cells were labeled with Cy3-transferrin and chased in wortmannin for 2 min, we saw an accumulation of ligand in numerous peripheral punctate structures similar to those seen in control cells (Fig. 3C). The intensity of labeling was decreased in wortmannin-treated cells relative to control cells, reflecting the decrease in cell-surface transferrin receptors caused by preincubation with wortmannin (Fig. 2B). After 12 min of chase, the distribution of labeled transferrin in wortmannin-treated cells was significantly different than in control cells, with a persistence of label in peripheral punctate structures (Fig. 3D). After 16 min of chase, pericentriolar concentration could be seen in some wortmannin-treated cells (data not shown). However, the labeled structures in these cells were more vesiculated and less uniform in appearance that those in control cells (data not shown; but see Fig. 4, A and B).
Figure 4: Trafficking of Cy3-transferrin in wortmannin-treated cells. TRVb-1 cells were incubated with Cy3-transferrin for 15 min at 37 °C and then chased for 15 min (A-D) or 45 min (E-H) in FITC-transferrin in the absence or presence of 100 nM wortmannin. A and C, B and D, E and G, and F and H show the same field of cells viewed with either Cy3- or FITC-specific filters, respectively. Control cells are shown in A, C, E, and G; cells chased in wortmannin are shown in B, D, F, and H.
These data suggest that wortmannin reduces the rate of delivery of internalized transferrin from endosomes to the recycling compartment. Alternatively, the altered morphology of the labeled compartment in wortmannin-treated cells could reflect a change in the fidelity of sorting, with delivery of internalized transferrin to a different compartment. To differentiate these possibilities, we examined the degree to which transferrin internalized prior to wortmannin treatment mixed with transferrin internalized in the presence of wortmannin. Cells were preincubated to equilibrium by a 45-min incubation with Cy3-transferrin in the absence of wortmannin; under these conditions, label accumulates in the pericentriolar recycling compartment. The cells were then washed and chased in medium containing FITC-transferrin in the absence or presence of 100 nM wortmannin. During this incubation, Cy3-transferrin effluxes from the cells, whereas FITC-transferrin accumulates.
In untreated cells labeled with Cy3-transferrin and then chased with FITC-transferrin for 15 min, residual Cy3 labeling of the recycling compartment was easily observable (Fig. 4A). Moreover, FITC-transferrin could be seen to accumulate in the same intracellular compartment that contains Cy3-transferrin (Fig. 4C). In cells chased with FITC-transferrin in the presence of wortmannin for 15 min, the residual Cy3 labeling of the recycling compartment is much brighter than in control cells (Fig. 4B), indicating that wortmannin slowed the recycling of transferrin receptors back to the cell surface. Moreover, the vesiculation of the pericentriolar compartment in wortmannin-treated cells is evident (Fig. 4B). However, the altered compartment is still clearly labeled with FITC-transferrin (Fig. 4D), indicating that this compartment is in communication with early endosomal structures containing newly internalized transferrin.
The wortmannin-induced slowing of efflux from the recycling compartment is even more striking after a 45-min chase. In control cells chased for 45 min, Cy3-transferrin in the pericentriolar structures has been substantially washed out (Fig. 4E), replaced by FITC-transferrin that entered during the chase (Fig. 4G). In contrast, the punctate pericentriolar structures in wortmannin-treated cells are still brightly labeled with Cy3-transferrin after 45 min (Fig. 4F). Again, Cy3-transferrin colocalizes with newly internalized FITC-transferrin (Fig. 4H), suggesting that the punctate pericentriolar structures in wortmannin-treated cells are in communication with early endosomal structures. The increased labeling of the recycling compartment during the FITC-transferrin chase in wortmannin-treated cells (Fig. 4, compare C with D and G with H) reflects the wortmannin-induced redistribution of transferrin-receptor complexes into the cell interior (Fig. 2A). Taken together, the data in Fig. 3and Fig. 4show that wortmannin has a pronounced inhibitory effect on both entry into and efflux from the recycling compartment.
Figure 5:
Degradation of endocytosed Semliki Forest
virus. CHO cells were pretreated with 100 nM wortmannin or
carrier and then incubated with S-labeled Semliki Forest
virus for 1.5 h at 4 °C. The cells were washed and warmed to 37
°C in the continued absence or presence of wortmannin. At various
times, aliquots of the medium were removed to determine the release of
trichloroacetic acid-soluble radioactivity. Trichloroacetic
acid-soluble radioactivity is expressed as a percentage of initial
cell-associated radioactivity.
Although these data suggest that wortmannin had significant
effects on the kinetics of late endosomal sorting, the altered kinetics
of SFV degradation could be due to effects on the acidification of the
endosomal lumen. We therefore examined the endocytic uptake of S-labeled SFV using a conformation-specific antibody to
the SFV E1 spike protein. The E1 spike protein undergoes a
conformational change when exposed to the acidic conditions of the
early endosome (pH 6.2 or lower), exposing an epitope that is
recognized by monoclonal antibody E1a-1(40) .
Untreated (Fig. 6A) or wortmannin-treated (Fig. 6B) cells were allowed to bind S-labeled SFV at 4 °C and then warmed rapidly to 37
°C. At various times, the cells were lysed, and proteins were
precipitated with either anti-spike antibody or conformation-specific
antibody E1a-1. No difference in the total amount of cell-associated
virus was detected in control versus wortmannin-treated cells (Fig. 6, A and B, lanes a). In both
treated and untreated cells, a decrease in cell-associated virus due to
lysosomal degradation was apparent by 15-30 min (Fig. 6, A and B, lanes d and e). Similarly,
in both untreated and wortmannin-treated cells, the E1 spike protein
was first recognized by antibody E1a-1 after 5 min at 37 °C (Fig. 6, A and B, lanes j), with
levels of E1a-1-precipitable protein reaching maximal levels by 15 min (lanes l). In control experiments, treatment of cells with
monensin completely blocked the conversion of E1 to the acidic
conformation (Fig. 6, A and B, lanes
o), whereas brief treatment of bound virus with acidic buffer (pH
5.5) caused maximal conversion of the E1 protein to its acidic
conformation (lanes p). We conclude that endosomal
acidification is not grossly affected by wortmannin.
Figure 6:
Effect of wortmannin on endosomal
acidification. TRVb-1 cells pretreated with carrier (A) or 100
nM wortmannin (B) were allowed to bind S-labeled SFV on ice for 90 min. After washes to remove
unbound virus, the cells were warmed to 37 °C in the continued
absence or presence of wortmannin. At various times, the cells were
lysed, and
S-labeled viral proteins were
immunoprecipitated with anti-spike antibody (A and B, lanes a-h) or antibody E1a-1 (lanes i-p),
which recognizes the E1 spike protein after it has undergone an
acid-dependent conformational change. Lanes g and o show immunoprecipitates from incubations that included monensin
(10 µM) to block acidification. Lanes h and p show cells treated with pH 5.5 buffer for 1 min prior to
lysis.
Figure 7:
Secretion of cathepsin D in
wortmannin-treated cells. A, TRVb-1 cells were incubated in
100 nM wortmannin or carrier for 30 min in methionine-free
medium plus an additional 30 min with
[S]methionine, followed by a chase in the
continued absence or presence of wortmannin. At various times, the
medium was harvested, the cells were lysed, and intracellular or
secreted
S-labeled cathepsin D was immunoprecipitated and
analyzed by SDS-polyacrylamide gel electrophoresis. B, cells
were incubated in the absence or presence of 2 µM wortmannin or 50 µM chloroquine for 30 min and then
processed as described above. The fluorographic exposure for B was one-third that used in A, which was intentionally
overexposed to visualize secreted cathepsin
D.
In contrast to the data obtained with 100 nM wortmannin, we saw a significant effect on the secretion of cathepsin D after treatment of cells with 2 µM wortmannin. This concentration of drug is 20-fold higher than that required for maximal effects on p85/p110 PI 3`-kinase activity or transferrin receptor redistribution. Fig. 7B shows identical exposures of intracellular and secreted cathepsin D from control cells, cells treated with 2 µM wortmannin, or cells treated with 50 µM chloroquine. Treatment of cells with 2 µM wortmannin had no significant effect on the synthesis or stability of intracellular cathepsin D, whereas treatment of cells with chloroquine increased the intracellular half-life of newly synthesized cathepsin D, presumably by inhibiting lysosomal degradation (Fig. 7B, upper panel). Notably, both 2 µM wortmannin and 50 µM chloroquine had pronounced effects on the secretion of newly synthesized cathepsin D. Secreted cathepsin D was undetectable in control cells (Fig. 7B, lower panel) using a fluorographic exposure time one-third of that used in Fig. 7A. In contrast, secreted cathepsin D was easily seen after 3 h in cells treated with 2 µM wortmannin (Fig. 7B, lower panel). 2 µM wortmannin induced a level of cathepsin D secretion similar to that seen in cells treated with chloroquine, a drug that is known to divert lysosomal enzymes into the secretory pathway(56) .
Figure 8: Morphology of the sorting compartment in cells treated with micromolar concentrations of wortmannin. TRVb-1 cells were incubated for 45 min in the presence of 5 µg/ml Cy3-transferrin, followed by an additional 45 min in the presence of Cy3-transferrin and 1 µM wortmannin. A and B represent two different optical sections from the same field.
Studies from several different laboratories suggest that PI 3`-kinase plays a role in endocytic or other trafficking events in mammalian cells. In this paper, we examined trafficking events in CHO cells treated with 100 nM wortmannin(27, 57, 58) , which maximally inhibits the p85/p110 PI 3`-kinase in these cells. We find that 100 nM wortmannin causes a significant redistribution of the transferrin receptor due to changes in both the endocytic and recycling rate constants. Moreover, wortmannin induces a vesicularization of the recycling compartment labeled with fluorescent transferrin and reduces the rate at which transferrin enters and exits this compartment. Transferrin receptor trafficking and p85/p110 PI 3`-kinase activity show similar sensitivities to wortmannin, consistent with the involvement of p85/p110 or a related enzyme in the regulation of endocytosis and recycling.
The effects of 100 nM wortmannin appear to be specific for the endocytic system, as we could not detect any changes in biosynthetic trafficking of SFV or VSV spike proteins. We did detect an enhancement of the secretion of cathepsin D, a phenotype similar to that observed in yeast containing mutant VPS-34 alleles, but only at micromolar concentrations of wortmannin. These data are consistent with those of Balch and co-workers(54) , who reported missorting of cathepsin D at 1-2 µM wortmannin in CHO and normal rat kidney cells, but differ from those of Davidson(55) , who observed effects on cathepsin D sorting at 100 nM wortmannin in K-532 cells. Presumably, the discordance reflects different sensitivities of the lysosomal enzyme-sorting apparatus in different cell types. However, our CHO cell data strongly suggest that the wortmannin-sensitive enzymes involved in lysosomal sorting are distinct from those involved in transferrin recycling and are unlikely to be the p85/p110 PI 3`-kinase. Furthermore, treatment of cells with 1 µM wortmannin causes an extensive tubulation and vesiculation of the transferrin-labeled compartment, which is barely visible at 100 nM wortmannin. The disruption of transferrin receptor cycling in CHO cells by 100 nM wortmannin therefore defines a sorting event(s) that is pharmacologically distinct from the wortmannin-sensitive steps in lysosomal enzyme delivery.
The
identity of the wortmannin-sensitive enzymes involved in transferrin
endocytosis and recycling is not clear, as the specificity of
wortmannin with regard to PI 3`-kinase is extremely problematic.
Wortmannin was initially described as an inhibitor of the myosin light
chain kinase, but it irreversibly inhibits PI 3`-kinase at 100-fold
lower
concentrations(26, 27, 57, 58) .
Both - and
- isoforms of the p110 PI 3`-kinase are
half-maximally inhibited by low nanomolar concentrations of wortmannin,
as is the recently cloned PI-specific PI 3`-kinase
; the
sensitivity of the
-stimulated PI 3`-kinase is not yet
clear(16, 18, 59) . In addition, a PI
4-kinase important in the regulation of intracellular PI(4)P and
PI(4,5)P
levels is half-maximally inhibited by 100 nM wortmannin and completely inhibited by 1 µM wortmannin(32) . This finding is particularly important;
PI(4,5)P
has been implicated in the regulation of AP-2
adaptin complexes and cytoskeletal components such as profillin and
plays a role in regulated secretion (60, 61, 62) . Moreover, PIP
has
been shown to bind to pleckstrin homology domains, suggesting that
disruption of PIP
synthesis could affect numerous cellular
functions (63) . The existence of multiple wortmannin-inhibited
enzymes, which are regulated by different mechanisms and may exist in
distinct intracellular locations, compromises the use of wortmannin as
a putatively specific inhibitor of PI 3`-kinase. Studies currently in
progress, involving the microinjection of inhibitory anti-p110
antibodies into TRVb-1 cells, will address the question of whether the
wortmannin-sensitive enzyme involved in transferrin trafficking is the
p85/p110 PI 3`-kinase.
The kinetic analysis of transferrin receptor
trafficking and Semliki Forest virus degradation in wortmannin-treated
cells demonstrates that three distinct steps in endocytic trafficking
are affected by the drug. First, we see an increase in the rates of I-labeled transferrin endocytosis and
Fe
accumulation, both of which measure the rate of constitutive coated
pit-mediated internalization. It is therefore possible that a
wortmannin-sensitive enzyme is important in regulating the early
rate-limiting steps governing coated pits. We did not see changes in
the rates of internalization of either
I-labeled insulin
or surface-bound
S-labeled SFV. However, both insulin and
SFV initially bind to receptors located in the microvillous regions of
the cell membrane and then move to coated
pits(47, 49) . Furthermore, the rate of insulin
receptor endocytosis appears to be limited by the rate of receptor
diffusion through the plasma membrane (48) and therefore is
probably not a reflection of the absolute rate of coated pit
endocytosis.
A second site of wortmannin action may be the sorting endosome. In CHO cells, recycled ligands are sorted from material destined for degradation by an iterative fractionation process in which the sorting endosome matures into a late endosome(38) . Consequently, if wortmannin affects the maturation of the sorting endosome, then both transport of the recycling membrane to the pericentriolar recycling compartment and delivery of material to lysosomes could be affected. Consistent with this possibility, wortmannin slows the movement of internalized receptor-bound transferrin from endosomes to the pericentriolar recycling compartment as well as the degradation of internalized SFV, which requires delivery to the lysosome. These changes do not appear to be due to defects in luminal acidification, which is known to affect intracellular trafficking of the transferrin receptor(64, 65) , as the acid-dependent conformational changes in the SFV E1 spike protein are normal in wortmannin-treated cells. Although our data could be explained by a common wortmannin-sensitive step in the sorting compartment, we cannot rule out the possibility of independent wortmannin-sensitive steps involved in transit of ligands between the sorting endosome and the recycling compartment or the lysosome. These steps could involve Rab5 or another Rab isoform, as has been recently suggested by Li et al.(66) .
A third potential site of action is the recycling compartment, defined as the pericentriolar structure that is labeled by fluorescent transferrin, but not by ligands like low density lipoprotein that are destined for lysosomal degradation(38) . Transport from the pericentriolar recycling compartment is the rate-limiting step in transferrin recycling(38) . The morphology of the recycling compartment is significantly altered by 100 nM wortmannin, and efflux of transferrin out of the compartment is slowed in wortmannin-treated cells. Although the mechanism by which wortmannin affects the recycling compartment is not yet clear, there is precedent for the regulation of transit from the recycling compartment. Thus, transferrin receptor recycling from the compartment to the plasma membrane is slowed by either alkalinization of the lumen or oligomerization of transferrin receptors in the presence of multivalent transferrin(64, 67) .
The redistribution of transferrin receptors caused by wortmannin is similar to that seen in a mutant liver line (Trf1) that expresses only State 1 receptors(68, 69) , defined by Weigel (68) as receptors resistant to down-regulation by metabolic and cytoskeletal inhibitors. However, residual surface receptors in Trf1 cells show unchanged endocytosis and accelerated recycling(69) . Receptor redistributions in wortmannin-treated cells therefore involve different mechanisms than those operative in Trf1 cells.
For the most part, the studies presented here examine trafficking under basal conditions of PI 3`-kinase activity. Some activation of PI 3`-kinase may occur during insulin-stimulated endocytosis in CHO/IR cells, although the studies were performed at insulin concentrations well below the half-maximal concentrations required for activation of PI 3`-kinase(70, 71) . Moreover, we have previously shown that mutant insulin receptors that do not activate PI 3`-kinase still undergo normal ligand-stimulated endocytosis(72, 73) . Siddle and co-workers (74) recently reported that wortmannin blocks insulin-stimulated up-regulation of the transferrin receptor in 3T3-L1 cells, and Joly et al.(23) have shown that treatment of cells with wortmannin or mutation of PI 3`-kinase-binding sites alters the post-endocytic routing of internalized platelet-derived growth factor receptors. Both of these studies examined cells under conditions of mitogen-induced PI 3`-kinase activation and may not be strictly comparable to the studies presented here.
In summary, we have evaluated the effects of wortmannin on vesicular trafficking in CHO cells. We find that concentrations of wortmannin that maximally inhibit the p85/p110 PI 3`-kinase have pronounced effects on three distinct steps in the endocytic cycle: internalization, transit from early endosomes to the recycling and degradative compartments, and transit from the recycling compartment back to the cell surface. A substantial change in the morphology of the sorting compartment is also seen under these conditions. However, we find no effects on the biosynthetic trafficking of viral glycoproteins or lysosomal enzymes at these concentrations. In contrast, wortmannin-induced secretion of cathepsin D was observed at significantly higher doses of wortmannin. We have therefore identified two thresholds of wortmannin-sensitive events in vesicular sorting, suggesting that the effects on endocytic and lysosomal trafficking are mechanistically distinct.