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doi: 10.1242/10.1242/jcs.00047

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Improperly folded green fluorescent protein is secreted via a non-classical pathway

Molecular Medicine Unit, Department of Medicine, Beth Israel, Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA

View larger version (26K): [in a new window]   Fig. 1. GFP is secreted via a non-classical pathway in CHO cells. (A) Untransfected CHO cells (Ctrl) and cells transiently transfected with either GFP (GFP) or preprolactin-myc (PPL) were labeled with [35S]-methionine and chased for 6 hours. The cell lysates (C) and media (M) were subjected to immunoprecipitation with either antibodies against GFP (lanes 1-8) or myc (lanes 9-12), and the washed immunoprecipitates were displayed by SDS-PAGE and fluorography. Some cells (+BFA) were treated with 1 µg/ml brefeldin A for 1 hour prior to labeling (lanes 3-4, 7-8 and 11-12). `% Sec.' denotes the percentage of secretion of GFP into the media as determined by a phosphorimager. (B) CHO cells transfected with plasmids encoding either mouse dihydrofolate reductase (mDHFR) or Schistosoma japonicum glutathione S-transferase (GST) both tagged with the FLAG epitope. The cells were labeled with [35S]-methionine and chased in serum-free medium. The cell lysate (C) and media (M) were immunoprecipitated with anti-FLAG antibodies and processed as above. (C) Plasmids coding for mDHFR and GFP were used to co-transfect CHO cells. The cells were labeled, chased and processed as described in B.

View larger version (19K): [in a new window]   Fig. 2. GFP is secreted by various cells including cancer cells. NIH 3T3, HEK-293, COS, MCF-7, A375 and HT-29 were transiently transfected with a plasmid encoding GFP. The day after transfection, the cells were labeled with [35S]-methionine and chased in the presence of BFA. After 6 hours, the cell lysates (C) and media (M) were harvested and immunoprecipitated with anti-GFP antibodies. The washed immunoprecipitates were resolved by SDS-PAGE followed by fluorography. `% Sec.' denotes the percentage of GFP secreted by transfected cells.

View larger version (21K): [in a new window]   Fig. 3. Kinetics of GFP secretion from CHO cells. CHO cells transiently expressing GFP were starved, metabolically labeled with 35S-methionine and chased for various times as indicated. At each time point, the cell lysate (C) and medium (M) were harvested and immunoprecipitated with anti-GFP antibodies and analyzed by SDS-PAGE followed by fluorography. The graph depicts the percentage of secretion at each time point.

View larger version (17K): [in a new window]   Fig. 4. GFP secretion is a temperature-dependent process. CHO cells transiently expressing GFP were starved, labeled with [35S]-methionine at 37°C and chased for 6 hours at various temperatures as indicated above each set of lanes. The cell lysates (C) and media (M) were harvested and immunoprecipitated with anti-GFP antibodies. `% Sec.' denotes the percentage of secretion.

View larger version (14K): [in a new window]   Fig. 5. GFP secretion is independent of factors present in serum. CHO cells transiently expressing GFP were starved, labeled with [35S]-methionine and chased for 6 hours in the presence of various concentrations of serum as indicated. The cell lysates (C) and media (M) were harvested and immunoprecipitated with anti-GFP antibodies. `% Sec.' denotes the percentage of secretion.

View larger version (9K): [in a new window]   Fig. 6. Externalized membrane vesicles are not involved in GFP secretion. CHO cells transiently transfected with the plasmid encoding GFP were starved, labeled with [35S]-methionine and chased for 6 hours. At the end of the chase period, the cell lysate (C) and medium (M) were collected, centrifuged at 2,000 g for 30 minutes, and each was divided into two equal aliquots. One aliquot was immunoprecipitated with anti-GFP antibodies. The second aliquot of the medium was subjected to further centrifugation at 90,000 g for 2 hours to pellet vesicles. The supernatant (S) was collected and the pellet (P) was resuspended in 1x TXSWB buffer and immunoprecipitated with anti-GFP antibodies. The samples were separated by SDS-PAGE and analyzed by autoradiography.

View larger version (20K): [in a new window]   Fig. 7. Non-classically secreted GFP is not fluorescent. Untransfected (CHO) and GFP-transfected CHO cells (CHO+GFP) were washed and incubated in fresh medium for 6 hours. (A) The cell lysate (C) and media (M) were immunoprecipitated with anti-GFP antibodies, separated by SDS-PAGE and immunoblotted with anti-GFP antibodies. (B) The fluorescence was measured from 200 µl aliquots of the cell lysate and the media harvested as above. The graph shows the relative fluorescence units (RFU) per ml for the samples. The fluorescence was also measured from the lysate of GFP-expressing cells diluted 1:1 in fresh medium or PBS (CHO+GFP lysate diluted). Each bar on the graph represents the average value derived from at least three independent samples. (C) CHO cells expressing GFP were lyzed in medium supplemented with 1x Triton X-100. The fluorescence was measured from 200 µl aliquots of the medium after 0, 3 and 6 hours of incubation at 37°C. The values are plotted as RFU per ml.

View larger version (25K): [in a new window]   Fig. 8. Two different forms of GFP are present in CHO cells but only the improperly folded form is secreted. CHO cells expressing GFP (CHO+GFP) were incubated at 37°C in the presence of 100 µM cycloheximide for 0 or 6 hours. (A) The cell lysate (C) and media (M) were harvested at the end of the incubation, immunoprecipitated and immunoblotted with anti-GFP antibodies. (B) Spectro-fluorometric measurements of 200 µl aliquots of the cell lysate and media harvested as above are plotted as RFU per ml. Each bar on the graph represents the average value derived from five independent samples.

View larger version (14K): [in a new window]   Fig. 9. The two-pool model of GFP secretion. See text for details.