Calcium- and pH-dependent aggregation and membrane association of the precursor of the prohormone convertase PC2

K I Shennan, N A Taylor, K Docherty

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

PC2 is a subtilisin-like protease which is thought to be involved in the processing of prohormones and proneuropeptides in neuroendocrine cells. The mature 68-kDa enzyme is generated by intracellular proteolytic processing of a 75-kDa pro-PC2 polypeptide. Neuroendocrine cells contain at least two secretory pathways: the regulated pathway whereby secreted products are concentrated, stored in granules, and released in response to external stimulation of the cell, and the constitutive pathway, whereby secretory and plasma membrane proteins are continuously transported to the cell surface without prior concentration or storage. An important step in the sorting of proteins into these pathways is thought to involve the aggregation of proteins destined for storage granules. To define the mechanisms in the intracellular sorting of PC2 to secretory vesicles, the present study was undertaken to investigate the aggregation and membrane association properties of precursor and mature forms of PC2. Using material expressed in microinjected Xenopus oocytes, it was demonstrated that the 75-kDa pro-PC2 polypeptide undergoes calcium- and acid pH-dependent aggregation. Calcium exhibited an effect on the aggregation of pro-PC2 at pH 7.0 and 6.5, whereas below 6.5 aggregation was independent of calcium. Association of pro-PC2 with membranes was observed at pH 5.5, but not at pH 7.0, 6.5, nor 6.0. The mature 68-kDa PC2 polypeptide remained soluble under conditions that caused aggregation and membrane association of the 75-kDa propolypeptide. Deletion of COOH-terminal sequences had no effect on the association of pro-PC2 with membranes, whereas a peptide corresponding to amino acids 57-85 of the propeptide was able to partially compete the membrane associated pro-PC2 away from the membranes.
Original languageEnglish
Pages (from-to)18646-50
Number of pages5
JournalThe Journal of Biological Chemistry
Volume269
Issue number28
Publication statusPublished - 15 Jul 1994

Fingerprint

Proprotein Convertase 2
Agglomeration
Calcium
Membranes
Neuroendocrine Cells
Peptides
Secretory Pathway
Sorting
Subtilisin
Secretory Vesicles
Protein Transport
Cell membranes
Processing
Xenopus
Oocytes
Blood Proteins
Membrane Proteins
Proteins
Peptide Hydrolases
Cell Membrane

Keywords

  • Amino Acid Sequence
  • Animals
  • Calcium
  • Cell Membrane
  • Chromatography, Affinity
  • Cytoplasmic Granules
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Precursors
  • Female
  • Humans
  • Hydrogen-Ion Concentration
  • Immunoblotting
  • Macromolecular Substances
  • Molecular Sequence Data
  • Molecular Weight
  • Mutagenesis, Site-Directed
  • Neurosecretory Systems
  • Oocytes
  • Peptides
  • Proprotein Convertase 2
  • Protein Processing, Post-Translational
  • RNA, Messenger
  • Subtilisins
  • Xenopus laevis

Cite this

Calcium- and pH-dependent aggregation and membrane association of the precursor of the prohormone convertase PC2. / Shennan, K I; Taylor, N A; Docherty, K.

In: The Journal of Biological Chemistry, Vol. 269, No. 28, 15.07.1994, p. 18646-50.

Research output: Contribution to journalArticle

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TY - JOUR

T1 - Calcium- and pH-dependent aggregation and membrane association of the precursor of the prohormone convertase PC2

AU - Shennan, K I

AU - Taylor, N A

AU - Docherty, K

PY - 1994/7/15

Y1 - 1994/7/15

N2 - PC2 is a subtilisin-like protease which is thought to be involved in the processing of prohormones and proneuropeptides in neuroendocrine cells. The mature 68-kDa enzyme is generated by intracellular proteolytic processing of a 75-kDa pro-PC2 polypeptide. Neuroendocrine cells contain at least two secretory pathways: the regulated pathway whereby secreted products are concentrated, stored in granules, and released in response to external stimulation of the cell, and the constitutive pathway, whereby secretory and plasma membrane proteins are continuously transported to the cell surface without prior concentration or storage. An important step in the sorting of proteins into these pathways is thought to involve the aggregation of proteins destined for storage granules. To define the mechanisms in the intracellular sorting of PC2 to secretory vesicles, the present study was undertaken to investigate the aggregation and membrane association properties of precursor and mature forms of PC2. Using material expressed in microinjected Xenopus oocytes, it was demonstrated that the 75-kDa pro-PC2 polypeptide undergoes calcium- and acid pH-dependent aggregation. Calcium exhibited an effect on the aggregation of pro-PC2 at pH 7.0 and 6.5, whereas below 6.5 aggregation was independent of calcium. Association of pro-PC2 with membranes was observed at pH 5.5, but not at pH 7.0, 6.5, nor 6.0. The mature 68-kDa PC2 polypeptide remained soluble under conditions that caused aggregation and membrane association of the 75-kDa propolypeptide. Deletion of COOH-terminal sequences had no effect on the association of pro-PC2 with membranes, whereas a peptide corresponding to amino acids 57-85 of the propeptide was able to partially compete the membrane associated pro-PC2 away from the membranes.

AB - PC2 is a subtilisin-like protease which is thought to be involved in the processing of prohormones and proneuropeptides in neuroendocrine cells. The mature 68-kDa enzyme is generated by intracellular proteolytic processing of a 75-kDa pro-PC2 polypeptide. Neuroendocrine cells contain at least two secretory pathways: the regulated pathway whereby secreted products are concentrated, stored in granules, and released in response to external stimulation of the cell, and the constitutive pathway, whereby secretory and plasma membrane proteins are continuously transported to the cell surface without prior concentration or storage. An important step in the sorting of proteins into these pathways is thought to involve the aggregation of proteins destined for storage granules. To define the mechanisms in the intracellular sorting of PC2 to secretory vesicles, the present study was undertaken to investigate the aggregation and membrane association properties of precursor and mature forms of PC2. Using material expressed in microinjected Xenopus oocytes, it was demonstrated that the 75-kDa pro-PC2 polypeptide undergoes calcium- and acid pH-dependent aggregation. Calcium exhibited an effect on the aggregation of pro-PC2 at pH 7.0 and 6.5, whereas below 6.5 aggregation was independent of calcium. Association of pro-PC2 with membranes was observed at pH 5.5, but not at pH 7.0, 6.5, nor 6.0. The mature 68-kDa PC2 polypeptide remained soluble under conditions that caused aggregation and membrane association of the 75-kDa propolypeptide. Deletion of COOH-terminal sequences had no effect on the association of pro-PC2 with membranes, whereas a peptide corresponding to amino acids 57-85 of the propeptide was able to partially compete the membrane associated pro-PC2 away from the membranes.

KW - Amino Acid Sequence

KW - Animals

KW - Calcium

KW - Cell Membrane

KW - Chromatography, Affinity

KW - Cytoplasmic Granules

KW - Electrophoresis, Polyacrylamide Gel

KW - Enzyme Precursors

KW - Female

KW - Humans

KW - Hydrogen-Ion Concentration

KW - Immunoblotting

KW - Macromolecular Substances

KW - Molecular Sequence Data

KW - Molecular Weight

KW - Mutagenesis, Site-Directed

KW - Neurosecretory Systems

KW - Oocytes

KW - Peptides

KW - Proprotein Convertase 2

KW - Protein Processing, Post-Translational

KW - RNA, Messenger

KW - Subtilisins

KW - Xenopus laevis

M3 - Article

VL - 269

SP - 18646

EP - 18650

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

SN - 0021-9258

IS - 28

ER -