Novel proteins that interact with the COOH-terminal cytosolic routing determinants of an integral membrane peptide-processing enzyme.
The steady state distribution of membrane forms of peptidylglycine alpha-amidating monooxygenase (PAM) in the secretory pathway of neurons and endocrine cells depends on signals in its cytosolic COOH-terminal domain (CD). Mutagenesis studies yielded catalytically active PAM proteins that are not properly localized or internalized. Employing the yeast two-hybrid system, we isolated two distinct cDNAs whose protein products showed a strong interaction with the CD of PAM. The interaction of these novel PAM COOH-terminal interactor proteins (P-CIPs) did not occur with a misrouted CD mutant as bait in the yeast system. Both proteins, P-CIP2 and P-CIP10, were expressed as fusion proteins that interacted in vitro with solubilized integral membrane PAM. P-CIP2 was homologous to several serine/threonine and dual specificity protein kinases, while P-CIP10 contained spectrin-like repeats. Endogenous P-CIP2 was localized to the Golgi region of AtT-20 corticotrope tumor cells, and expression of integral membrane PAM disrupted the distribution of endogenous P-CIP2. Both P-CIP2 and P-CIP10 mRNAs were found to be expressed in rat brain neurons also expressing PAM proteins. P-CIP2 and P-CIP10 may be members of a family of cytosolic proteins involved in the routing of membrane proteins that function in the regulated secretory pathway.
Alam
M R
MR
Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Caldwell
B D
BD
Johnson
R C
RC
Darlington
D N
DN
Mains
R E
RE
Eipper
B A
BA
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases; D003600:Cytosol / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D014018:Tissue Distribution
D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; C102365:Kalrn protein, rat; D009097:Multienzyme Complexes; C102364:Uhmk1 protein, rat; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases