The G-protein Regulator AGS3 Controls an Early Event during Macroautophagy in Human Intestinal HT-29 Cells*

Sophie Pattingre {ddagger} §, Luc De Vries ¶ ||, Chantal Bauvy {ddagger}, Isabelle Chantret {ddagger}, Françoise Cluzeaud **, Eric Ogier-Denis {ddagger} {ddagger}{ddagger}, Alain Vandewalle ** and Patrice Codogno {ddagger} §§

From the {ddagger}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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 DISCUSSION
 REFERENCES
 
AGS3 contains GoLoco or G-protein regulatory motifs in its COOH-terminal half that stabilize the GDP-bound conformation of the {alpha}-subunit of the trimeric Gi3 protein. The latter is part of a signaling pathway that controls the lysosomal-autophagic catabolism in human colon cancer HT-29 cells. In the present work we show that the mRNA encoding for AGS3 is expressed in human intestinal cell lines (Caco-2 and HT-29) whatever their state of differentiation. Together with the full-length form, minute amounts of the mRNA encoding a NH2-terminal truncated form of AGS3, previously characterized in cardiac tissues, were also detected. Both the endogenous form of AGS3 and a tagged expressed form have a localization compatible with a role in the G{alpha}i3–dependent control of autophagy. Accordingly, expressing its non-G{alpha}i3-interacting NH2-terminal domain or its G{alpha}i3-interacting COOH-terminal domain reversed the stimulatory role of AGS3 on autophagy. On the basis of biochemical and morphometric analysis, we conclude that AGS3 is involved in an early event during the autophagic pathway probably prior to the formation of the autophagosome. These data demonstrate that AGS3 is a novel partner of the G{alpha}i3 protein in the control of a major catabolic pathway.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 DISCUSSION
 REFERENCES
 
Macroautophagy or autophagy is a general and evolutionary conserved response to starvation and stress activated in eucaryotic cells (1, 2, 3, 4, 5). During the induction of autophagy, portions of the cytoplasm are rapidly sequestered to form an autophagosome by a membrane of unknown origin (6, 7). After receiving input from endocytic vesicles (8, 9, 10), autophagic vacuoles ultimately fuse with the lysosomal compartment where the sequestered material is degraded.

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 G{alpha}i3 protein is in the GTP-bound form and becomes stimulated when GDP is bound to the G{alpha}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{alpha}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{alpha}i3 protein (29).

Recently a protein named AGS3 has been shown to interact with the GDP-bound form of the G{alpha}i3 (30). Soon thereafter AGS3 was demonstrated to stabilize the GDP-bound form of G{alpha}i proteins (31, 32, 33). In fact AGS3 is a guanine dissociation inhibitor that inhibits the dissociation of GDP bound to the {alpha} subunit and at the same time inhibits the association of the GDP-bound form of G{alpha}i proteins with {beta}{gamma} 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{alpha}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 G{alpha}i3-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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 DISCUSSION
 REFERENCES
 
Reagents
3-MA, monodansylcadaverine, anti-FLAG-M2, anti-p58, anti-HA antibodies and all other chemicals were purchased from Sigma. Anti-protein-disulfide isomerase and anti-giantin antibodies were kindly provided by S. Fuller (Heidelberg, Germany) and H. P. Hauri (Biozentrum, University of Basel, Switzerland), respectively. mRNA from HT-29 MTX cells (treated with 10-5 M methotrexate) and HT-29 FUra cells (treated with 10-5 M fluorouracil) were kindly provided by M. Rousset and T. Lesuffleur (INSERM U505, Paris, France). Cell culture reagents, enzymes for RT-PCR, the eucaryote expression vectors pcDNA3.0, pcDNA3.1/c-myc/HA/FLAG, and XPressTM System Synthetic oligonucleotides were from Invitrogen (Cergy Pontoise, France). Nitrocellulose membranes were from Schleicher & Schüll (Dassel, Germany). Rabbit polyclonal antibody anti-calnexin and rabbit polyclonal anti-G{alpha}i3 were from Stressgen (Glandford, Canada) and BIOMOL, respectively.

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-(1–650), 7TPR-(1–348), 4GoLoco-(424–650) motif, wild-type 1GoLoco-(591–650) 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 PCR—The forward PCR primers used were (P1): 5'-ATGGAGGCCTCCTGTCTGGAG-3' and (P2) 5'-TCTTCGGACGAGGAGTGCTTC-3'. The reverse primer used was (P3): 5'-TGCACCCTCTGAATGAGGCTG-3'.

Northern Blot—Poly(A)+ RNAs were isolated from HT-29 and Caco-2 cells using the direct Oligotex mini kit according the supplier's instructions. The P2–P3 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 Domains—HT-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 Apparatus—HT-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 G{alpha}i3 protein, HT-29 cells were first incubated with a rabbit anti-G{alpha}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{alpha}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 Staining—When 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 Degradation—HT-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 Sequestration—Briefly, 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 Morphometry—Morphometric 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 Cells—The 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).



View larger version (27K):
[in this window]
[in a new window]
 
FIG. 1.
Expression of AGS3 in human intestinal cell lines. A, schematic representation of human AGS3. P1,P2, and P3 are the primers used for RT-PCR. The sequences of primers are indicated in boxes. B, 5 µg of total mRNA were submitted to RT-PCR using either P1–P3 or P2–P3 primers. Lane 1, undifferentiated HT-29 cells; lane 2, HT-29 FUra cells (cells expressing an enterocytic phenotype); lane 3, HT-29 MTX cells (cells expressing a mucus-secreting phenotype); lane 4, undifferentiated Caco-2 cells (day 4 of culture). C, poly(A)+ RNA were run and hybridized with the P2–P3-specific probe. HT-29 cells (undifferentiated cells), Caco-2 D4 (day 4, undifferentiated cells), Caco-2 D21 (day 21, differentiated cells: enterocytic phenotype).

 

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 P2–P3 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 G{alpha}i3 Protein Have a Partially Overlapping Membrane Distribution—We have previously shown that the G{alpha}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).



View larger version (61K):
[in this window]
[in a new window]
 
FIG. 2.
Localization of AGS3 proteins in HT-29 cells. A, immunofluorescence. HT-29 cells (left panels) and AGS3-FL-expressing HT-29 cells (right panels) were fixed with paraformaldehyde and permeabilized with Triton X-100 and double labeled with either ER markers (monoclonal anti-protein-disulfide isomerase or polyclonal anti-calnexin) or a Golgi marker (anti-giantin) and a polyclonal anti-AGS3 (to detect the endogenous and the transfected forms of AGS3 in anti-giantin co-staining experiments) or a monoclonal anti-HA (to detect AGS3-FL in transfected cells in anti-calnexin co-staining experiments). The anti-HA was used to detect AGS3-FL in transfected cells in double staining experiments with the polyclonal anti-G{alpha}i3. The anti-AGS3 was used in HT-29 cells in double staining experiments with the polyclonal anti-G{alpha}i3 following the protocol detailed under "Experimental Procedures." Bars, 10 µm. B, distribution of AGS3-FL after subcellular fractionation of HT-29 cells. Subcellular fractionation was performed on a sucrose gradient as detailed under "Experimental Procedures." The Golgi marker p58 was associated with fractions 2–6, whereas the ER marker calnexin was detected in fractions 9–15. Fractions were lysed in Laemmli buffer, and proteins from each fraction were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and revealed by the relevant antibody. Fractions 1 and 16 represent the top and bottom of the gradient, respectively.

 

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 2–6 while the ER marker calnexin was detected in fractions 9–15. AGS3-FL codistributed with p58 in fractions 5–6 and with calnexin in fractions 9–14, respectively. This distribution is reminiscent to that of the G{alpha}i3 protein in HT-29 cells (35). Immunofluorescence examination indicated that both AGS3-FL and the endogenous form of AGS3 partially overlap with the G{alpha}i3 protein (Fig. 2A). As the G{alpha}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 Cells—In 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).



View larger version (12K):
[in this window]
[in a new window]
 
FIG. 3.
Effect of AGS3-FL in HT-29 cell populations expressing different autophagic capacities. Proliferating HT-29 cells (panel A) and Q204L-expressing cells (panel B) were transfected with 5 µg of cDNA encoding AGS3-FL. After 72 h, the rate of degradation of [14C]valine-labeled long-lived proteins was measured in cells incubated in nutrient-free medium (HBSS) in the presence or absence of 10 mM 3-MA. The control was performed in cells transfected with the empty vector (vector). The values reported are the mean ± S.D. of four experiments. *, p < 0.005 significant increase of the protein degradation compared with control cells (vector).

 

View this table:
[in this window]
[in a new window]
 
TABLE I
Autophagic sequestration in HT-29 cells expressing different cDNA of AGS3

 

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 G{alpha}i3 protein (Q204L) (26). This is in line with the fact that AGS3 does not interact with the GTP{gamma}S-bound form of the G{alpha}i3 protein (31, 44) and with our previous results showing the importance of the GDP-bound conformation of the G{alpha}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{alpha}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{alpha}i3 protein in an early step during the macroautophagic pathway.



View larger version (35K):
[in this window]
[in a new window]
 
FIG. 4.
AGS3 controls an early step during macroautophagy. A, morphometric analysis. Upper panel, the relative volume occupied by autophagic vacuoles in four different HT-29 cell populations is expressed as a percentage of the cell volume as described under "Experimental Procedures." HT-29 cells, HT-29 cells transfected with an empty vector; HT-29 AGS3-FL, HT-29 cells transfected with the cDNA encoding the full-length AGS3. Q204L cells (HT-29 cells expressing the Q204L mutant of the G{alpha}i3 protein), Q204L cells transfected with an empty vector; Q2040L AGS3-FL, Q204L-expressing cells transfected with the cDNA encoding the full-length AGS3. Values are mean ± S.E. of 20 analyzed micrographs in each condition tested. Lower panels, electron micrographs of HT-29 cells expressing AGS3-FL (AGS3-FL) (left panel) and HT-29 cells transfected with an empty vector (HT-29) (right panel). Magnification, x10,000. B, monodansylcadaverine and AGS3 staining. AGS3-FL (transfected HA-AGS3), AGS3 (endogenous AGS3), and monodansylcadaverine (MDC). Bars, 10 µm. *, p < 0.005 significant increase or decrease of the volume occupied by autophagic vacuoles compared with HT-29 cells transfected with an empty vector (HT-29 cells).

 

Effect of AGS3-FL Domains on Autophagy—To 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 G{alpha}i3 protein because this domain does not interact with the G{alpha}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{alpha}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{gamma}S to the G{alpha}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{alpha}i3 binding.



View larger version (33K):
[in this window]
[in a new window]
 
FIG. 5.
Distribution and effect on autophagic proteolysis of TPR repeats and GoLoco domains of AGS3. A, subcellular localization of AGS3-7TPR and AGS3-4GoLoco in HT-29 cells. Left panels, after 72 h of transfection with either the cDNA encoding AGS3-TPR or the cDNA encoding AGS3-4GoLoco, cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and labeled with anti-HA or anti-FLAG to detect AGS3-7TPR and AGS3-4GoLoco, respectively. The bar represents 10 µm. Right panels, after 72 h of transfection with either the cDNA encoding AGS3-7TPR or the cDNA encoding AGS3-4GoLoco, HT-29 cells were fractionated by centrifugation. The postnuclear supernatant (PNS) was centrifuged at 100,000 x g. The resulting supernatant represents the cytosol (S), and the pellet (P) represents the membrane fraction. Anti-HA was used to detect AGS3-7TPR (upper panel) and anti-FLAG was used to detect AGS3-4GoLoco (lower panel). Postnuclear supernatant of untransfected cells was used as a control (U). B, effect of AGS3 domains on autophagy. Proliferating HT-29 cells were transfected with increasing amounts of cDNA (from 2.5 to 20 µg) encoding different domains of AGS3 (7TPR repeats, 4GoLoco motifs, 1GoLoco motif, and R624F-1GoLoco motif). After 72 h, the rate of degradation of [14C]valine-labeled long-lived proteins was measured in cells incubated in nutrient-free medium (HBSS) in the presence or absence of 10 mM 3-MA. Cells transfected with the empty vector were used as a control. Autophagy corresponds to the 3-MA-sensitive proteolysis. The rate of 3-MA-sensitive proteolysis in HT-29 cells transfected with an empty vector was set as 100%. The values reported are the mean ± S.D. of four experiments. *, p < 0.005 significant decrease of the protein degradation compared with control cells (vector).

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 DISCUSSION
 REFERENCES
 
AGS3 belongs to a new family of proteins that inhibits the dissociation of GDP from the G{alpha}i and G{alpha}t proteins in the absence of the {beta}{gamma} dimer (30, 31, 32, 33). In the present work we show that AGS3 is expressed in different human intestinal cell lines whatever their state of differentiation. Northern blot analysis suggests that the short form of AGS3 truncated of its NH2-terminal TPR repeats recently described in cardiac tissue (34) represents a minor form of AGS3 in intestinal cells. The absence of detection of AGS3 polypeptides by Western blotting in human intestinal cell extracts was probably the consequence of two phenomena: (i) the use of an antibody directed against the COOH-terminal part of the rat protein that is different from its human counterpart (Ref. 45 and our data), or (ii) the limitation of protein detection by Western blot analysis (44). This last possibility would be in line with the low level of AGS3 observed in human tissues other than brain and heart (34).

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 G{alpha}i3 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{alpha}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{alpha}i3 protein.

Together with the G{alpha}i3 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{alpha}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{alpha}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{alpha}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{alpha}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 G{alpha}i3 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{alpha}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 G{alpha}i 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{alpha}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{alpha}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{alpha}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{alpha}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 {beta}{gamma} 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{alpha}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 G{alpha}i3 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 G{alpha}i3 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{alpha}i3–dependent 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{alpha}i3 protein in controlling macroautophagy.


    FOOTNOTES
 
* This work was supported in part by INSERM and Association pour la Recherche sur le Cancer Grant 4622. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a fellowship from the Association pour la Recherche sur le Cancer. Back

|| Present address: Institut de Recherche Pierre Fabre, Département de Biologie Cellulaire et Moléculaire, 17 avenue Jean Moulin, 81106 Castres, France. Back

{ddagger}{ddagger} Present address: INSERM U479, 16 rue Henri Huchard, 75018 Paris, France. Back

§§ 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{alpha} 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{gamma}S, guanosine 5'-3-O-(thio)triphosphate. Back


    ACKNOWLEDGMENTS
 
We thank Ph. Coullin (CNRS UMR 1599, Villejuif, France) for the acquisition of immunofluorescence images and Z. Mishal (Institut André Lwoff, Villejuif, France) for confocal microscopy analysis. We also thank M. Rousset and T. Lesuffleur (INSERM U505) for providing the mRNAs isolated from HT-29 MTX and HT-29 FUra cells, S. Fuller (Heidelberg, Germany) and H. P. Hauri (Biozentrum, University of Basel, Switzerland) for providing antibodies, and S. E. H. Moore for critical reading of the manuscript.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 DISCUSSION
 REFERENCES
 

  1. Seglen, P. O., and Bohley, P. (1992) Experientia 48, 158-172[Medline] [Order article via Infotrieve]
  2. Dunn, W. A., Jr. (1994) Trends Cell Biol. 4, 139-143[CrossRef]
  3. Mortimore, G. E., and Kadowaki, M. (1994) in Cellular Proteolytic Systems (Ciechanover, A. J., and Schwartz, A. L., eds) pp. 65-87, Wiley-Liss, New York
  4. Kim, J., and Klionsky, D. J. (2000) Annu. Rev. Biochem. 69, 303-342[CrossRef][Medline] [Order article via Infotrieve]
  5. Klionsky, D. T., and Ohsumi, Y. (1999) Annu. Rev. Cell Dev. Biol. 15, 1-32[CrossRef][Medline] [Order article via Infotrieve]
  6. Baba, M., Osumi, M., and Ohsumi, Y. (1995) Cell Struct. Funct. 20, 465-471[Medline] [Order article via Infotrieve]
  7. Fengsrud, M., Erichsen, E. S., Berg, T. O., Raiborg, C., and Seglen, P. O. (2000) Eur. J. Cell Biol. 79, 871-882[Medline] [Order article via Infotrieve]
  8. Stromhaug, P. E., and Seglen, P. O. (1993) Biochem. J. 291, 115-121[Medline] [Order article via Infotrieve]
  9. Berg, T. O., Fengsrud, M., Stromhaug, P. E., Berg, T., and Seglen, P. O. (1998) J. Biol. Chem. 273, 21883-21892[Abstract/Free Full Text]
  10. Liou, W., Geuze, H. J., Geelen, M. J. H., and Slot, J. W. (1997) J. Cell Biol. 136, 61-70[Abstract/Free Full Text]
  11. Hariri, M., Millane, G., Guimond, M. P., Guay, G., Dennis, J. W., and Nabi, I. R. (2000) Mol. Biol. Cell 11, 255-268[Abstract/Free Full Text]
  12. Holm, T. M., Braun, A., Trigatti, B. L., Brugnara, C., Sakamoto, M., Krieger, M., and Andrews, N. C. (2002) Blood 99, 1817-1824[Abstract/Free Full Text]
  13. Sulzer, D., Bogulavsky, J., Larsen, K. E., Karatekin, E., Kleinman, M., Turro, N., Krantz, D., Edwards, R., Greene, L. A., and Zecca, L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11869-11874[Abstract/Free Full Text]
  14. Liang, X. H., Jackson, S., Seaman, M., Brown, K., Kempkes, B., Hibshoosh, H., and Levine, B. (1999) Nature 402, 672-676[CrossRef][Medline] [Order article via Infotrieve]
  15. Tanaka, Y., Guhde, G., Suter, A., Eskelinen, E. L., Hartmann, D., Lullmann-Rauch, R., Janssen, P. M. L., Blanz, J., von Figura, K., and Saftig, P. (2000) Nature 406, 902-906[CrossRef][Medline] [Order article via Infotrieve]
  16. Nishino, I., Fu, J., Tanji, K., Yamada, T., Shimojo, S., Koori, T., Mora, M., Riggs, J. E., Oh, S. J., Koga, Y., Sue, C. M., Yamamoto, A., Murakami, N., Shanske, S., Byrne, E., Bonilla, E., Nonaka, I., Di Mauro, S., and Hirano, M. (2000) Nature 406, 906-910[CrossRef][Medline] [Order article via Infotrieve]
  17. Villard, L., des Portes, V., Levy, N., Louboutin, J. P., Recan, D., Coquet, M., Chabrol, B., Figarella-Branger, D., Chelly, J., Pellissier, J. F., and Fontes, M. (2000) Eur. J. Hum. Genet. 8, 125-129[CrossRef][Medline] [Order article via Infotrieve]
  18. Anglade, P., Vyas, S., Javoy-Agid, F., Herrero, M. T., Michel, P. P., Marquez, J., Mouatt-Prigent, A., Ruberg, M., Hirsch, E. C., and Agid, Y. (1997) Histol. Histopathol. 12, 25-31[Medline] [Order article via Infotrieve]
  19. Petersen, Å., Larsen, K. E., Behr, G. G., Romero, N., Przedborski, S., Brundin, P., and Sulzer, D. (2001) Hum. Mol. Genet. 10, 1243-1254[Abstract/Free Full Text]
  20. Dorn, B. R., Dunn, W. A., Jr., and Progulske-Fox, A. (2002) Cell Microbiol. 4, 1-10[CrossRef][Medline] [Order article via Infotrieve]
  21. Suhy, D. A., Giddings, T. H., Jr., and Kirkegaard, K. (2000) J. Virol. 74, 8953-8965[Abstract/Free Full Text]
  22. Tallóczy, Z., Jiang, W., Virgin, H. W., IV, Leib, D. A., Scheuner, D., Kaufman, R. J., Eskelinen, E. L., and Levine, B. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 190-195[Abstract/Free Full Text]
  23. Bursch, W., Ellinger, A., Gerner, C., Fröhwein, U., and Schulte-Hermann, R. (2000) Ann. N. Y. Acad. Sci. 926, 1-12[Abstract/Free Full Text]
  24. Klionsky, D. J., and Emr, S. D. (2000) Science 290, 1717-1721[Abstract/Free Full Text]
  25. Ogier-Denis, E., Couvineau, A., Maoret, J. J., Houri, J. J., Bauvy, C., De Stefanis, D., Isidoro, C., Laburthe, M., and Codogno, P. (1995) J. Biol. Chem. 270, 13-16[Abstract/Free Full Text]
  26. Ogier-Denis, E., Houri, J. J., Bauvy, C., and Codogno, P. (1996) J. Biol. Chem. 271, 28593-28600[Abstract/Free Full Text]
  27. De Vries, L., Zheng, B., Fischer, T., Elenko, E., and Farquhar, M. G. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 235-271[CrossRef][Medline] [Order article via Infotrieve]
  28. Ogier-Denis, E., Petiot, A., Bauvy, C., and Codogno, P. (1997) J. Biol. Chem. 272, 24599-24603[Abstract/Free Full Text]
  29. Ogier-Denis, E., Pattingre, S., El Benna, J., and Codogno, P. (2000) J. Biol. Chem. 275, 39090-39095[Abstract/Free Full Text]
  30. Takesono, A., Cisnmowski, M. J., Ribas, C., Bernard, M., Chung, P., Hazard, S., III, Duzic, E., and Lanier, S. M. (1999) J. Biol. Chem. 274, 33202-33205[Abstract/Free Full Text]
  31. De Vries, L., Fischer, T., Tronchere, H., Brothers, G. M., Strockbine, B., Siderovski, D. P., and Farquhar, M. G. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 14364-14369[Abstract/Free Full Text]
  32. Natochin, M., Lester, B., Peterson, Y. K., Bernard, M. L., Lanier, S. M., and Artemyev, N. O. (2000) J. Biol. Chem. 275, 40981-40985[Abstract/Free Full Text]
  33. Peterson, Y. K., Bernard, M. L., Ma, H. Z., Hazard, S., Graber, S. G., and Lanier, S. M. (2000) J. Biol. Chem. 275, 33193-33196[Abstract/Free Full Text]
  34. Pizzinat, N., Takesono, A., and Lanier, S. M. (2001) J. Biol. Chem. 276, 16601-16610[Abstract/Free Full Text]
  35. Petiot, A., Ogier-Denis, E., Bauvy, C., Cluzeaud, F., Vandewalle, A., and Codogno, P. (1999) Biochem. J. 337, 289-295[CrossRef][Medline] [Order article via Infotrieve]
  36. Biederbick, A., Kern, H. F., and Elsasser, H. P. (1995) Eur. J. Cell Biol. 66, 3-14[Medline] [Order article via Infotrieve]
  37. Houri, J. J., Ogier-Denis, E., De Stefanis, D., Bauvy, C., Baccino, F. M., Isidoro, C., and Codogno, P. (1995) Biochem. J. 309, 521-527[Medline] [Order article via Infotrieve]
  38. Seglen, P. O., and Gordon, P. B. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1889-1892[Abstract]
  39. Weibel, E. R., Stäubli, W., Gnägi, H. R., and Hess, F. A. (1969) J. Cell Biol. 42, 68-91[Abstract/Free Full Text]
  40. Lawrence, P. B., and Brown, W. J. (1992) J. Cell Sci. 102, 515-526[Abstract]
  41. Lesuffleur, T., Barbat, A., Dussaulx, E., and Zweibaum, A. (1990) Cancer Res. 50, 6334-6343[Abstract]
  42. Lesuffleur, T., Kornowski, A., Luccioni, C., Muleris, M., Barbat, A., Beaumatin, J., Dussaulx, E., Dutrillaux, B., and Zweibaum, A. (1991) Int. J. Cancer 49, 721-730[Medline] [Order article via Infotrieve]
  43. Pinto, M., Robine-Léon, S., Appay, M. D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J., and Zweibaum, A. (1983) Biol. Cell 47, 323-330
  44. Bernard, M. L., Peterson, Y. K., Chung, P., Jourdan, J., and Lanier, S. M. (2001) J. Biol. Chem. 276, 1585-1593[Abstract/Free Full Text]
  45. Blumer, J. B., Chandler, L. J., and Lanier, S. M. (2002) J. Biol. Chem. 277, 15897-15903[Abstract/Free Full Text]
  46. Dunn, W. A., Jr. (1990) J. Cell Biol. 110, 1923-1933[Abstract]
  47. Yamamoto, A., Masaki, R., and Tashiro, Y. (1990) J. Histochem. Cytochem. 38, 573-580[Abstract]
  48. Munafò, D. B., and Colombo, M. I. (2002) Traffic 3, 472-482[CrossRef][Medline] [Order article via Infotrieve]
  49. Ishihara, N., Hamasaki, M., Yokota, S., Suzuki, K., Kamada, Y., Kihara, A., Yoshimori, T., Noda, T., and Ohsumi, Y. (2001) Mol. Biol. Cell 12, 3690-3702[Abstract/Free Full Text]
  50. Kihara, A., Noda, T., Ishihara, N., and Ohsumi, Y. (2001) J. Cell Biol. 152, 519-530[Abstract/Free Full Text]
  51. Nuoffer, C., and Balch, W. E. (1994) Annu. Rev. Biochem. 63, 949-990[CrossRef][Medline] [Order article via Infotrieve]
  52. Fengsrud, M., Roos, N., Berg, T., Liou, W. L., Slot, J. W., and Seglen, P. O. (1995) Exp. Cell Res. 221, 504-519[CrossRef][Medline] [Order article via Infotrieve]
  53. Stow, J. L., de Almeida, J. B., Narula, N., Holtzman, E., Ercolani, L., and Ausiello, D. (1991) J. Cell Biol. 114, 1113-1124[Abstract]
  54. Wylie, F., Heimann, K., Le, T. L., Brown, D., Rabnott, G., and Stow, J. L. (1999) Am. J. Physiol. 276, C497-C506[Medline] [Order article via Infotrieve]
  55. Lin, P., Yao, Y., Hofmeister, R., Tsien, R. Y., and Farquhar, M. G. (1999) J. Cell Biol. 145, 279-289[Abstract/Free Full Text]
  56. Weiss, T. S., Chamberlain, C. E., Takeda, T., Lin, P., Hahn, K. M., and Farquhar, M. G. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 14961-14966[Abstract/Free Full Text]
  57. Ballif, B. A., Mincek, N. V., Barratt, J. T., Wilson, M. T., and Simmons, D. L. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5544-5549[Abstract/Free Full Text]
  58. Gordon, P. B., Holen, I., Fosse, M., Rotnes, J. S., and Seglen, P. O. (1993) J. Biol. Chem. 268, 26107-26112[Abstract/Free Full Text]
  59. Blatch, G. L., and Lässle, M. (1999) BioEssays 21, 932-939[CrossRef][Medline] [Order article via Infotrieve]
  60. Harano, T., Nose, S., Uezu, R., Shimizu, N., and Fujiki, Y. (2001) Biochem. J. 357, 157-165[CrossRef][Medline] [Order article via Infotrieve]
  61. Natochin, M., Gasimov, K. G., and Artemyev, N. O. (2001) Biochemistry 40, 5322-5328[Medline] [Order article via Infotrieve]
  62. Kimple, R. J., De Vries, L., Tronchère, H., Behe, C. I., Morris, R. A., Farquhar, M. G., and Siderovski, D. P. (2001) J. Biol. Chem. 276, 29275-29281[Abstract/Free Full Text]
  63. Murayama, T., and Ui, M. (1983) J. Biol. Chem. 258, 3319-3326[Abstract/Free Full Text]
  64. van Sluijters, D. A., Dubbelhuis, P. F., Blommaart, E. F. C., and Meijer, A. J. (2000) Biochem. J. 351, 545-550[CrossRef][Medline] [Order article via Infotrieve]