From the
INSERM U504, 16 avenue Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France, the ¶Department of Cellular and Molecular Medicine, University of California, La Jolla, California 92093-0651, and **INSERM U478, 16 rue Henri Huchard, Paris 75018, France
Received for publication, January 28, 2003 , and in revised form, March 10, 2003.
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ABSTRACT |
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INTRODUCTION |
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There is increasing evidence for the importance of autophagy in tissue-specific functions such as the generation of pulmonary surfactant (11), the maturation of erythrocytes (12), and the production of neuromelanin in brain (13). Dysregulation of autophagy is associated with a variety of disease including tumor progression (14), cardiomyopathy and myopathy (15, 16, 17), neurodegenerative diseases (18, 19), and bacterial and viral infections (20, 21, 22). Recent progress, including the identification of the molecular machinery and signaling pathway involved in autophagy have brought some clues on its role in proliferation, differentiation, and cell death (23, 24).
Previous studies from our laboratory have shown that a cytoplasmic heterotrimeric Gi3 protein regulates autophagy in the human colon cancer HT-29 cell line (25). The rate of autophagy is minimal when the Gi3 protein is in the GTP-bound form and becomes stimulated when GDP is bound to the G
i3 protein (26). In agreement with these results, GAIP,1 a RGS protein (regulator of G protein signaling) (27), that activates the hydrolysis of GTP by the G
i3 protein has been shown to increase the rate of autophagy (28). More recently, we have shown that an extracellular signal-regulated kinase 1/2-dependent phosphorylation of GAIP stimulates its activity toward the GTP-bound conformation of the G
i3 protein (29).
Recently a protein named AGS3 has been shown to interact with the GDP-bound form of the Gi3 (30). Soon thereafter AGS3 was demonstrated to stabilize the GDP-bound form of G
i proteins (31, 32, 33). In fact AGS3 is a guanine dissociation inhibitor that inhibits the dissociation of GDP bound to the
subunit and at the same time inhibits the association of the GDP-bound form of G
i proteins with
dimers. AGS3 is a bimodular protein, its amino-terminal half contains seven TPR repeats and its carboxyl-terminal half contains four GoLoco motifs that interact with G
i proteins. Recently, a NH2-terminal truncated AGS3 containing only GoLoco motifs has been shown to be the major AGS3 form present in heart while the full-length form (7 TPR + 4 GoLoco) is more widely distributed (34). Few data are available concerning the involvement of AGS3 in a cell biological context.
In the present work we show that the mRNA encoding for the full-length form of AGS3 is expressed in different human intestinal cell lines (Caco-2, HT-29) whatever their state of differentiation. Together with the full-length form, a low expression of the mRNA encoding the NH2-terminal truncated form of AGS3 was detected. The distribution of the tagged form of AGS3 and that of the endogenous suggests that AGS3 could be part of the Gi3-dependent control of autophagy in intestinal cells. Both biochemical and morphometric analysis show that AGS3 is involved in the control of an early step of macroautophagy prior to the formation of the autophagosome.
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EXPERIMENTAL PROCEDURES |
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The radioisotopes L-[U-14C]valine (288.5 mCi/mmol) and [32P]dCTP (6000 Ci/mmol) were from PerkinElmer Life Sciences (Paris, France). The ECLTM Western blotting detection kit, secondary antibodies, and protein A-Sepharose were from Amersham Biosciences (Les Ulis, France). Alkaline phosphatase detection kit was from Bio-Rad (Marne la Coquette, France) and Superfect kit and Oligotex direct mRNA mini kit were purchased from Qiagen (Les Ulis, France).
Cell Culture and Transfection of HT-29 Cells
HT-29 cells were cultured as previously described (26). Constructions of pcDNA3 vectors encoding AGS3-FL-(1650), 7TPR-(1348), 4GoLoco-(424650) motif, wild-type 1GoLoco-(591650) motif and its mutant R624F, were obtained as previously described (31). Five µg of HA-AGS3-FL, FLAG-AGS3-4GoLoco, wild-type and mutant FLAG-AGS3-1GoLoco or HA-AGS3-7TPR cDNA constructions were transfected into exponentially growing HT-29 cells by Superfect kit according to the supplier's instructions. Cells were used 72 h after transfection. Hereafter in the text, proteins encoded by rat cDNAs will be referred to as AGS3-FL, AGS3-7TPR, AGS3-4GoLoco, and AGS3-1GoLoco to discriminate them from the endogenous full-length and short forms of AGS3.
Reverse Transcription PCR and Northern Blot
Reverse Transcription PCRThe forward PCR primers used were (P1): 5'-ATGGAGGCCTCCTGTCTGGAG-3' and (P2) 5'-TCTTCGGACGAGGAGTGCTTC-3'. The reverse primer used was (P3): 5'-TGCACCCTCTGAATGAGGCTG-3'.
Northern BlotPoly(A)+ RNAs were isolated from HT-29 and Caco-2 cells using the direct Oligotex mini kit according the supplier's instructions. The P2P3 probe obtained by RT-PCR was 32P-labeled by random priming. Hybridizations were conducted at 42 °C and high-stringency washes were performed in 0.5x SSC, 0.1% SDS at 60 °C.
Cell Fractionation
Seventy-two hours after transfection, cells were incubated for 10 min at 4 °C in hypotonic buffer (1 mM Hepes, pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). After scraping, cells were centrifuged for 20 min at 4 °C at 500 x g. The supernatant was then centrifuged at 100,000 x g for 30 min at 4 °C to generate a cytosolic fraction and a membrane fraction.
Subcellular Fractionation
Subcellular fractionation was performed as previously described (35). Cells were scraped into 1 ml of ice-cold PBS containing phenylmethylsulfonyl fluoride (1 mM) and aprotinin (100 units/ml). All the following steps were performed at 4 °C. The cells were homogenized in a Potter homogenizer until greater than 90% of the cells had been broken, and a postnuclear supernatant was prepared by centrifugation for 10 min at 500 x g. Five-hundred microliters were layered on a discontinuous sucrose gradient (0.2, 0.4, 0.6, 1.0, 1.4, and 1.8 M: 750 µl each) and centrifuged for 2 h at 100,000 x g (Beckman 70 Ti rotor). Sixteen fractions (400 µl) were collected from the top and centrifuged for 1 h at 100,000 x g. The resulting pellets were solubilized in Laemmli sample buffer, and the proteins were separated by SDS-PAGE.
Immunoblotting
HT-29 cells were scraped in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.25 M sucrose, 5 mM EDTA, 5 mM EGTA, 0.5% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin). The lysate was clarified by centrifugation at 500 x g for 15 min at 4 °C. One-hundred µg of proteins were submitted to SDS-PAGE and transferred to nitrocellulose. The membrane was incubated for 1 h in Tris-buffered saline (25 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) containing 5% nonfat milk. Primary antibodies were incubated overnight at 4 °C in Tris-buffered saline supplemented with 2% BSA. Anti-FLAG (1:4000) and anti-HA, (1:2000) were used to detect AGS3-FL and AGS3-4GoLoco, respectively. After 3 washes in Tris-buffered saline, membranes were incubated for 1 h at room temperature with the appropriate horseradish peroxidase-labeled secondary antibody. Bound antibodies were detected by enhanced chemiluminescence (ECL).
Immunofluorescence
Staining of AGS3 DomainsHT-29 cells transfected with either cDNA encoding for AGS3-7TPR or AGS3-4GoLoco were grown on glass slides. Seventy-two hours post-transfection, cells were washed with PBS and fixed with 3% paraformaldehyde diluted in PBS for 30 min. Next, they were incubated for 10 min in 50 mM NH4Cl diluted in PBS. Cells were then permeabilized for 30 min with 0.1% Triton X-100 and washed with PBS, 2% BSA. Samples were first incubated with a mouse anti-HA (1:100) or a mouse anti-FLAG (1:200) for 60 min at room temperature. After rinses with PBS, 2% BSA, samples were incubated with a CY3-conjugated secondary anti-mouse antibody for 60 min at room temperature. As controls, the primary antibody was omitted. Coverslips were mounted with glycergel and examined by fluorescence microscopy (Axioplan, Zeiss).
Staining of AGS3-FL, Endoplasmic Reticulum, and Golgi ApparatusHT-29 cells or HT-29 cells transfected with cDNA encoding for AGS3-FL were grown on glass slides. Seventy-two hours post-transfection, cells were washed with PBS and fixed with 3% paraformaldehyde diluted in PBS for 30 min. Next, they were incubated for 10 min in 50 mM NH4Cl diluted in PBS. Thereafter, cells were then permeabilized for 30 min with 0.1% Triton X-100 and washed with PBS, 2% BSA. AGS3-FL-expressing cells were first incubated with a mouse monoclonal anti-HA antibody (1:100) and a rabbit anti-calnexin antibody (1:200) for 60 min at room temperature. After rinsing with PBS, 2% BSA, cells were incubated with a CY3-conjugated anti-mouse IgG (1:200) and a FITC-conjugated anti-rabbit antibody (1:200) for 60 min at room temperature. HT-29 cells were first incubated with a mouse monoclonal anti-protein-disulfide isomerase antibody (1:100) and a rabbit polyclonal anti-AGS3 antibody (1:200) for 60 min at room temperature. After rinsing with PBS, 2% BSA, cells were incubated with a FITC-conjugated anti-mouse IgG (1:200) and a CY3-conjugated anti-rabbit antibody (1:200) for 60 min at room temperature. For co-staining with anti-giantin, HT-29 cells and AGS3-FL-expressing cells were first incubated with a rabbit anti-AGS3 antibody (1:200), directed against the last 14 amino acids at the COOH terminus of rat AGS3 (31), and a mouse anti-giantin antibody (1:1000) for 60 min at room temperature. After rinsing with PBS, 2% BSA, cells were incubated with a CY3-conjugated anti-rabbit antibody (1:200) and a FITC-conjugated anti-mouse antibody (1:200) for 60 min at room temperature. For co-staining with the Gi3 protein, HT-29 cells were first incubated with a rabbit anti-G
i3 polyclonal antibody (1:50) for 60 min at room temperature. After rinsing with PBS, 2% BSA, samples were incubated with a FITC-conjugated anti-rabbit IgG (1:200) for 60 min at room temperature. Samples were then incubated with a goat anti-rabbit F(ab')2 IgG fragment to saturate the first anti-rabbit FITC-conjugated antibody. After rinsing with PBS, 2% BSA, samples were incubated with a rabbit anti-AGS3 for 60 min at room temperature. In AGS3-FL-expressing cells, co-staining experiments were performed with a rabbit anti-G
i3 polyclonal antibody and a mouse anti-HA with the respective secondary antibody. In all the above described conditions, coverslips were mounted with glycergel and examined by confocal laser scanning microscopy equipped with epifluorescent optics (Leica TCS SP).
Monodansylcadaverine StainingWhen necessary, AGS3-FL-expressing cells were used 72 h after transfection. Both HT-29 cells and AGS3-FL-expressing cells were first washed with HBSS and then incubated for 3 h at 37 °C in HBSS. For staining of autophagic vacuoles a monodansylcadaverine stock solution (0.1 M in Me2SO) was diluted 1:1000 in HBSS and applied to the cells for 60 min at 37 °C (36). After washes with PBS, cells were fixed with 3% paraformaldehyde for 30 min. After washes with PBS, cells were incubated for 10 min with 50 mM NH4Cl and then permeabilized with 0.1% Triton X-100 for 30 min. Rabbit anti-AGS3 antibody (HT-29 cells) or mouse monoclonal anti-HA antibody (AGS3-FL-expressing cells) were incubated for 60 min at room temperature to detect AGS3 and AGS3-FL, respectively. The appropriate secondary CY3-conjugated antibody was then incubated for 60 min at room temperature. Coverslips were mounted with glycergel and examined by fluorescence microscopy (Axioplan, Zeiss).
Macroautophagic Parameters
Measurement of the degradation of [14C]valine-labeled long-lived proteins and LDH sequestration were monitored as reported previously (26, 37).
Analysis of Protein DegradationHT-29 cells were incubated for 18 h at 37 °C with 0.2 µCi/ml L-[14C]valine. Unincorporated radioisotope was removed by three rinses with PBS (pH 7.4). Cells were then incubated in nutrient-free medium (without amino acids and in the absence of fetal calf serum) plus 0.1% BSA and 10 mM cold valine. When required 10 mM 3-MA, a potent inhibitor of the formation of autophagic vacuoles (38), was added throughout the chase period. After the first hour of incubation, at which time short-lived proteins were being degraded, the medium was replaced with the appropriate fresh medium and the incubation continued for an additional 4-h period. Cells and radiolabeled proteins from the 4-h chase medium were precipitated in 10% trichloroacetic acid (v/v) at 4 °C. The precipitated proteins were separated from the soluble radioactivity by centrifugation at 600 x g for 10 min then dissolved in 250 µl of Soluene 350. The rate of protein degradation was calculated as acid-soluble radioactivity recovered from both cells and media.
Analysis of LDH SequestrationBriefly, cells were gently washed three times with PBS (pH 7.4) and then twice with homogenization buffer (50 mM potassium phosphate, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 300 mM sucrose, 100 µg/ml BSA, 0.01% Tween 20, pH 7.5). Cells were homogenized in 1 ml of cold homogenization buffer by 13 strokes in a glass/Teflon homogenizer on ice. A postnuclear supernatant was prepared by centrifugation at 300 x g for 10 min at 4 °C. Postnuclear material was layered on 4 ml of buffered metrizamide/sucrose (10% sucrose, 8% metrizamide, 1 mM EDTA, 100 µg/ml BSA, 0.01% Tween 20, pH 7.5) and centrifuged at 7000 x g for 60 min. Finally, the pellet was washed once with homogenization buffer and resuspended in buffer containing 2 mM Tris-HCl (pH 7.4), 50 mM mannitol, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 0.1 µg/ml aprotinin, 0.7 µg/ml pepstatin. The suspension was sonicated (VibraCellTM sonicator model 72434, power setting 3, microtip, for 20 s at 20% charge) and centrifuged (10 min, 10,000 x g). The LDH activity was assayed by measuring the oxidation of NADH with pyruvate as substrate at 340 nm.
Electron Microscopy and MorphometryMorphometric analysis was performed as previously described (35). Confluent cells grown on Petri dishes were cultured for 3 h in a nutrient-free medium to stimulate autophagy in the presence of 50 µM vinblastine before being fixed for 2 h with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, embedded in Epon, and processed for transmission electron microscopy by standard procedures. For morphometric analysis 20 micrographs per condition were used. The fractional volume represented by autophagic vacuoles was quantified by using the method of Weibel et al. (39) assuming a spherical shape for the calculation. Morphological criteria were used to identify autophagic vacuoles (40).
Statistical Analysis
Statistical analysis of differences between the groups was performed using Student's t test. p < 0.005 was considered statistically significant.
RESULTS
AGS3 Expression in Intestinal CellsThe use of primers P1 and P3 (Fig. 1A) in RT-PCR experiments allowed us to detect a product with the expected size for the human full-length AGS3 in undifferentiated HT-29 cells (Fig. 1B, left panel). The product was also detected in HT-29 MTX and HT-29 FUra cells with goblet cell-like (41) and enterocyte-like (42) phenotypes, respectively. The expression of the full-length AGS3 was not restricted to HT-29 cells. In Caco-2 cells, which spontaneously differentiate in enterocyte-like cells in a growth dependent manner (43), the RT-PCR product was detected before (day 4) and after differentiation (day 21) (Fig. 1B, left panel). When oligonucleotides P2 and P3 were used, the same RT-PCR product of 496 bp was detected in all cell populations tested (Fig. 1B, right panel).
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As a short form of AGS3 truncated of its NH2-terminal part and encompassing the sequence amplified between P2 and P3 has been detected in rat heart (34), we checked by Northern blot analysis whether the fragment of 496 bp corresponds to the short form of AGS3. The P2P3 RT-PCR product was used as probe in the Northern blot experiment (Fig. 1C). Both the 4-kb mRNA encoding the full-length AGS3 and the 2-kb mRNA encoding the 5' truncated form were detected in poly(A)+ RNA isolated from undifferentiated HT-29 cells (thereafter referred to HT-29 cells) and from undifferentiated and differentiated Caco-2 cells. Whatever the cell population considered, mRNA encoding the full-length AGS3 is more expressed than the mRNA encoding the short form AGS3 (Fig. 1C). The amount of poly(A)+ RNA available was not sufficient to investigate the short form AGS3 expression in HT-29 MTX cells and HT-29 FUra cells.
AGS3 and the Gi3 Protein Have a Partially Overlapping Membrane DistributionWe have previously shown that the G
i3 protein is associated with Golgi and ER membranes in HT-29 cells (35). The intracellular localization of the endogenous AGS3 protein has been investigated by immunofluorescence. A heterogeneous cytoplasmic staining pattern of AGS3 was observed by immunofluorescence examination (Fig. 2A). However, double staining experiments revealed a limited overlapping staining with the ER marker protein-disulfide isomerase and with the Golgi marker giantin (Fig. 2A).
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Because the anti-AGS3 antibody only works in immunofluorescence detection we have used the tagged form of the protein (AGS3-FL) to confirm the ER and Golgi localization of AGS3. After transfection, the intracellular distribution of AGS3-FL was analyzed by immunofluorescence (Fig. 2A) and subcellular fractionation (Fig. 2B). Similarly to the endogenous AGS3, a heterogeneous cytoplasmic staining pattern of ASG3-FL was observed by immunofluorescence examination with a limited overlapping staining with the ER marker calnexin and with the Golgi marker giantin (Fig. 2A). These morphological observations were complemented by subcellular fractionation experiments on sucrose density gradients (Fig. 2B). The Golgi marker p58 was present in fractions 26 while the ER marker calnexin was detected in fractions 915. AGS3-FL codistributed with p58 in fractions 56 and with calnexin in fractions 914, respectively. This distribution is reminiscent to that of the Gi3 protein in HT-29 cells (35). Immunofluorescence examination indicated that both AGS3-FL and the endogenous form of AGS3 partially overlap with the G
i3 protein (Fig. 2A). As the G
i3 protein and its GTPase activating protein, GAIP, which control macroautophagy in HT-29 cells (26, 28) are also present in ER and Golgi membranes (35), we next investigated whether AGS3 is involved in the control of this process.
AGS3-FL Stimulates Autophagy in HT-29 CellsIn a first series of experiments, the rate of autophagy was measured in HT-29 cells transfected with the cDNA encoding HA-AGS3-FL. 3-MA-sensitive degradation of [14C]valine-labeled proteins was stimulated 1.9-fold after AGS3-FL overexpression when compared with that measured in HT-29 cells transfected with an empty vector (Fig. 3A). In line with these results, the sequestration of the cytosolic enzyme LDH in sedimentable material was also increased in AGS3-FL-expressing cells (Table I).
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In contrast to the results reported above, overexpression of AGS3-FL did not increase either the low rate of autophagic proteolysis (Fig. 3B) or sequestration (data not shown) in HT-29 cells overexpressing a GTPase-deficient Gi3 protein (Q204L) (26). This is in line with the fact that AGS3 does not interact with the GTP
S-bound form of the G
i3 protein (31, 44) and with our previous results showing the importance of the GDP-bound conformation of the G
i3 protein to stimulate autophagy (26). A morphometric analysis has demonstrated that the fractional volume occupied by autophagic vacuoles is significantly increased in HT-29 cells expressing AGS3-FL when compared with control cells (Fig. 4A). By contrast the expression of AGS3-FL did not modify the low fractional volume occupied by autophagic vacuoles in Q204L-expressing cells (Fig. 4A). However, neither AGS3-FL nor the endogenous form of AGS3 co-localized with autophagic vacuoles labeled with monodansylcadaverine (Fig. 4B). In addition neither the intracellular distribution of AGS3 (endogenous and transfected) nor that of the G
i3 protein is changed during the induction of macroautophagy by nutrient deprivation (data not shown). These results suggest that AGS3 can be involved together with the G
i3 protein in an early step during the macroautophagic pathway.
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Effect of AGS3-FL Domains on AutophagyTo establish that the endogenous AGS3 protein controls autophagy we have generated different domains of AGS3 and thereafter analyzed their effect on autophagy. First we investigated the effect of the NH2-terminal TPR repeats (AGS3-7TPR). As previously shown by Pizzinat et al. (34) and on the basis of immunofluorescence staining and membrane distribution (Fig. 5A), AGS3-7TPR is associated with intracellular membranes. A dose-dependent inhibition of autophagy was observed in HT-29 cells transfected with increasing amounts of the cDNA encoding AGS3-7TPR (Fig. 5B). This result strongly suggests that AGS3-7TPR is dominant-negative toward the endogenous AGS3. In addition, the inhibitory effect of AGS3-7TPR is not related to a sequestration of the Gi3 protein because this domain does not interact with the G
i3 protein. Next, we have investigated the effect of COOH-terminal GoLoco domains of AGS3 on autophagy. AGS3-4GoLoco behaved as a cytosolic protein based on immunofluorescence staining and membrane distribution (Fig. 5A). This domain has a very potent inhibitory effect on autophagy (Fig. 5B). Accordingly AGS3-1GoLoco also has an inhibitory effect on autophagy (Fig. 5B). This effect was specific because expression of a mutated form of AGS3-1GoLoco (R624F) unable to interact with the G
i3 protein (31) did not inhibit the rate of autophagy (Fig. 5B). The observation that the inhibition of autophagy was more pronounced in AGS3-4GoLoco-expressing cells than in AGS3-1GoLoco-expressing cells is in line with the fact that AGS3-4GoLoco is more potent than AGS3-1GoLoco in inhibiting the in vitro binding of GTP
S to the G
i3 protein (31, 32). These results suggest that AGS3-1GoLoco and AGS3-4GoLoco are able to compete with the endogenous form of AGS3 for G
i3 binding.
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DISCUSSION |
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However, both the tagged form (AGS3-FL) and the endogenous form of AGS3 have a distribution compatible with a function in early events of the macroautophagic pathway. Although the intracellular localization of AGS3 remains to be firmly established, one can assume that a fraction of AGS3 co-localizes with the Gi3 protein and GAIP in ER and Golgi membranes (35), which are possible sources for the delimiting membrane of the autophagosome (46, 47). The localization of AGS3 did not change after the induction of autophagy by amino acid deprivation. Similar observations have been made for the G
i3 protein and GAIP (data not shown). Interestingly the localization of the monomeric G-protein Rab24 that is involved in the macroautophagic pathway change during the induction of autophagy (48). These results suggest that Rab24 is involved in a later step of the macroautophagic pathway than the G
i3 protein.
Together with the Gi3 protein and GAIP, AGS3 controls the cytoplasmic fractional volume occupied by autophagic vacuoles (this work and Ref. 35), thus we can hypothesize that the G
i3 protein and its partners (GAIP and AGS3) have a regulatory function prior to the formation of the autophagosome. It has been reported that the autophagosomal membrane could derive from ER and Golgi (46, 47). According to these studies, recent data suggest that membrane elements emanating from both the ER and Golgi apparatus can contribute to the genesis of the preautophagosomal membrane (49, 50). As trimeric G proteins gate membrane movements in the endo/exocytic pathway (51), it can be hypothesized that a G
i3 protein-based mechanism could control membrane fluxes emanating from the ER and Golgi. These membrane pools could converge to form a preautophagosomal compartment reminiscent of the unique autophagic organelle phagophore identified by Seglen's group (see Ref. 52 for discussion). Accordingly, it is worth noting that when the G
i3-dependent transport of proteoglycans along the exocytic pathway in epithelial cells is stimulated (53, 54), the macroautophagic pathway is severely impaired (25, 26, 28) and the reciprocal is true when the macroautophagic pathway is stimulated. This suggests that the G
i3 protein and its cohort proteins can control the balance between the flow of membrane in the exocytic pathway and the delivery of membrane constituents to the macroautophagic pathway.
According to its guanine dissociation inhibitor capacity, AGS3-FL was co-immunoprecipitated with the Gi3 protein from HT-29 cell homogenates in a GDP-dependent manner (data not shown) suggesting that AGS3 forms a membrane-bound complex with the GDP-bound G
i3 required to stimulate macroautophagy. This complex could recruit proteins from the cytosol including the Ca2+ sensor CalNuc (nucleobindin) (55, 56, 57). This last observation is noteworthy because autophagic sequestration is known to be sensitive to the release of intracellular calcium pools (58). In addition, the NH2-terminal TPR repeats of AGS3 are probably involved in the control of autophagy. The expression of AGS3-7TPR has an inhibitory effect on autophagy (see Fig. 5) suggesting that this domain interferes with the function of the endogenous AGS3 protein. TPR repeats, which are required for membrane association of AGS3 (Ref. 34 and this study), have properties that could be relevant to autophagy such as the formation of chaperone complexes and protein import complexes through organelle membranes (59, 60). Studies are now aimed in our laboratory at the identification of the molecular partners of the NH2-terminal domain of AGS3.
GoLoco motifs are involved in the recognition and stabilization of the GDP-bound form of Gi proteins (31, 32, 33, 61, 62). Our data demonstrate that AGS3-(1/4)GoLoco inhibits autophagy by interfering with the formation of the autophagosome because of the reduced autophagic sequestration of LDH (Table I). This effect was dependent upon the guanine dissociation inhibitor activity of AGS3 GoLoco motifs for the following reasons: (i) an inactive form of AGS3-1GoLoco motif has no effect on autophagy, and (ii) AGS3-4GoLoco interacts with the G
i3 protein in a GDP-dependent manner in HT-29 cells. At first glance these results could be difficult to reconcile with our previous data showing that the GDP-bound form of the G
i3 protein stimulates autophagy (26, 28). However, we have also demonstrated that pertussis toxin, which stabilizes the GDP-bound form of the trimeric Gi3 protein, inhibits autophagic sequestration (25, 26). ADP-ribosylation of G
i proteins by pertussis toxin is known to block their interaction with receptors (63). From these data we proposed that the GDP-bound form of the G
i3 protein should interact with a putative membrane-bound partner to control autophagy. In addition, the results presented here together with our previous data (26) confirm that the
dimer is dispensable in the control of autophagy. We can hypothesize that AGS3-4GoLoco, which stabilizes the GDP-bound form of the protein, disrupts G
i3 interaction with a membrane partner or interferes with the function of endogenous full-length AGS3.
From our present results, we conclude that the GDP-bound form of the Gi3 protein is necessary but not sufficient to stimulate the autophagic pathway. In fact only the full-length form of AGS3 has the capacity to stimulate autophagy. Whether or not the NH2-terminal-truncated form of AGS3 detected in intestinal cells (this study) and in cardiac cells (34) has the capacity to inhibit autophagy remains to be investigated.
Our previous studies have shown that GAIP by means of its GTPase activating capacity toward the Gi3 protein primes the stimulation of autophagy (28). This priming is regulated by amino acids that are physiological regulators of autophagy (reviewed in Ref. 64). Amino acids keep the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2-dependent GAIP Ser151 phosphorylation at a low level, which consequently decreases the rate of GDP accumulation and reduces autophagic capacities (29). Together with GAIP, AGS3 is a novel element in the G
i3dependent control of autophagy. Identification of cytoplasmic partners of the NH2-terminal half of full-length AGS3 would bring important information about the function of the G
i3 protein in controlling macroautophagy.
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FOOTNOTES |
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Supported by a fellowship from the Association pour la Recherche sur le Cancer.
|| Present address: Institut de Recherche Pierre Fabre, Département de Biologie Cellulaire et Moléculaire, 17 avenue Jean Moulin, 81106 Castres, France.
Present address: INSERM U479, 16 rue Henri Huchard, 75018 Paris, France.
To whom correspondence should be addressed: INSERM U504, Glycobiologie et Signalisation Cellulaire, 16 avenue Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France. Tel.: 33-1-45-59-50-42; Fax: 33-146-77-02-33; E-mail: codogno{at}vjf.inserm.fr.
1 The abbreviations used are: GAIP, G interacting protein; AGS3, activator of G protein signaling 3; AGS3-FL, AGS3 full-length; HA-AGS3-FL, AGS3 full-length NH2-terminal-tagged with the hemagglutinin epitope; AGS3-4GoLoco, AGS3 deleted of its 423 NH2-terminal amino acids; FLAG-AGS3-4GoLoco, AGS3 deleted of its 423 NH2-terminal amino acids and NH2-terminal tagged with the FLAG-M2 epitope; AGS3-1GoLoco, AGS3 deleted of its 590 NH2-terminal amino acids; AGS3-TPR, AGS3 deleted of its 302 COOH-terminal amino acids; BSA, bovine serum albumin; ER, endoplasmic reticulum; HBSS, Hanks' balanced salt solution; LDH, lactate dehydrogenase; 3-MA, 3-methyladenine; PBS, phosphate-buffered saline; RT, reverse transcriptase; TPR, tetratricopeptide regulatory; HA, hemagglutinin; FITC, fluorescein isothiocyanate; GTP
S, guanosine 5'-3-O-(thio)triphosphate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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