Inhibition of the vacuolar H+-ATPase perturbs the transport, sorting, processing and release of regulated secretory proteins.
Vacuolar H+-ATPases (V-ATPases) are multisubunit enzymes that acidify various intracellular organelles, including secretory pathway compartments. We have examined the effects of the specific V-ATPase inhibitor bafilomycin A1 (Baf) on the intracellular transport, sorting, processing and release of a number of neuroendocrine secretory proteins in primary Xenopus intermediate pituitary cells. Ultrastructural examination of Baf-treated intermediate pituitary cells revealed a reduction in the amount of small dense-core secretory granules and the appearance of vacuolar structures in the trans-Golgi area. Pulse-chase incubations in combination with immunoprecipitation analysis showed that in treated cells, the proteolytic processing of the newly synthesized prohormone proopiomelanocortin, prohormone convertase PC2 and secretogranin III (SgIII) was inhibited, and an intracellular accumulation of intact precursor forms and intermediate cleavage products became apparent. Moreover, we found that treated cells secreted considerable amounts of a PC2 processing intermediate and unprocessed SgIII in a constitutive fashion. Collectively, these data indicate that in the secretory pathway, V-ATPases play an important role in creating the microenvironment that is essential for proper transport, sorting, processing and release of regulated secretory proteins.
Schoonderwoert
V T
VT
Department of Animal Physiology, University of Nijmegen, the Netherlands.
Holthuis
J C
JC
Tanaka
S
S
Tooze
S A
SA
Martens
G J
GJ
D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism; D002451:Cell Compartmentation; D004705:Endocytosis; D006056:Golgi Apparatus / Q000378:metabolism; D006868:Hydrolysis; D018942:Macrolides; D010902:Pituitary Gland / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D025262:Vacuolar Proton-Translocating ATPases; D014982:Xenopus laevis
D000900:Anti-Bacterial Agents; D018942:Macrolides; D011506:Proteins; C040929:bafilomycin A1; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases