Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
PLoS One
2007 Aug 08;28:e704. doi: 10.1371/journal.pone.0000704.
Show Gene links
Show Anatomy links
Disparate effects of p24alpha and p24delta on secretory protein transport and processing.
Strating JR
,
Bouw G
,
Hafmans TG
,
Martens GJ
.
???displayArticle.abstract???
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 pituitarymelanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC). 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. 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.
???displayArticle.pubmedLink???
17684551
???displayArticle.pmcLink???PMC1933603 ???displayArticle.link???PLoS One
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).
Belden,
Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport.
1996, Pubmed
Belden,
Erv25p, a component of COPII-coated vesicles, forms a complex with Emp24p that is required for efficient endoplasmic reticulum to Golgi transport.
1996,
Pubmed
Belden,
Deletion of yeast p24 genes activates the unfolded protein response.
2001,
Pubmed
Bouw,
A cell-specific transgenic approach in Xenopus reveals the importance of a functional p24 system for a secretory cell.
2004,
Pubmed
,
Xenbase
Braks,
7B2 is a neuroendocrine chaperone that transiently interacts with prohormone convertase PC2 in the secretory pathway.
1994,
Pubmed
,
Xenbase
Carney,
p24 proteins, intracellular trafficking, and behavior: Drosophila melanogaster provides insights and opportunities.
2004,
Pubmed
Chen,
TMP21 is a presenilin complex component that modulates gamma-secretase but not epsilon-secretase activity.
2006,
Pubmed
Chu,
The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus.
1989,
Pubmed
,
Xenbase
Collin,
Biosynthesis and differential processing of two pools of amyloid-beta precursor protein in a physiologically inducible neuroendocrine cell.
2005,
Pubmed
,
Xenbase
Denzel,
The p24 family member p23 is required for early embryonic development.
2000,
Pubmed
Dominguez,
gp25L/emp24/p24 protein family members of the cis-Golgi network bind both COP I and II coatomer.
1998,
Pubmed
Elrod-Erickson,
Genes that control the fidelity of endoplasmic reticulum to Golgi transport identified as suppressors of vesicle budding mutations.
1996,
Pubmed
Emery,
The trans-membrane protein p25 forms highly specialized domains that regulate membrane composition and dynamics.
2003,
Pubmed
Gommel,
p24 and p23, the major transmembrane proteins of COPI-coated transport vesicles, form hetero-oligomeric complexes and cycle between the organelles of the early secretory pathway.
1999,
Pubmed
Gupta,
Evidence for a role of transmembrane protein p25 in localization of protein tyrosine phosphatase TC48 to the ER.
2006,
Pubmed
Holthuis,
Molecular probing of the secretory pathway in peptide hormone-producing cells.
1995,
Pubmed
,
Xenbase
Jansen,
Transgene-driven protein expression specific to the intermediate pituitary melanotrope cells of Xenopus laevis.
2002,
Pubmed
,
Xenbase
Kroll,
Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation.
1996,
Pubmed
,
Xenbase
Kuiper,
Prohormone transport through the secretory pathway of neuroendocrine cells.
2000,
Pubmed
,
Xenbase
Kuiper,
Differential induction of two p24delta putative cargo receptors upon activation of a prohormone-producing cell.
2000,
Pubmed
,
Xenbase
Kuiper,
Localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell.
2001,
Pubmed
,
Xenbase
Lavoie,
Roles for alpha(2)p24 and COPI in endoplasmic reticulum cargo exit site formation.
1999,
Pubmed
Majoul,
KDEL-cargo regulates interactions between proteins involved in COPI vesicle traffic: measurements in living cells using FRET.
2001,
Pubmed
Majoul,
KDEL receptor (Erd2p)-mediated retrograde transport of the cholera toxin A subunit from the Golgi involves COPI, p23, and the COOH terminus of Erd2p.
1998,
Pubmed
Martens,
Biosynthesis of two structurally different pro-opiomelanocortins in the pars intermedia of the amphibian pituitary gland.
1982,
Pubmed
,
Xenbase
Martens,
Expression of two proopiomelanocortin genes in the pituitary gland of Xenopus laevis: complete structures of the two preprohormones.
1986,
Pubmed
,
Xenbase
Marzioch,
Erp1p and Erp2p, partners for Emp24p and Erv25p in a yeast p24 complex.
1999,
Pubmed
Mironov,
ER-to-Golgi carriers arise through direct en bloc protrusion and multistage maturation of specialized ER exit domains.
2003,
Pubmed
Monigatti,
The Sulfinator: predicting tyrosine sulfation sites in protein sequences.
2002,
Pubmed
Muñiz,
The Emp24 complex recruits a specific cargo molecule into endoplasmic reticulum-derived vesicles.
2000,
Pubmed
Palade,
Intracellular aspects of the process of protein synthesis.
1975,
Pubmed
Rojo,
Involvement of the transmembrane protein p23 in biosynthetic protein transport.
1997,
Pubmed
Rojo,
The transmembrane protein p23 contributes to the organization of the Golgi apparatus.
2000,
Pubmed
Rötter,
Cell-type-specific and selectively induced expression of members of the p24 family of putative cargo receptors.
2002,
Pubmed
,
Xenbase
Schimmöller,
The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi.
1995,
Pubmed
Simpson,
Biogenesis of tubular ER-to-Golgi transport intermediates.
2006,
Pubmed
Sparrow,
A simplified method of generating transgenic Xenopus.
2000,
Pubmed
,
Xenbase
Springer,
The p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae.
2000,
Pubmed
Stamnes,
An integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in budding.
1995,
Pubmed
Stark,
BioGRID: a general repository for interaction datasets.
2006,
Pubmed
Vetrivel,
Dual roles of the transmembrane protein p23/TMP21 in the modulation of amyloid precursor protein metabolism.
2007,
Pubmed
Wen,
p24 proteins and quality control of LIN-12 and GLP-1 trafficking in Caenorhabditis elegans.
1999,
Pubmed
van Kuppeveld,
Intracellular transport, sorting, and proteolytic processing of regulated secretory proteins does not require protein sulfation.
1997,
Pubmed
,
Xenbase
