Response of an integral granule membrane protein to changes in pH.
A key feature of the regulated secretory pathway in neuroendocrine cells is lumenal pH, which decreases between trans-Golgi network and mature secretory granules. Because peptidylglycine alpha-amidating monooxygenase (PAM) is one of the few membrane-spanning proteins concentrated in secretory granules and is a known effector of regulated secretion, we examined its sensitivity to pH. Based on antibody binding experiments, the noncatalytic linker regions between the two enzymatic domains of PAM show pH-dependent conformational changes; these changes occur in the presence or absence of a transmembrane domain. Integral membrane PAM-1 solubilized from rat anterior pituitary or from transfected AtT-20 cells aggregates reversibly at pH 5.5 while retaining enzyme activity. Over 35% of the PAM-1 in anterior pituitary extracts aggregates at pH 5.5, whereas only about 5% aggregates at pH 7.5. PAM-1 recovered from secretory granules and endosomes is highly responsive to low pH-induced aggregation, whereas PAM-1 recovered from a light, intracellular recycling compartment is not. Mutagenesis studies indicate that a transmembrane domain is necessary but not sufficient for low pH-induced aggregation and reveal a short lumenal, juxtamembrane segment that also contributes to pH-dependent aggregation. Taken together, these results demonstrate that several properties of membrane PAM serve as indicators of granule pH in neuroendocrine cells.
Bell-Parikh
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LC
Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Eipper
B A
BA
Mains
R E
RE
D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D011992:Endosomes / Q000378:metabolism; D005091:Exons; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010902:Pituitary Gland / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism; D014162:Transfection
D003902:Detergents; D009097:Multienzyme Complexes; D020033:Protein Isoforms; D013395:Sucrose; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase