BackgroundThe p24 family is thought to be somehow involved in endoplasmic reticulum (ER)-to-Golgi protein transport. A subset of the p24 proteins (p24α3, -β1, -γ3 and -δ2) 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 FindingsHere we find that transgene expression of p24α3 or p24δ2 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 p24α3 greatly reduces POMC transport and leads to accumulation of the prohormone in large, ER-localized electron-dense structures, whereas p24δ2-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/SignificanceTransgenic 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.

The p24 family consists of type I transmembrane proteins that are present abundantly in transport vesicles, may play a role in endoplasmic reticulum-to-Golgi cargo transport, and have been classified into subfamilies named p24alpha, -beta, -gamma, and -delta. We previously identified a member of the p24delta subfamily that is coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of the intermediate pituitary during black background adaptation of the amphibian Xenopus laevis ( approximately 30-fold increase in POMC mRNA). In this study, we report on the characterization of this p24delta member (Xp24delta(2)) and on the identification and characterization of a second member (Xp24delta(1)) that is also expressed in the melanotrope cells and that has 66% amino acid sequence identity to Xp24delta(2). The two p24delta members are ubiquitously expressed, but Xp24delta(2) is neuroendocrine enriched. During black background adaptation, the amount of the Xp24delta(2) protein in the intermediate pituitary was increased approximately 25 times, whereas Xp24delta(1) protein expression was increased only 2.5 times. Furthermore, the level of Xp24delta(2) mRNA was approximately 5-fold higher in the melanotrope cells of black-adapted animals than in those of white-adapted animals, whereas Xp24delta(1) mRNA expression was not induced. Therefore, the expression of Xp24delta(2) specifically correlates with the expression of POMC. Together, our findings suggest that p24delta proteins have a role in selective protein transport in the secretory pathway.

The p24␣, -␤, -␥, and -␦ proteins are major multimeric constituents of cycling endoplasmic reticulum-Golgi transport vesicles and are thought to be involved in protein transport through the early secretory pathway. In this study, we targeted transgene overexpression of p24␦ 2 specifically to the Xenopus intermediate pituitary melanotrope cell that is involved in background adaptation of the animal and produces high levels of its major secretory cargo proopiomelanocortin (POMC). The transgene product effectively displaced the endogenous p24 proteins, resulting in a melanotrope cell p24 system that consisted predominantly of the transgene p24␦ 2 protein. Despite the severely distorted p24 machinery, the subcellular structures as well as the level of POMC synthesis were normal in these cells. However, the number and pigment content of skin melanophores were reduced, impairing the ability of the transgenic animal to fully adapt to a black background. This physiological effect was likely caused by the affected profile of POMC-derived peptides observed in the transgenic melanotrope cells. Together, our results suggest that in the early secretory pathway an intact p24 system is essential for efficient secretory cargo transport or for supplying cargo carriers with the correct protein machinery to allow proper secretory protein processing. INTRODUCTIONTransport of cargo proteins through the early secretory pathway involves cargo selection, transport vesicle formation, quality control to recycle misfolded cargo, and cycling of the COPI-and COPII-coated vesicles between the endoplasmic reticulum (ER) and Golgi (Barlowe, 2000). One of the major constituents of the transport vesicles is the p24 family of type I transmembrane proteins that can be classified into four main subfamilies, designated p24␣, -␤, -␥, and -␦ (Schimmö ller et al., 1995;Stamnes et al., 1995;Sohn et al., 1996;Nickel et al., 1997;Dominguez et al., 1998). The p24 proteins share a number of structural characteristics, such as a relatively large lumenal putative cargo-binding domain, a coiled-coil region thought to be involved in the formation of multimeric p24 complexes, a transmembrane region, and a short cytoplasmic tail containing COPI-and COPII-binding motifs that are used for p24 traveling from the ER to the Golgi and back (for review, see Kaiser, 2000). In yeast and mammalian cells, p24 proteins form functional heterotetrameric complexes containing one representative of each subfamily, whereby the composition of the complex may differ in various cell types (Dominguez et al., 1998;Fü llekrug et al., 1999;Marzioch et al., 1999;Ciufo and Boyd, 2000;Emery et al., 2000;Belden and Barlowe, 2001). Furthermore, the stability of the p24 members seems to be compromised when cells are deficient in the expression of a single p24 protein (Marzioch et al., 1999;Denzel et al., 2000). Recent evidence suggests a complex and dynamic p24 system of mostly monomers and homo-/heterodimers and that the degree of oligomerization constantly alters and largely depends on the s...

Members of the p24 family of putative cargo receptors (subdivided into p24-alpha, -beta, -gamma and -delta) are localized in the intermediate-and cis-Golgi compartments of the early secretory pathway, and are thought to play an important role in protein transport. In the present study, we wondered what effect increased biosynthetic cell activity with resulting high levels of protein transport would have on the subcellular localization of p24. We examined p24 localization in Xenopus intermediate pituitary melanotrope cells, which in black- and white-adapted animals are biosynthetically highly active and virtually inactive respectively. In addition, p24 localization was studied in Xenopus anterior pituitary cells whose activity is not changed during background adaptation. Using organelle fractionation, we found that in the inactive melanotropes and moderately active anterior pituitary cells of white-adapted animals, the p24-alpha, -beta, -gamma and -delta proteins are all located in the Golgi compartment. In the highly active melanotropes, but not in the anterior cells of black-adapted animals, the steady-state distribution of all four p24 members changed towards the intermediate compartment and subdomains of the endoplasmic reticulum (ER), most probably the ER exit sites. In the active melanotropes, the major cargo protein pro-opiomelanocortin was mostly localized to ER subdomains and partially co-localized with the p24 proteins. Furthermore, in the active cells, in vitro blocking of protein biosynthesis by cycloheximide or dispersion of the Golgi complex by brefeldin A led to a redistribution of the p24 proteins, indicating their involvement in ER-to-Golgi protein transport and extensive cycling in the early secretory pathway. We conclude that the subcellular localization of p24 proteins is dynamic and depends on the biosynthetic activity of the cell.