Intracellular sorting of peptide hormone precursors and the mechanism of secretory vesicle formation

Abstract

This thesis is divided into two related parts: (i) Prohormone processing. Most peptide hormones are synthesized as precursors possessing a propeptide. To test that the propeptide may function in intracellular sorting of prohormones to the trans-Golgi network (TGN), a chimeric construct was made which comprises the signal peptide and proregion of anglerfish prosomatostatin-I (SRIF-I) which is processed in rat pituitary GH{dollar}\sb3{dollar} cells, fused to proSRIF-II which degraded in these cells. When the chimera was expressed in GH{dollar}\sb3{dollar} cells, it was found that the SRIF-I propeptide rescued proSRIF-II from degradation and diverted it to secretory vesicles. The chimera was processed to SRIF-28, the physiological product of proSRIF-II. The SRIF-I propeptide functioned only in cis, but not in trans. These data suggest that the SRIF-I propeptide may possess a sorting signal for sequestration into the secretory pathway rather than functioning as an intramolecular chaperone to promote protein folding. (ii) Secretory vesicle formation from the TGN. ADP-ribosylation factor (ARF), a small GTP-binding protein, regulates the budding of vesicles that mediate endoplasmic reticulum (ER)-to-Golgi and intra-Golgi transport. It also plays an important role in maintaining the morphology of the Golgi apparatus. Using a permeabilized cell system derived from GH{dollar}\sb3{dollar} cells, the recombinant wild type human ARF-1 enhanced secretory vesicle budding about two-fold. A mutant lacking the first 17 N-terminal residues, as well as a mutant in the GDP-binding form did not stimulate vesicle formation. In contrast, a mutant defective in GTP hydrolysis promoted vesicle budding. Strikingly, a synthetic peptide corresponding to the N-terminus of human ARF-1 (amino acids 2-17) stimulated secretory vesicle budding, this is in marked contrast to its inhibitory effect on E.R. to Golgi transport. These data demonstrate that in endocrine cells, ARF-1 and particularly its N-terminus plays an essential role in the formation of secretory vesicles. Preliminary evidence suggests that phospholipase D, a downstream effector of ARF, also plays a essential role in promoting release of secretory vesicles from the TGN.