Immunocytochemical evidence for intragranular processing of pro-opiomelanocortin in the melanotropic cells of the rabbit

Abstract

The immunogold technique, employing antisera with clear-cut specificities, was used to localise different processing stages of pro-opiomelanocortin (POMC) in rabbit melanotropic cells. While the antiserum against gamma 3-MSH labelled all the secretory granules including intrasaccular condensations in the Golgi apparatus, antisera against alpha-MSH only labelled extra-Golgi secretory vesicles (SV). All extra-Golgi SV were likewise labelled with the three antisera against alpha-MSH used, despite their different specificities for the deacetylated, N-acetylated or C-amidated forms of the peptide. The antibody against beta-endorphin also labelled the extra-Golgi SV, while only some SV were labelled with the antibody against gamma-endorphin. These results correlate with biochemical data in favour of mainly--if not exclusively--intragranular processing of POMC. Except for gamma 3-MSH, the cleavage of which could coincide with Golgi packaging of secretory material, other post-translational modifications of the precursor seem to occur when SV are discharged outside the Golgi area. The cleavage of gamma-endorphin appears to be a later step in POMC processing, occurring in some mature SV.

Proinsulin Endoproteolysis Confers Enhanced Targeting of Processed Insulin to the Regulated Secretory Pathway

“…In most if not all of these classic studies, no special mechanistic significance had been attributed to the efficient storage of fully processed hormones. Rather, this was felt to reflect the fact that most processed hormones are generated after the proproteins are contained within granules (Orci et al, 1985(Orci et al, , 1987bStoeckel et al, 1985;Steiner et al, 1987;Tooze et al, 1987;Benjannet et al, 1992;Zhou et al, 1993;Huang and Arvan, 1994;Schmidt and Moore, 1995;Fernandez et al, 1997;Jutras et al, 1997;Tanaka et al, 1997;Urbe et al, 1997), whereas a failure to capture prohormones into forming granules would be coupled to constitutive secretion of these precursors (Bauerfeind et al, 1994). However, the more recent sorting-by-retention model of granule-based sorting (Arvan and Castle, 1998;Dumermuth and Moore, 1998;Thiele and Huttner, 1998b;Tooze, 1998) leaves open the possibility that some prohormones (as well as partially processed prohormones and other luminal proteins) may be removed from maturing secretory granules, with important consequences for the efficiency of regulated secretory protein targeting.…”

Preferential nuclear localization of the human papillomavirus type 16 E6 oncoprotein in cervical carcinoma cells

“…Approximately 10 5 cells from the CaSki, SiHa and C33A cell lines, cultured in parallel, were fixed with PBS containing 4 % paraformaldehyde and 0?1 % glutaraldehyde. After dehydratation in graded ethanol, the cells were embedded in Araldite-Epon resin (Stoeckel et al, 1985). Ultra-thin sections of these preparations were collected on Maxtaform grids and processed essentially as for immunofluorescence microscopy, except that the secondary antibodies used were goat anti-mouse immunoglobulins conjugated to colloidal gold particles (12 nm; Chemicon).…”

Ultrastructural Characterization of Adrenocorticotrope Hormone (ACTH) Immunoreactive Fibres in the Mesencephalic Central Grey Substance of the Rat

“…Immunohistochemical studies showed a co-localized distribution of two or more of the POMC-derived peptides in the same corticotropic and melanotropic cells of the anterior and intermediate lobe of the pituitary, and in the POMC neurons in the basal hypothalamus, at the light and electron microscopical level (Akil et al, 1978;Bloch et al, 1978Bloch et al, , 1979Crine et al, 1978;Dub6 et al, 1978;Sofroniew, 1979;Weber et al, 1979;Chatelain et al. 1979;Nilaver et al, 1979;Vaudri et al, 1980;Guy et al, 1980;Loh et al, 1982;Cantin et al, 1983;Stoeckel et al, 1983Stoeckel et al, , 1985Mains et al, 1983;Tanaka and Kurosumi, 1986;Powel and Baker, 1987;Kantin et al, 1988). Pelletier and Leclerc (1979) have shown at the ultrastructural level that ACTH is present in dense-core secretory granules in the brain.…”