Trafficking of a secretory granule membrane protein is sensitive to copper.
We explored the effect of copper availability on the synthesis and trafficking of peptidylglycine alpha-amidating monooxygenase (PAM), an essential cuproenzyme whose catalytic domains function in the lumen of peptide-containing secretory granules. Corticotrope tumor cell lines expressing integral membrane and soluble forms of PAM were depleted of copper using bathocuproinedisulfonic acid or loaded with copper by incubation with CuCl(2). Depleting cellular copper stimulates basal secretion of soluble enzyme produced by endoproteolytic cleavage of PAM in secretory granules and transit of membrane PAM though the endocytic pathway and back into secretory granules. Unlike many cuproenzymes, lack of copper does not lead to instability of PAM. Copper loading decreases cleavage of PAM in secretory granules, secretion of soluble enzyme, and the return of internalized PAM to secretory granules. The trafficking and stability of the soluble, luminal domain of PAM and truncated membrane PAM lacking a cytosolic domain are not affected by copper availability. Taken together, our data demonstrate a role for copper-sensitive cytosolic machinery in directing endocytosed membrane PAM back to secretory granules or to a degradative pathway. The response of PAM to lack of copper suggests that it facilitates copper homeostasis.
De
Mithu
M
University of Connecticut Health Center, Farmington, Connecticut 06030-3401, USA.
Ciccotosto
Giuseppe D
GD
Mains
Richard E
RE
Eipper
Betty A
BA
D000818:Animals; D002122:Calcium Chloride / Q000737:chemistry; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis; D051379:Mice; D008856:Microscopy, Fluorescence; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D022142:Secretory Vesicles / Q000378:metabolism*; D014168:Transferrin / Q000737:chemistry
D009097:Multienzyme Complexes; D014168:Transferrin; D003300:Copper; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D002122:Calcium Chloride