Localization of p24 putative cargo receptors in the early secretory pathway depends on the biosynthetic activity of the cell

Abstract

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.

The major cellulases CBH‐1 and CBH‐2 ofNeurospora crassarely on distinct ER cargo adaptors for efficient ER‐exit

“…As expected, the fluorescently tagged p24 and ERV‐29 cargo adaptors localized to ER membranes in conidial cells. This cellular localization is consistent with ER localization of GFP‐tagged versions of Erv25p and Erp3p in yeast cells, but distinct from the predominantly Golgi localization of p24 proteins in metazoan cells (Barr et al ., ; Kuiper et al ., ). Interestingly, in hyphal cells, EMP‐24 showed a differential localization that was dependent on the proximity to the hyphal tip, with the protein localizing to the early Golgi close to the tip and at the ER at regions that were distal from the tip.…”

Disparate Effects of p24α and p24δ on Secretory Protein Transport and Processing

“…The rabbit polyclonal antibodies against a region in the lumenal part of Xenopus laevis p24α 3 , against the C-terminal tail of Xenopus p24γ 3 , against part of the lumenal region of Xenopus p24δ 1 (anti-RH6), against portions of the lumenal and C-terminal regions of Xenopus laevis p24δ 2 (anti-1262N and anti-1262C respectively) and against the C-terminal region of Xenopus APP (C87) have been described previously [19], [24], [37]. We raised a polyclonal antibody against Xenopus p24β 1 by injecting rabbits with a recombinant protein encompassing the lumenal domain of Xenopus p24β 1 fused to GST.…”

Cell type‐specific transgene expression of the prion protein in Xenopus intermediate pituitary cells

“…Sy [50], and rabbit polyclonal anti‐human PrP IgG Sal1 was kindly provided by T. Sklaviadis (Aristotle University of Thessaloniki, Greece)[51]. The rabbit polyclonal antibody raised against the C‐terminal region of Xenopus p24δ 1/2 (anti1262CH) has been described previously [52,53]. A polyclonal antiserum against GFP was kindly provided by B. Wieringa [54], against Xenopus POMC (ST62, recognizing only the precursor isoform) by S. Tanaka (Shizuoka University, Hamamatsu, Japan [55]), against recombinant mature human PC2 by W.J.M.…”