Mechanism of the facilitation of PC2 maturation by 7B2: involvement in ProPC2 transport and activation but not folding.
Among the members of the prohormone convertase (PC) family, PC2 has a unique maturation pattern: it is retained in the ER for a comparatively long time and its propeptide is cleaved in the TGN/ secretory granules rather than in the ER. It is also unique by its association with the neuroendocrine protein 7B2. This interaction results in the facilitation of proPC2 maturation and in the production of activatable proPC2 from CHO cells. In the present study, we have investigated the mechanism of this interaction. ProPC2 binds 7B2 in the ER, but exits this compartment much more slowly than 7B2. We found that proPC2 was also slow to acquire the capacity to bind 7B2, whereas 7B2 could bind proPC2 rapidly after synthesis. This indicated that proPC2 folding was the limiting step in the formation of the complex. Indeed, sensitivity of native proPC2 to N-glycanase F digestion and inhibition of proPC2 folding supported the notion that 7B2 is not involved in the early steps of proPC2 folding, and that proPC2 must fold before binding 7B2. Under experimental conditions that prevent propeptide cleavage, 7B2 expression increased proPC2 transport to the Golgi. This increase exhibited the same kinetics as the facilitation of the removal of the propeptide. Finally, proPC2 activation could be reconstituted in Golgi- enriched subcellular fractions. In vitro, 7B2 was required for proPC2 activation at an acidic pH. Taken together, our results demonstrate that rather than promoting proPC2 folding, 7B2 acts as a helper protein involved in proPC2 transport and is required in the proPC2 activation process. We propose, therefore, that 7B2 stabilizes proPC2 in a conformation already competent for these two events.
Muller
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Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112, USA.
Zhu
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X
Lindberg
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I
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000502:physiology; D006056:Golgi Apparatus / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D051859:Neuroendocrine Secretory Protein 7B2; D009490:Neurosecretory Systems / Q000737:chemistry / Q000166:cytology / Q000502:physiology; D010907:Pituitary Hormones / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D045706:Proprotein Convertase 2; D011485:Protein Binding / Q000502:physiology; D017510:Protein Folding; D013347:Subcellular Fractions / Q000378:metabolism / Q000502:physiology; D013381:Subtilisins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
D009419:Nerve Tissue Proteins; D051859:Neuroendocrine Secretory Protein 7B2; D010907:Pituitary Hormones; D013381:Subtilisins; D045706:Proprotein Convertase 2; D002118:Calcium