©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Heterotrimeric G-protein Controls Autophagic Sequestration in the Human Colon Cancer Cell Line HT-29 (*)

(Received for publication, August 17, 1994; and in revised form, October 25, 1994)

Eric Ogier-Denis (1) Alain Couvineau (1) Jean José Maoret (1) Jean Jacques Houri (1) Chantal Bauvy (1) Daniela De Stefanis (2) Ciro Isidoro (2) Marc Laburthe (1) Patrice Codogno (1)(§)

From the  (1)From INSERM U410, Neuroendocrinologie et Biologie Cellulaire Digestives, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France and (2)Dipartimento di Medicina ed Oncologia Sperimentale, Sez. Patologia Generale, Università di Torino, Corso Raffaello 30, 10125 Torino, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(s) and G(i)) in these cells. After pertussis toxin treatment which ADP-ribosylates heterotrimeric G(i)-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(s)-proteins. Further insights on the nature of the pertussis toxin-sensitive alpha subunit controlling autophagic sequestration were obtained by cDNA transfections of alpha(i) subunits. Overexpression of the alpha subunit increased autophagic sequestration and degradation in undifferentiated cells, whereas overexpression of the alpha subunit, the only other pertussis toxin-sensitive alpha subunit expressed in HT-29 cells, did not alter the rate of autophagy.


INTRODUCTION

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) (^1)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.


EXPERIMENTAL PROCEDURES

Reagents

CTX, PTX, leupeptin, dMM, asparagine, and 3-MA were from Sigma. Cell culture reagents and Geneticin (G418) were from Life Technologies, Inc. (Eragny, France). Nitrocellulose membrane was from Schleicher & Schuell (Dassel, Germany). BCA kit was from Pierce. Pronase grade CB and endo-beta-N-acetylglucosaminidase H were from Calbiochem (Meudon, France) and Genzyme (Cambridge, MA), respectively. Rat cDNAs encoding the alpha and alpha subunits were kindly provided by Dr. R. Reed (John Hopkins University, Baltimore, MD). Polyclonal rabbit antibodies to the alpha subunits of G-, G-proteins were from Euromedex (Souffelweyersheim, France). Each antibody was raised to decapeptides from the C termini of the respective alpha subunit(9) . The radioisotopes [P]NAD (1000 Ci/mmol), [^14C]leucine (312 mCi/mmol), and I-labeled sheep anti-rabbit IgG (13 mCi/mg) were from Amersham (Les Ulis, France). D-[2-^3H]mannose (20-30 Ci/mmol) was from ICN Biomedicals (Orsay, France). [^3H]Raffinose (5-15 Ci/mmol) was from NEN Dupont de Nemours (Les Ulis, France).

Cell Labeling and Glycoprotein Analysis

HT-29 cells were cultured as described previously(5, 10) . Cells were radiolabeled with 400 µCi/ml D-[2-^3H]mannose for 10 min and then chased for the indicated times(5) . PTX (200 ng/ml) was added 18 h before the labeling period and was present throughout the pulse-chase experiment. When used, 2 mM dMM was added 6 h before the labeling period and was present throughout the pulse-chase experiment (6) . N-Linked glycoproteins were isolated from delipidated cell homogenates, and N-glycans were analyzed after Pronase digestion as described(5, 6) . PTX does not affect either the synthesis of lipid-linked oligosaccharides or their transfer en bloc to polypeptides.

Autophagic Sequestration of [^3H]Raffinose

[^3H]Raffinose sequestration was monitored using a modification of the method of Seglen et al.(11) . Briefly, the cells were resuspended at a density of 5 times 10^6/500 µl with 2 µCi of [^3H]raffinose, after which they were incubated for 15 min at 37 °C and submitted to electroinjection by a single voltage pulse (330 V, 1000 millifarads). After electroinjection, the suspension was maintained at 4 °C for 30 min and incubated at 37 °C for 15 min. At the end of the incubation period, cells were washed twice with PBS and resuspended in complete medium with or without 5 mM 3-methyladenine. When used toxins were added 18 h before the electroinjection and present during the incubation period. Subsequently, at different times (see ``Results''), the cells were washed twice with 10% sucrose at 4 °C, resuspended in 0.5 ml of 10% sucrose and homogenized by 5 strokes in a glass/Teflon homogenizer on ice. Immediately after homogenization, 0.5 ml ice-cold phosphate buffer (100 mM potassium phosphate, 2 mM EDTA and 2 mM dithiothreitol, 100 µg/ml bovine serum albumin, 0.01% Tween 20, pH 7.5) was added, and 1 ml of cell homogenate was layered on the top of a 4 ml density cushion of buffered metrizamide/sucrose (10% sucrose, 8% metrizamide, 1 mM EDTA, 100 µg/ml bovine serum albumin, 0.01% Tween 20, pH 7.5) and centrifuged at 7000 times g for 60 min. The radioactivity associated with the pellet and total homogenate was measured by liquid scintillation counting.

DNA Transfections

Rat cDNAs encoding either the alpha or alpha subunits were subcloned into the expression vector pBK/CMV (Stratagene). Plasmids were introduced into undifferentiated HT-29 cells by the calcium phosphate precipitation method(12) . Twenty-four hours after transfection, cells were grown in selective medium containing 400 µg/ml G418 for at least 3 weeks. Resistant cells were cloned by serial dilution. Eight and 12 clones were selected and screened for their level of alpha and alpha expression, respectively.

Immunoblotting of G-protein alpha Subunits

Cell homogenates, crude membranes, and cytosolic fractions were prepared exactly according to Wilson et al.(13) . One hundred micrograms of protein were resolved by SDS-polyacrylamide gel electrophoresis (10% gel) and were transferred onto a nitrocellulose membrane. Thereafter the membrane was blocked in blotting buffer (5% nonfat dry milk in 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 2 mM CaCl(2), 1% Nonidet P-40), incubated with either anti-alpha (1/500) or anti-alpha (1/1000). After washing, bound IgG was labeled with I-labeled sheep anti-rabbit IgG.

ADP-ribosylation of HT-29 Membranes

Cell-free ADP-ribosylation was conducted as described by van den Berg et al.(14) . Briefly, cells were incubated in either the absence or presence of 200 ng/ml PTX for 18 h and ADP-ribosylation was performed by incubating 150 µg of membrane proteins for 60 min at 32 °C in the substrate mix(14) . Control incubations in the absence of PTX were also included. Reactions were terminated by addition of 400 µl of 20% trichloroacetic acid.

Protein Degradation

Cells were labeled for 6 h with 0.2 µCi of [^14C]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(4)Cl (10 mM) were added at the beginning of the chase period. Degradation of [^14C]leucine-labeled proteins was measured after trichloroacetic acid-phosphotungstic acid precipitation as described previously(7) .

Immunofluorescence Microscopy

Cells grown on 12-mm glass coverslips were fixed at room temperature with 2% paraformaldehyde in PBS for 15 min, washed with PBS, quenched in 50 mM NH(4)Cl in PBS, and then blocked and permeabilized in 0.2% gelatin and 0.075% saponin in PBS for 20 min. The coverslips were then incubated with anti-alpha (diluted 1/50) or anti-alpha (diluted 1/50) for 45 min. Antibodies were diluted in gelatin-saponin-PBS. After washing, the coverslips were incubated for 45 min with a fluorescein isothiocyanate goat anti-rabbit antibody (diluted 1/500). The coverslips were mounted in Glycergel.


RESULTS AND DISCUSSION

When undifferentiated HT-29 cells (hereafter referred to as HT-29 cells) are treated with PTX, which ADP-ribosylates the alpha(i) 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 [^14C]leucine incorporation, or modification of Golgi complex-associated glycosyltransferase activities (evaluated by the activity of beta1,4 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 alpha-mannosidase and Golgi complex alpha-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(i)-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 [^3H]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 [^3H]raffinose was insensitive to CTX, which ADP-ribosylates the alpha(s) subunit of G-proteins(25) , although this toxin is able to activate G(s)-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 alpha(i) subunits in homogenates from PTX-treated cells confirmed that PTX could modify the G(i)-proteins in vivo (Fig. 2b). In order to determine the identity of the PTX-sensitive G-protein alpha(i) subunit that controls the autophagic sequestration we have transfected HT-29 cells with cDNA encoding either the alpha or the alpha 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 alpha(i) subunits were selected. Whatever the alpha(i) 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 [^3H]raffinose (Fig. 3a) and the degradation of [^14C]leucine-labeled proteins (Fig. 3b). As shown in Fig. 3, the overexpression of alpha (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 alpha (data not shown). In contrast we observed a relationship between the overexpression of alpha 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 [^3H]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 [^3H]raffinose sequestration was correlated to an increase in protein degradation in overexpressing alpha cells: by 2.6- and 4.0-fold in clones 1 and 2, respectively (Fig. 3b). The degradation of proteins in overexpressing alpha 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(4)Cl). These results underscore the involvement of alpha in the control of the autophagic pathway. As shown in Fig. 4a, the overexpression of alpha (3-fold) does not change the cellular localization of both alpha and alpha 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 alpha(i) subunits was found in the cytosolic fraction, which was more evident for alpha subunit (Fig. 4b). Whether this distribution of alpha is related to the autophagic capacity of HT-29 cells remains to be explored. Nevertheless the absolute amount of membrane-bound alpha was increased in overexpressing alpha 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 alpha 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 GTPS inhibits the stimulated autophagic sequestration in rat hepatocytes, suggesting a role for GTP-binding proteins in the regulation of autophagy. Since GTPS 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-^3H]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 [^3H]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 alpha stimulates autophagic sequestration and degradation. a, autophagic sequestration of [^3H]raffinose was determined as detailed in the legend to Fig. 2in overexpressing alpha cells (3-fold overexpression), overexpressing alpha cells (clone 1: 1.5-fold overexpression, clone 2: 3-fold overexpression), and control cells. b, protein degradation in untransfected cells (control) and alpha and alpha overexpressing cells (clone 1 and clone 2). c, inhibition of degradation by drugs in overexpressing alpha cells (clone 2). For protein degradation studies, cells were labeled for 6 h with 0.2 µCi of [^14C]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(4)Cl (10 mM) were added at the beginning of the chase period. Degradation of [^14C]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 alpha(i) subunits. a, immunofluorescent localization of alpha and alpha in untransfected cells and in alpha overexpressing cells (clone 2); bar represents 15 µm. b, Western blot of cytosolic (C) and membrane-bound (M) alpha and alpha subunits in HT-29 cells (untransfected) and overexpressing alpha cells (clone 2). A 3.0-fold increase (measured by densitometry) in the amount of alpha subunit was found in the membrane-bound fraction of clone 2.




FOOTNOTES

*
This work was supported by institutional funding from INSERM and from the Consiglio Nazionale delle Ricerche (CNR), and by grants from the Association pour la Recherche sur le Cancer (Grant 6014 to P. C.), AIRC (Milan), MURST (Rome), special project ACRO of the CNR (Rome), and a fellowship from the Ligue Nationale contre le Cancer (to J. J. H.). This work is part of a bilateral project of INSERM/CNR. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-1-44-85-61-34; Fax: 33-1-42-28-87-65.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; CTX, cholera toxin; dMM, 1-deoxymannojirimycin; 3-MA, 3-methyladenine; PTX, pertussis toxin; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Dr. R. Reed for the gift of rat cDNA encoding the alpha and alpha subunits. We also thank Dr. S. E. H. Moore for critical reading of the manuscript.


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