(Received for publication, August 17, 1994; and in revised form, October 25, 1994)
From the
Human colon cancer HT-29 cells exhibit a
differentiation-dependent autophagic-lysosomal pathway that is
responsible for the degradation of a pool of newly synthesized N-linked glycoproteins in undifferentiated cells. In the
present study, we have investigated the molecular control of this
degradative pathway in undifferentiated HT-29 cells. For this purpose,
we have modulated the function and expression of the heterotrimeric
G-proteins (G and G
) in these cells. After
pertussis toxin treatment which ADP-ribosylates heterotrimeric
G
-proteins, we observed an inhibition of autophagic
sequestration and the complete restoration of the passage of N-linked glycoproteins through the Golgi complex. In contrast,
autophagic sequestration was not reduced by cholera toxin, which acts
on heterotrimeric G
-proteins. Further insights on the
nature of the pertussis toxin-sensitive
subunit controlling
autophagic sequestration were obtained by cDNA transfections of
subunits. Overexpression of the
subunit increased autophagic sequestration and degradation in
undifferentiated cells, whereas overexpression of the
subunit, the only other pertussis toxin-sensitive
subunit expressed in HT-29 cells, did not alter the rate of
autophagy.
Although recent reports indicate that the autophagic-lysosomal
route could be modulated by intracellular signals including
mobilization of calcium pools(1) , protein
phosphorylation(2) , and GTP hydrolysis(3) , little
information on the molecular control of this process has yet to emerge.
We have previously shown that the N-glycan trimming, which
reflects endoplasmic reticulum (ER) ()to Golgi complex
traffic(4) , is dependent on the state of enterocytic
differentiation of HT-29 cells (5) . The conversion of high
mannose oligosaccharides to their complex counterparts is observed in
differentiated cells, whereas a partial blockade of high mannose
oligosaccharide trimming is a characteristic of undifferentiated cells.
This partial impairment of N-linked glycoprotein processing is
the consequence of a bypass of the Golgi complex and delivery of high
mannose type glycoproteins to the lysosomal compartment (6) via
an autophagic pathway(7) . We have taken advantage of our
previous data to investigate the function of heterotrimeric G-proteins,
pivotal players in membrane dynamics (reviewed in (8) ), in the
molecular control of autophagy.
When undifferentiated HT-29 cells (hereafter referred to as
HT-29 cells) are treated with PTX, which ADP-ribosylates the
subunits of heterotrimeric G-proteins, resulting in
the inhibition of GDP/GTP exchange(15) , a quantitative
trimming of high mannose to complex-type oligosaccharides was observed (Fig. 1, a and b). This PTX-induced processing
is not a consequence of either an increase in protein synthesis, as
determined by [
C]leucine incorporation, or
modification of Golgi complex-associated glycosyltransferase activities
(evaluated by the activity of
1,4 galactosyltransferase activity:
1.58 and 1.37 nmol/h/mg protein in control and PTX-treated cells,
respectively). Moreover, PTX treatment was also associated with the
cessation of N-linked glycoprotein degradation. This was
demonstrated after treatment of cells with dMM (Fig. 1a, inset), an inhibitor of ER
-mannosidase and Golgi complex
-mannosidase I(16) .
Under these conditions we have shown previously that high mannose
glycoproteins were unstable in HT-29 cells ( (10) and Fig. 1a, inset). In contrast, in the presence
of PTX high mannose glycoproteins were stable during a pulse-chase
experiment (Fig. 1a, inset). The above
described effect of PTX suggests that the restoration of the trimming
of N-glycan chains in HT-29 cells could be due to either a
cessation of the autophagic sequestration that impairs the Golgi
delivery of N-linked glycoproteins (6, 7) or
to an accelerated rate of ER to Golgi transport that prevents
glycoproteins from entering the autophagic pathway. Indeed the presence
of PTX-sensitive heterotrimeric G
-proteins in ER and Golgi
membranes is compatible with a function in protein transport from ER to
Golgi(17, 18, 19) . We therefore examined the
rate of maturation of the N-linked glycans of either Lamp 1, a
glycoprotein associated with lysosomal membranes (20) or
dipeptidylpeptidase IV, a plasma membrane glycoprotein(21) . In
both cases the kinetics of high mannose oligosaccharide maturation to
complex oligosaccharides was not changed by PTX treatment (data not
shown), in agreement with other results(22, 23) ,
indicating that ER to Golgi complex transport is unaffected by PTX. In
contrast, PTX dramatically inhibits the autophagic sequestration of
[
H]raffinose electroloaded into HT-29 cells (Fig. 2a), the level of PTX inhibition is similar to
that observed in the presence of 3-MA (an inhibitor of autophagic
sequestration, (24) ). This sequestration of electroloaded
[
H]raffinose was insensitive to CTX, which
ADP-ribosylates the
subunit of
G-proteins(25) , although this toxin is able to activate
G
-proteins in HT-29 cells as determined by the increase of
cAMP production(26, 27) . In addition, the absence of in vitro PTX-dependent ADP-ribosylation of
subunits in homogenates from PTX-treated cells confirmed that PTX
could modify the G
-proteins in vivo (Fig. 2b). In order to determine the identity of
the PTX-sensitive G-protein
subunit that controls the
autophagic sequestration we have transfected HT-29 cells with cDNA
encoding either the
or the
subunits. We focused our study on these two subunits since as in
normal intestinal cells(14, 28) , they are the only
PTX-sensitive subunits expressed in HT-29 cells (data not shown).
Several stably transfected clones expressing different levels of
subunits were selected. Whatever the
subunit considered, Western blot analysis showed that these
clones could be classified into two groups: the first one has a
1.5-fold increase in the level of the expression and the second a
3-fold increase. All the screened clones have phenotypic
characteristics similar to that of parental untransfected cells. The
rate of autophagy was measured in representative clones by assaying the
sequestration of electroloaded [
H]raffinose (Fig. 3a) and the degradation of
[
C]leucine-labeled proteins (Fig. 3b). As shown in Fig. 3, the
overexpression of
(3-fold) does not change
autophagic sequestration/degradation in HT-29 cells. Similar results
were obtained using other clones with lower (1.5-fold) overexpression
of
(data not shown). In contrast we observed a
relationship between the overexpression of
and the
rate of autophagy. Clones 1 (1.5-fold increase) and 2 (3-fold increase)
have 2.2- and 3.8-fold increases, respectively, in the sequestration of
[
H]raffinose when compared to that observed in
untransfected cells. In both clones the autophagic sequestration was
inhibited by PTX and 3-MA treatment (Fig. 3a). This
increase in the [
H]raffinose sequestration was
correlated to an increase in protein degradation in overexpressing
cells: by 2.6- and 4.0-fold in clones 1 and 2,
respectively (Fig. 3b). The degradation of proteins in
overexpressing
cells was inhibited by PTX and drugs
that impair the autophagic-lysosomal pathway at different steps (Fig. 3c), i.e. sequestration (3-MA), fusion
of autophagic vacuoles with lysosomes (asparagine), lysosomal
degradation (NH
Cl). These results underscore the
involvement of
in the control of the autophagic
pathway. As shown in Fig. 4a, the overexpression of
(3-fold) does not change the cellular localization
of both
and
subunits as observed
previously in other cell lines(19, 29) . In contrast
to most of the cell lines studied so
far(13, 18, 19) , a high amount of
subunits was found in the cytosolic fraction, which
was more evident for
subunit (Fig. 4b). Whether this distribution of
is related to the autophagic capacity of HT-29 cells remains to
be explored. Nevertheless the absolute amount of membrane-bound
was increased in overexpressing
cells by 1.5-3.0-fold (Fig. 4b). This
increase was correlated with changes in autophagic degradation and
autophagic sequestration (see above), which is compatible with an
amplified response for heterotrimeric G-protein pathways (see (29) and (30) , and references therein). The
PTX-dependent inhibition of autophagic sequestration/degradation would
suggest that the stabilization of membrane-bound G
-protein
in association with GDP is a key regulatory step in autophagy. As
autophagic sequestration is constitutively expressed in HT-29 cells,
our results suggest, by analogy with the functioning of plasma
membrane-bound G-proteins(31) , that an endomembraneassociated
effector is under a conformational state that permanently activates
G
-protein. This putative effector could be in an inactive
state in differentiated HT-29 cells where autophagic sequestration is
down-regulated (6, 7) despite the fact that the
subunit is expressed at the same level as in
undifferentiated HT-29 cells (data not shown). Such a change in the
conformation of a membrane-bound effector would fit with the rapidity
of G-protein mediated signal transduction (31) and induction of
autophagic sequestration in stimulated cells, e.g. hepatocytes(32, 33, 34, 35) .
Modifications of the effector conformation can be induced by either the
binding of a diffusible ligand or changes in its phosphorylation
status, two stimuli known to affect the G-protein
response(30, 36) . The latter possibility would be
interesting to consider, since the phosphorylation status of proteins
has been shown to modulate the induction of autophagy in
hepatocytes(2) . Recently, Kadowaki et al.(3) reported that GTP
S inhibits the stimulated
autophagic sequestration in rat hepatocytes, suggesting a role for
GTP-binding proteins in the regulation of autophagy. Since GTP
S
acts as well on heterotrimeric and monomeric G-proteins(22) ,
it cannot be concluded from this study on the identity of G-proteins
involved in the autophagic sequestration. However, from the results
reported in the present work and the possible localization of Rab24 (37) , a monomeric GTP-binding protein, along the autophagic
pathway it could be suggested that autophagy is under the control of
multiple GTP-binding proteins as already observed for membrane dynamics
along the exocytic and endocytic
routes(26, 38, 39, 40) .
Figure 1:
Effect of PTX on the processing and
stability of high mannose oligosaccharides. a, high mannose
oligosaccharides; b, complex oligosaccharides. Inset,
fate of high mannose oligosaccharides in dMM-treated cells. HT-29 cells
were radiolabeled with 400 µCi/ml D-[2-H]mannose for 10 min and then
chased for the indicated times. PTX and dMM were used as detailed under
``Experimental Procedures.''
Figure 2:
The effect of toxins on the autophagic
sequestration of electroloaded [H]raffinose in
undifferentiated HT-29 cells. a, autophagic sequestration was
determined either in the absence or presence of 200 ng/ml PTX, 500
ng/ml CTX, or 5 mM 3-MA. After homogenization and
centrifugation in a sucrose/metrizamide gradient, the radioactivity was
measured in the sedimentable material. Values are the mean ±
S.D. (n = 5). b, cell-free PTX-catalyzed
ADP-ribosylation were performed with [
P]NAD on
cell homogenates prepared from PTX-treated (+) or control(-)
cells. Proteins were subsequently analyzed by SDS-polyacrylamide gel
electrophoresis (10% gel).
Figure 3:
Overexpression of
stimulates autophagic sequestration and degradation. a,
autophagic sequestration of [
H]raffinose was
determined as detailed in the legend to Fig. 2in overexpressing
cells (3-fold overexpression), overexpressing
cells (clone 1: 1.5-fold overexpression, clone 2:
3-fold overexpression), and control cells. b, protein
degradation in untransfected cells (control) and
and
overexpressing cells (clone 1 and clone 2). c, inhibition of degradation by drugs in overexpressing
cells (clone 2). For protein degradation studies,
cells were labeled for 6 h with 0.2 µCi of
[
C]leucine and chased for different times in
medium containing 5 mM leucine. PTX (200 ng/ml) was added 18 h
before the labeling period and was present throughout the chase period;
3-MA (5 mM), asparagine (10 mM), and NH
Cl
(10 mM) were added at the beginning of the chase period.
Degradation of [
C]leucine-labeled proteins was
measured as described previously(11) . In panels
a-c, values are the mean ± S.D. (n =
5).
Figure 4:
Localization and distribution of
subunits. a, immunofluorescent localization
of
and
in untransfected cells and
in
overexpressing cells (clone 2); bar represents 15 µm. b, Western blot of cytosolic (C) and membrane-bound (M)
and
subunits in HT-29 cells (untransfected) and
overexpressing
cells (clone 2). A 3.0-fold increase
(measured by densitometry) in the amount of
subunit
was found in the membrane-bound fraction of clone
2.