Alpha amidating monooxygenase
To distinguish between these two possibilities, we undertook construction of point mutants of PAM that could not interact with known cytosolic domain interactors.
Elimination of this paradoxical effect of PAM by mutation would suggest selective interference with a normal trafficking step.
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.
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.
We suggest that P-CIP2, through its interaction with and phosphorylation of the cytosolic domain of PAM, plays a key role in the formation of immature secretory granules from , 1996) was used.
A set of primers (sense, 5′-AGGTCGACCCGGTGGAAAAAATCAAG-3′; and antisense, 5′-GGACTAGTAAGACTCAGTTCCGTCGTC-3′), complementary to the c DNA at both the 5′ and 3′ ends of the truncated PAM-CD, was used to perform low-fidelity polymerase chain reaction (PCR) random mutagenesis (Leung, 1989).