Abstract：
:Propeptide processing occurs in specific compartments of the secretory pathway, but how these processing-competent organelles are generated from their processing-incompetent precursor compartments is unknown. To dissect the process biochemically, we have developed a novel cell-free system reconstituting the production of processing-competent secretory granules in AtT-20 cells. Using donor membranes containing [(35)S]sulfate labeled pro-opiomelanocortin (POMC)(5) in the trans-Golgi, we can reconstitute cytosol- and ATP-dependent prohormone processing as well as incorporation of processed ACTH into immature secretory granules (ISGs). Under limiting cytosol conditions, both reactions are greatly stimulated by ADP-ribosylation factor 1 (ARF1) but not by the GDP-bound ARF1 T31N mutant. pH studies show that lumenal acidification, most likely due to ARF-mediated sorting of proton pumps and leaks during budding, confers processing competency to the resulting organelle. Surprisingly, comparison of onset of processing and ISG release reveals that they are distinct biochemical processes with different kinetics and separate cytosolic requirements. Moreover, ARF regulates the onset of prohormone processing but not ISG release. Our data suggest a two-step mechanism (onset of processing followed by ISG release) for the production of processing-competent organelles from the trans-Golgi and provide the first system with which these two steps may be individually dissected.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Andresen JM,Moore HPdoi
10.1021/bi0112762subject
Has Abstractpub_date
2001-10-30 00:00:00pages
13020-30issue
43eissn
0006-2960issn
1520-4995pii
bi0112762journal_volume
40pub_type
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