Disparate effects of p24alpha and p24delta on secretory protein transport and processing

Abstract

Background: The p24 family is thought to be somehow involved in endoplasmic reticulum (ER)-to-Golgi protein transport. A subset of the p24 proteins (p24alpha(3), -beta(1), -gamma(3) and -delta(2)) is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC).

Methodology/principal findings: Here we find that transgene expression of p24alpha(3 )or p24delta(2) specifically in the Xenopus melanotrope cells in both cases causes an effective displacement of the endogenous p24 proteins, resulting in severely distorted p24 systems and disparate melanotrope cell phenotypes. Transgene expression of p24alpha(3) greatly reduces POMC transport and leads to accumulation of the prohormone in large, ER-localized electron-dense structures, whereas p24delta(2)-transgenesis does not influence the overall ultrastructure of the cells nor POMC transport and cleavage, but affects the Golgi-based processes of POMC glycomaturation and sulfation.

Conclusions/significance: Transgenic expression of two distinct p24 family members has disparate effects on secretory pathway functioning, illustrating the specificity and non-redundancy of our transgenic approach. We conclude that members of the p24 family furnish subcompartments of the secretory pathway with specific sets of machinery cargo to provide the proper microenvironments for efficient and correct secretory protein transport and processing.

Figure 1. Generation of Xenopus with transgene expression of p24α₃ or p24δ₂ specifically in the melanotrope cells.

(A and B) Schematic representation of the linear injection fragments pPOMC-p24α₃-GFP (A) and pPOMC-p24δ₂-GFP (B) containing a Xenopus POMC gene promoter fragment (pPOMC) and the protein-coding sequence of p24α₃-GFP (transgenic lines #605, #55 and #602) or p24δ₂-GFP (lines #125, #115, #124 and #224); pPOMC drives transgene expression specifically to the melanotrope cells. (C) Pituitary-specific GFP-fluorescence (arrows) in living tadpoles transgenic for p24α₃ (line #55) or p24δ₂ (line #224); G, gut; E, eye; N, nose. (D) Fluorescence in the intermediate lobe (IL) and not in the anterior lobe (AL) of the pituitary of adult frogs transgenic for p24α₃ (#55) or p24δ₂ (#224).

Figure 2. Analysis of p24α₃ or p24δ₂ fusion protein expression in transgenic Xenopus intermediate pituitary melanotrope cells.

(A and B) Western blot analysis of neurointermediate lobe lysates from wild-type frogs (wt) and frogs transgenic for p24α₃ (#55) or p24δ₂ (#224) with anti-p24 antibodies (A) or an anti-GFP antibody (B). Tubulin was used as a control for equal loading. (C and D) Immuno-electron microscopy analysis of intermediate pituitary melanotrope cells from frogs transgenic for p24α₃ (#55; C) or p24δ₂ (#224; D1 and D2) using an anti-GFP antibody. The p24α₃-transgene product was mainly present in ER-localized electron-dense structures (EDS; C), whereas the p24δ₂-transgene product was found on the ER (D1) and on the Golgi (D2). G, Golgi; ER, endoplasmic reticulum. Bars equal 200 nm.

Figure 3. Steady-state levels of secretory cargo proteins in wild-type and transgenic Xenopus intermediate pituitary cells.

Western blot analysis of neurointermediate lobe (NIL) lysates from wild-type frogs (wt) and frogs transgenic for p24α₃ (#55) or p24δ₂ (#224) using antibodies directed against the soluble cargo proteins proopiomelanocortin (POMC) and prohormone convertase 2 (PC2), and the transmembrane cargo amyloid-β precursor protein (APP). Tubulin was used as a control for equal loading.

Figure 4. The effect of p24α₃- or p24δ₂-transgene expression on POMC biosynthesis and processing in Xenopus melanotropes.

(A–C) Neurointermediate lobes (NILs) from wild-type frogs (wt) and frogs transgenic for p24α₃ (#55) or p24δ₂ (#224) were pulse labeled with [³⁵S]-Met/Cys for 30 min and subsequently chased for 3 hrs. Newly synthesized proteins extracted from the NILs (Cells; 5% of extract) and secreted into the incubation medium (Media; 20%) were resolved by 15% SDS-PAGE and visualized by autoradiography. (A) The analysis was performed in six independent experiments and a representative autoradiogram is shown. (B) The amount of newly synthesized 37K POMC in wild-type (n = 16) and the p24α₃-transgenic (n = 10) and p24δ₂-transgenic (n = 6) cells was quantified and is shown relative to the wild-type cells. (C) The amounts of newly synthesized 18K and 18K* POMC in wild-type (n = 12) and the p24α₃-transgenic (n = 5) and p24δ₂-transgenic (n = 6) cells were quantified and are shown relative to wild-type 18K POMC. Indicated are the 18K/18K* ratios and their statistical evaluations. Data are shown as means +/− SEM. n.s., not significant; **, p<0.01; ***, p<0.001.

Figure 5. Newly synthesized 18K and 18K* POMC differ in N-glycosylation.

Neurointermediate lobes (NILs) from wild-type frogs (wt) and frogs transgenic for p24α₃ (#55) or p24δ₂ (#224) were pulse labeled with [³⁵S]-Met/Cys for 30 min and subsequently chased for 3 hrs. Newly synthesized proteins extracted from the NILs were deglycosylated with PNGaseF (F) or control-treated (C), resolved by 20% SDS-PAGE and visualized by autoradiography; the #55 lanes were exposed three times longer than the other lanes.

Figure 6. Sulfation of newly synthesized POMC in wild-type and transgenic Xenopus intermediate pituitary cells.

Neurointermediate lobes (NILs) from wild-type frogs (wt) and frogs transgenic for p24α₃ (#55) or p24δ₂ (#224) were pulse labeled with ³⁵S-sulfate and ³H-lysine for 15 min. Newly synthesized proteins extracted from the NILs were resolved by 15% SDS-PAGE and the amount of [³⁵S]SO₄ and ³H-lysine incorporated into newly-synthesized 37K POMC was determined. Shown are the amounts of newly synthesized sulfated 37K POMC produced in the transgenic relative to wt NILs. Data are shown as means +/− SEM (wt, n = 7; transgenics, n = 5). *, p<0.05; n.s., not significant.

Figure 7. Electron microscopy analysis of wild-type and transgenic melanotrope cells.

(A–C) Wild-type (wt; A1 and A2) intermediate pituitary cells showed a well-developed rough endoplasmic reticulum and extensive Golgi-ribbons. The p24α₃-transgenic cells (#55; B1 and B2) contained Golgi mini-stacks and large electron-dense structures (EDS). The p24δ₂-transgenic cells (#224; C1 and C2) showed an ultrastructure similar to that of wild-type cells. The dotted lines highlight the outline of the Golgi. (D–F) Immuno-electron microscopy analysis of intermediate pituitary melanotrope cells from wt frogs (D), and frogs transgenic for p24α₃ (#55; E) or p24δ₂ (#224; F) using an anti-POMC antiserum. Immunoreactivity was found in dense-core secretory granules (filled arrowheads) in wt and p24α₃- and p24δ₂-transgenic cells and occasionally in newly forming secretion granules still attached to the trans-Golgi network (open arrowheads). In addition, in the p24α₃-transgenic cells a strong POMC-immunolabeling was observed in the EDS, which were localized to the ER lumen (arrow) and occasionally in EDS newly forming within the ER lumen (open arrow). G, Golgi; L, lysosome; M, mitochondrion; N, nucleus; RER, rough endoplasmic reticulum; PM, plasma membrane; sg, immature secretory granules. Bars equal 1 µm (A–C); 500 nm (D–F).