A Protease Processing Site Is Essential for Prorenin Sorting to the Regulated Secretory Pathway

Véronique Brechler,William N Chu,John D Baxter,Gaétan Thibault,Timothy L Reudelhuber

doi:10.1074/jbc.271.34.20636

Abstract

Transfected mouse pituitary AtT-20 cells were used to examine the sorting of human prorenin to dense core secretory granules and the regulated secretory pathway. These cells secrete prorenin constitutively and sort a portion of the prorenin to secretory granules, where it is converted to active renin by proteolytic processing. Pulse-chase labeling of transfected AtT-20 cells demonstrated that regulated secretion of prorenin was prevented by: 1) the mutagenic deletion of the prosegment, 2) the premature proteolytic removal of the prosegment by a Golgi-resident processing protease, or 3) the mutation of the native cleavage site so as to prevent removal of the prosegment. In addition, expression of fusion proteins containing portions of the prorenin prosegment demonstrated that exposure of potential proteolytic cleavage sites was sufficient to confer cleavage-dependent regulated secretion of the corresponding protein. These data implicate the protease cleavage event in the regulated secretion of prorenin and are consistent with the involvement of a subclass of processing proteases in the sorting of certain proteins to secretory granules in AtT-20 cells.

Highlights

Transfected mouse pituitary AtT-20 cells were used to examine the sorting of human prorenin to dense core secretory granules and the regulated secretory pathway
Prorenin Is Sorted to the Regulated Secretory Pathway—Fig. 1 shows the pattern of secretion of native human prorenin from AtT-20 cells transfected by electroporation with a preprorenin expression vector
It is noteworthy that all of the renin released by forskolin migrates with the expected size of active renin from which the 43-amino acid prosegment has been removed by proteolytic cleavage

Summary

EXPERIMENTAL PROCEDURES

Recombinant Plasmid Construction—Construction of the expression vectors for native human prorenin (proren, referred to as pRhR1100) and prosegment-deleted prorenin (pro-del) have been described previously [15] (Fig. 1). Acids in the prosegment of human prorenin was carried out by overlap extension polymerase chain reaction [17]. Expression vectors for fusion proteins containing portions of human prorenin and the mouse immunoglobulin heavy chain were constructed as follows. Deletions that would result in predicted frame fusions were ligated to the IgG fragment and inserted in the place of the native human preprorenin cDNA in the expression vector proren (Fig. 2). DNA Transfections and Pulse-Chase Experiments—Purified plasmid DNA (50 ␮g) was mixed with 107 AtT-20 cells resuspended in serumfree Dulbecco’s modified Eagle’s medium and electroporated with a single pulse of 300 V/4 mm, 1000 microfarads. Twenty-four hours later, parallel wells of transfected cells were depleted of methionine for 1 h in methionine-free Dulbecco’s modified Eagle’s medium containing 10% dialyzed fetal calf serum.

RESULTS

Regulated Secretion of Human Prorenin

DISCUSSION