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First published online 11 April 2006
doi: 10.1242/jcs.02905

Journal of Cell Science 119, 1833-1842 (2006)
Published by The Company of Biologists 2006
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Lumenal protein sorting to the constitutive secretory pathway of a regulated secretory cell

Roberto Lara-Lemus1,*, Ming Liu1,*, Mark D. Turner2,*, Philipp Scherer3, Gudrun Stenbeck4, Puneeth Iyengar3 and Peter Arvan1,{ddagger}

1 Division of Metabolism, Endocrinology and Diabetes, University of Michigan Medical Center, Ann Arbor, MI 48109, USA
2 Centre for Diabetes and Metabolic Medicine, Institute of Cell and Molecular Science, Queen Mary's School of Medicine and Dentistry, University of London, Whitechapel, London, E1 1BB, UK
3 Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
4 Bone and Mineral Centre, University College London, London, WC1E 6JJ, UK


Figure 1
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  		Fig. 1. Western blotting of secretory alkaline phosphatase (SEAP) expressed in INS-1 ß-cells. (A) A stably transfected clone of INS-1 cells (clone 12, Trnfxn) immunoblotted for alkaline phosphatase next to untransfected control cells (Con). In addition, a faster-migrating specific band is detected in the stably transfected cells. Upon glycan digestion (+), the steady state distribution of SEAP is divided into endo-H-sensitive (S) and endo-H-resistant (R) forms, the latter indicative of a significant Golgi and/or post-Golgi pool of the protein in these cells. (B) A comparison of SEAP expression level in the clone shown in panel A (C-12) to two other independently selected INS-1 clones (C-14 and C-15), each of which maintains a similar proportion of endo-H-resistant SEAP.

 

Figure 2
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  		Fig. 2. Acquisition of endo H resistance and secretion of SEAP at 2 hours of chase in INS-1 ß-cells. Cells were metabolically labeled for 30 minutes and the media (M), cells (C) or total (T, media + cells) were immunoprecipitated with anti-alkaline-phosphatase antibody. At the zero chase time, SEAP had not yet appeared in the medium and all intracellular SEAP was sensitive to digestion with endo H (S). At 2 hours of chase, more than half of labeled SEAP had acquired endo H resistance (R); 35% of labeled SEAP reached the medium (all of which is endo H resistant), and 25% of intracellular SEAP accumulated as an endo-H-resistant form. The data are representative of three experiments.

 

Figure 3
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  		Fig. 3. Phase-contrast microscopy and confocal indirect immunofluorescence distribution of insulin and SEAP in transfected INS-1 ß-cells. Insulin secretory granules tend to concentrate in a subplasmalemmal distribution and are not found in the stippled areas seen by phase contrast. All cells in the clonal population express SEAP (green), which exhibits large areas of overlapping distribution with that of insulin (red). A small bracket in each fluorescence image encloses three puncta that are thought to represent subplasmalemmal secretory granules or granule clusters. The bracketed area is reproduced as an inset at higher magnification in the lower left corner of the merged image to highlight the degree of colocalization of SEAP and insulin. Bar, 10 µm.

 

Figure 4
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  		Fig. 4. Similar handling of proinsulin and SEAP during pulse-chase examination of transfected INS-1 ß-cells. Each of three independent clones expressing SEAP were pulse labeled for 30 minutes with 35S amino acids and unstimulated media were removed and replaced at 1 hour and 3 hour chase times. A final collection of medium was then performed from 3-7 hours of chase (7) under unstimulated or stimulated (Stim - or +) conditions, respectively. Secretion of insulin-containing peptides was analyzed by immunoprecipitation with anti-insulin (A, proinsulin conversion intermediates highlighted with an asterisk), whereas secretion of SEAP from the identical samples was analyzed by immunoprecipitation with anti-alkaline phosphatase (B). Note that SEAP enters and is stored within the stimulus-dependent secretory pathway, in parallel with insulin, in all clones of INS-1 cells.

 

Figure 5
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  		Fig. 5. Endogenous expression of Cab45, a Golgi lumenal-resident protein, in INS-1 cells. Cells were pulse labeled for 30 minutes with 35S amino acids and chased continuously for 4 hours in the absence (-) or presence (+) of secretagogue (Stim). In duplicate, media (Med.) were collected and cells lysed, and the samples were then analyzed by sequential immunoprecipitation with mAb anti-Cab45 (lower panel) and polyclonal anti-insulin (upper panel). The Cab45 immunoprecipitates were further divided either for mock digestion (-) or endoglycosidase H (Endo H) digestion (+). At 4 hours of chase, Cab45 appears as two intracellular endo-H-resistant bands; the position of the 45 kDa molecular mass marker is shown.

 

Figure 6
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  		Fig. 6. Secretion of Cab308Myc from transfected INS cells. (A) Scheme of the primary structure of Cab45 and the Cab308Myc construct. (B) Cells were pulse labeled for 30 minutes with 35S amino acids and chased continuously for 4 hours in the absence (-) or presence (+) of secretagogue (Stim). Media (M) were collected and cells lysed (C), and the samples were then analyzed by sequential immunoprecipitation with anti-Myc (upper panel) and anti-insulin (lower panel; proinsulin conversion intermediates highlighted with an asterisk). (C) Cells were labeled as in B and then either lysed immediately without chase (Time 0) or chased for 4.5 hours in the absence (-) or presence (+) of secretagogue (Stim). Media (M) were collected and cells lysed (C), and the samples were then analyzed by immunoprecipitation with anti-Myc followed by mock digestion (-) or endoglycosidase H (Endo H) digestion (+). By SDS-PAGE, Cab308Myc migrates as two bands ranging from 33 to 35 kDa. (D) Double-labeled immunofluorescence distribution of Cab308Myc in transfected INS-1 cells (using polyclonal rabbit anti-Myc, green) relative to the (Golgi) distribution of GM130 (in red).

 

Figure 7
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  		Fig. 7. Secretion of Cab308Myc from AtT20 cells. Two days after transient transfection, untransfected control cells (left panels) or transfected cells (right panel) were pulse-labeled as in Fig. 6B, and then chased sequentially for period 1 (3 hours), period 2 (30 minutes) and period 3 (30 minutes). Periods 1 and 2 were in the absence of secretory stimulus whereas period 3 included either the absence (-) or presence (+) of secretagogue (Stim) as indicated. The upper panels show immunoprecipitation with anti-ACTH from media and from cells (C) at the end of the experiment. A large quantity of constitutive-like release of POMC processing intermediate (asterisk) is seen in period 1 (Dumermuth and Moore, 1998Go). Stimulus-dependent secretion in period 3 is evident by increased ACTH secretion over that recovered in the medium of period 2 from the same cells, which serves as an internal control. From transfected AtT20 cells, Cab308Myc (lower panels) was immunoprecipitated only from the secretion during period 1, and no further release occurred upon subsequent stimulation of granule exocytosis. The data shown are representative of three independent experiments.

 

Figure 8
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  		Fig. 8. Short-term secretion of newly synthesized Cab308Myc and AAT from untransfected or transfected INS-832/13 cells. (A) Cells were pulse labeled for 30 minutes with 35S amino acids and chased for 40 minutes in the absence (-) or presence (+) of secretagogue (Stim). Media (M) were collected and cells lysed (C), and the samples were then analyzed by sequential immunoprecipitation with anti-Myc (upper panels) and anti-insulin (middle panels), or anti-AAT (lower panels). A representative clone transfected with the Cab308Myc construct (Clone 2) is shown on the right. Untransfected INS-832/13 cells (left) serve as a negative control for the anti-Myc and anti-AAT immunoprecipitations. The positions of proinsulin (Proins) and insulin are shown; higher bands represent proinsulin conversion intermediates. (B) Two additional independent clones transfected with the Cab308Myc construct (Clones 34 and 24) are shown with less Cab308Myc expression. The experimental protocol was identical to A except that cells were pulse labeled for 40 minutes. For clarity, lines have been added at bottom indicating the stimulus-dependent secretion of newly synthesized insulin. As shown, stimulus-dependent secretion of Cab308Myc was zero in these clones. These experiments have been repeated and confirmed.

 

Figure 9
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  		Fig. 9. Indirect immunofluorescence distribution of Cab308Myc in transfected INS cells. Triple immunofluorescence labeling with guinea pig anti-insulin (A, blue), rabbit anti-calnexin (B, red) and mouse anti-Myc (Cab308Myc, C, green) after 60 minutes of cycloheximide treatment. In the cell periphery, Cab308Myc primarily exists in organelles that are non-overlapping with anti-insulin (merge, D; an additional tenfold zoom of the inset from D is shown in E). (F) A different experiment using the same cells double labeled for Cab308Myc (with anti-rabbit conjugate in green) and the mAb GSA8 directed against the intact proinsulin cleavage site (with anti-mouse conjugate in red), which marks immature rather than mature secretory granules. Bar, 10 µm.

 

Figure 10
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  		Fig. 10. Behavior of SEAP, Cab308Myc and Cab45 after saponin permeabilizing intracellular membranes and sedimenting the resultant extract. Both non-aggregative (N-A.; neutral pH, low calcium) and aggregative (Agg.; low pH, high calcium) conditions were used for extraction as described in the Materials and Methods. (A) Western blotting of SEAP and Cab308Myc after saponin permeabilization of organelle membranes. SEAP, which enters secretory granules, appears largely (>90%) soluble under both non-aggregative and aggregative conditions, whereas Cab308Myc is almost completely (>95%) sedimentable under these conditions. (B) Immunoprecipitation of newly synthesized Cab308Myc after saponin permeabilization or Triton X-100 solubilization, followed by sedimenting the resultant extracts. INS-Cab308Myc cells were pulse labeled with 35S-amino acids for 100 minutes without chase. Organellar membranes from these cells were saponin permeabilized under non-aggregative conditions (control, lanes 1 and 2), or dissolved with 1.5% Triton X-100 (lanes 3 and 4). After centrifugation, the supernatants (S) and solubilized pellets (P) were immunoprecipitated with anti-Myc and analyzed by SDS-PAGE and fluorography. (C) Western blot of Cab45 as in A, using non-aggregative conditions in the absence (No Deterg.) or presence of detergents as shown. The data shown are representative of three independent experiments. (D) INS-Cab308Myc cells were pulse labeled as in panel B in the presence of brefeldin A (BFA, 10 µg/ml). Organellar membranes from these cells were saponin permeabilized under non-aggregative conditions, or dissolved with 1.5 % Triton X-100 (TX100), or mock treated without detergent (No deterg.). After centrifugation, the supernatants (S) and solubilized pellets (P) were immunoprecipitated with anti-Myc and analyzed by SDS-PAGE and fluorography.

 

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© The Company of Biologists Ltd 2006