Abstract
Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.
Highlights
From the Department of Cell and Molecular Biology, Divisionof Cell and Developmental Biology, Universityof California, ~~
Secretion is not affected by cycloheximide, sug- messenger RNA encoding antibodiesintoPC12 cells and gesting that operationof this pathwaydoes notrequire showed that binding of antibodies to a granule component components suppliedby new protein synthesis.In this diverted them from the constitutive pathway to rtehgeulated report we determined the effectsof protein synthesis secretory pathway
We found that treatment of AtT-20 cells with 100 pg/ml cyclowere centrifuged for 2 min in an Eppendorf microcentrifuge to heximide for 30 min inhibits 97% of their protein synthesis, remove any cells that may have detached during the chase, and the as measured by trichloroacetic acid-precipitable radioactivity supernatant was transferred to fresh tubes
Summary
DIFFERENTIALEFFECTS OF PROTEINSYNTHESISARREST ON TRANSPORTOF GLYCOSAMINOGLYCAN CHAINSTOTHETWOSECRETORYPATHWAYS*. Secretion is not affected by cycloheximide, sug- messenger RNA encoding antibodiesintoPC12 cells and gesting that operationof this pathwaydoes notrequire showed that binding of antibodies to a granule component components suppliedby new protein synthesis.In this diverted them from the constitutive pathway to rtehgeulated report we determined the effectsof protein synthesis secretory pathway Taken together, these experimseungtsgest inhibitor on membrane traffic to the regulated secre- thatpeptidehormonesare actively sortedintodense core tory pathway in themouse pituitary AtT-20 cells. These resultsimply that incorporation of the tripeptide into constitutive secretoryvesicles does not require the presencoef other newly synthesizedcargo proteins in the trans-Golgi.components of the vesicle machinery must recycle continuously, andhence no new protein this fact.
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