Passive sorting in maturing granules of AtT-20 cells: the entry and exit of salivary amylase and proline-rich protein

Abstract

Previous studies have suggested that salivary amylase and proline-rich protein are sorted differently when expressed in AtT-20 cells (Castle, A.M., L.E. Stahl, and J.D. Castle. 1992. J. Biol. Chem. 267:13093- 13100; Colomer, V., K. Lal, T.C. Hoops, and M.J. Rindler. 1994.EMBO (Eur. Mol. Biol. Organ.) J. 13:3711- 3719). We now show that both exocrine proteins behave similarly and enter the regulated secretory pathway as judged by immunolocalization and secretagogue- dependent stimulation of secretion. Analysis of stimulated secretion of newly synthesized proline-rich protein, amylase, and endogenous hormones indicates that the exogenous proteins enter the granule pool with about the same efficiency as the endogenous hormones. However, in contrast to the endogenous hormones, proline-rich protein and amylase are progressively removed from the granule pool during the process of granule maturation such that only small portions remain in mature granules where they colocalize with the stored hormones. The exogenous proteins that are not stored are recovered from the incubation medium and are presumed to have undergone constitutive-like secretion. These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage. Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.

Figure 1

Immunocytochemical localization of PRP, amylase, and ACTH in transfected AtT-20 cells. AtT-20 cells expressing PRP were stained with anti-PRP (A and B), or anti-ACTH (E and F); cells expressing amylase were stained with an anti-amylase antibody (C and D). Binding of primary antibodies was detected with Cy3- or Texas red–conjugated goat anti–rabbit IgG. The distribution of ACTH in the amylase expressing cells was highly similar to that shown in (E and F). Bar, 10 μm.

Figure 2

Localization of PRP and ACTH within cellular processes by double label immunofluorescence. Cells expressing PRP were labeled with anti-PRP antibody followed by Cy3-conjugated goat anti–rabbit IgG, and biotinylated anti-ACTH antibody followed by Cy5-conjugated avidin. Shown here are images of cellular processes (PRP in A, C, E, and G; ACTH in B, D, F, and H) obtained by confocal microscopy as a single optical section. The images in A–D were obtained with a clone expressing PRP at the same level as ACTH, and images in E–H were obtained with a clone expressing 1/20 as much PRP as ACTH. The pattern of staining of PRP shows many areas of colocalization with ACTH (arrowheads), but also areas where PRP and ACTH exhibit different relative intensities of staining (arrows). Bar, 5 μm.

Figure 3

Localization of amylase and ACTH within cellular processes by double label immunofluorescence. Cells expressing amylase were labeled with anti-amylase antibody followed by Cy3-conjugated goat anti– rabbit IgG, and biotinylated anti-ACTH antibody followed by Cy5-conjugated avidin. In single optical sections of cellular processes (amylase in A and C; ACTH in B and D) the arrowheads show examples of the extensive colocalization of amylase and ACTH, while the arrows point to areas with different relative intensities of the two antigens. Bar, 5 μm.

Figure 4

Fluorescent staining intensity ratios of PRP versus ACTH and amylase versus ACTH. (A–C) Staining intensities for PRP, amylase, and ACTH of fluorescent spots seen in Figs. 2 and 3 were evaluated using the intensity profile function. A section of each paired image (not including the cell body and the tip of the process) was chosen, and the intensities of all individual spots were quantitated. For each spot, the peak intensity of PRP or amylase was divided by the corresponding peak intensity of ACTH. The intensity ratios were then grouped into 7 bins: 1 (0–0.5), 2 (0.6–1), 3 (1.1–1.5), 4 (1.6–2), 5 (2.1–2.5), 6 (2.6–3), and 7 (>3). The frequency in each bin was expressed as a fraction of total spots counted. (A) High-expressing PRP (from Fig. 2, A and B); 110 spots counted. (B) Low-expressing PRP (from Fig. 2, E and F), 132 spots counted. (C) Amylase (from Fig. 3, A and B), 80 spots counted. (D) Staining intensity ratios for PRP versus mouse transferin receptor were evaluated as above (164 spots counted). The last distribution is indicative of two nonoverlapping compartments.

Figure 5

Dense-core granules contain variable amounts of PRP. (A) AtT-20 cells expressing PRP at the same level as ACTH were labeled with affinity-purified anti-ACTH antibody. (B–E) AtT-20 cells expressing PRP at the same level as ACTH double labeled with anti-PRP serum (10 nM gold), and biotinylated anti-ACTH antibody (5 nM gold). Granules labeled with both types of gold are observed near the plasma membrane (B and D) as well as in intracellular locations (C and E). Arrowheads point to a subset of granules which contain significant ACTH staining but where the gold particles are more difficult to visualize. Bar, 150 nm.

Figure 6

Distribution of endo H–resistant PRP and mature ACTH on a sucrose density gradient. Postnuclear supernates from cells labeled for 15 h (to approach steady state) with 0.4 mCi/ml [³H]proline and chased for 1 h, were subjected to centrifugation on sucrose density gradients. Endo H–resistant PRP (•) and mature ACTH (○) were quantitated from each fraction as described in Materials and Methods. The top and bottom of the gradient are in fractions 1 and 12, respectively. The data come from one of two independent experiments in which nearly identical profiles were obtained.

Figure 7

Stimulation of secretion of newly synthesized PRP and amylase. Cells expressing PRP (A) and amylase (B) were labeled for 1 h with 0.5 mCi/ml of [³H]lysine or 0.15 mCi/ml of Expre³⁵S³⁵S label, respectively. Cells were first chased for 1 or 5 h in the absence of secretagogue, and subsequently for 1 h in the absence (−) or presence (+) of 5 mM 8-Br-cAMP (8 Br). Immunoprecipitated PRP (A) and amylase (B) from the chases are shown. All of each immunoprecipitate was loaded except in A for the 5-h chase, where only 1/3 of the total immunoprecipitate was used.

Figure 8

Timecourse of stimulation of secretion of ACTH. Mock-transfected AtT-20 cells were labeled with 0.25 mCi/ml of [³⁵S]methionine for 20 min and chased for 0, 0.5, 1, 2, or 4 h. At each timepoint, chase medium was replaced with medium containing or lacking 5 mM 8-Br-cAMP and chased for an additional 30 min. Net stimulated secretion of mature ACTH peptides was determined as described in Table II.