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Proteolytic Processing of Pro-ACTH/Endorphin Begins in the Golgi Complex of Pituitary Corticotropes and AtT-20 Cells

Department of Cell Biology, Yale University School of Medicine New Haven, Connecticut 06510
The Johns Hopkins University School of Medicine Baltimore, Maryland 21205

Address requests for reprints to: Dr. Marilyn Farquhar, Yale University School of Medicine, Department of Cell Biology, 333 Cedar Street, New Haven, Connecticut 06510.

Abstract

The intracellular sites where proteolytic processing of pro-ACTH/endorphin or POMC take place have not yet been reliably identified. We have used affinity-purified antisera that recognize only the products of POMC processing and immunoelectron microscopy to identify the compartments of rat pituitary corticotropes and mouse AtT-20 cells in which cleavage occurs. Immunoperoxidase labeling of cryostat sections and immunogold labeling of ultrathin frozen sections were used for localization of the processing sites. By both procedures we detected processed peptides in Golgi cisternae and secretion granules. Within the Golgi, labeling was limited to the last or transmost cisterna and was most concentrated in its dilated rims which contain condensing secretory protein. No labeling of other Golgi cisternae was seen. All Golgi cisternae were labeled, however, when antisera that recognize unprocessed POMC were used for immunolabeling. We conclude that in AtT-20 and rat pituitary cells: 1) processing of POMC through at least two endo- and exoproteolytic cleavage steps and -amidation of joining peptide begin in the trans Golgi subcompartment; 2) no detectable processing takes place before POMC reaches the trans Golgi cisterna; and 3) this Golgi cisterna as well as secretion granules contain the active enzymes necessary for proteolytic processing and -amidation.

FOOTNOTES

This work was supported by NIH Research Grants DK-17780 (to M.G.F.) and DK-32948 and DA-00097 (to R.E.M.). Preliminary results of the work have been presented at the American Society of Cell Biology/American Society for Biochemistry and Molecular Biology Joint Meeting in San Francisco, CA, 1989.

^* Recipient of Fellowship 1986–1989 from the German Research Foundation.

Received for publication May 4, 1989. Revision received May 18, 1989. Accepted for publication May 18, 1989.

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