Alpha amidating monooxygenase
Independent colonies (1440) were picked and challenged for growth on medium deficient in Trp, Leu, and His plus 15 m M 3-amino-1,2,4-triazole (Sigma, St.Louis, MO) for 3 d at 30°C, and colonies having poor or no growth were identified.Initially, an aliquot of the 225-base pair (bp) PCR product was subcloned into p Bluescript (Stratagene, La Jolla, CA), and DNA sequence analysis of a representative sample of clones determined that the frequency of point mutations averaged one per truncated CD.The mutated PCR products were digested withstrain DH5α (BRL, Rockville, MD) was used for transformation of the ligation reaction, and the mutagenized p PC97.To distinguish between these two possibilities, we undertook construction of point mutants of PAM that could not interact with known cytosolic domain interactors.
Library DNA (0.9 μg) was used to transform the HF7c strain of, 1997) and selection for double transformants was performed using a synthetic dextrose medium deficient in Trp and Leu.
PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor.
We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2.
By examining At T-20 cells expressing PAM under control of an inducible promoter and after infection with adenovirus encoding PAM, we established a causal relationship between PAM overexpression and impaired operation of the regulated secretory pathway (Ciccotosto, 1999).
Knowing that overexpression of PAM caused these changes, we next sought to distinguish between two possibilities: first, high levels of membrane protein expression might nonspecifically inhibit protein trafficking; second, high levels of PAM expression might selectively interfere with a normal trafficking step.
PAM is one of the few peptide-processing enzymes that spans the secretory granule membrane.