MUTAGENESIS OF RAT DOPAMINE-BETA-HYDROXYLASE - EXAMINATION IN CELL-FREE SYSTEM

Dopamine beta-hydroxylase (DBH; EC 1.14.17.1) exists as membrane-bound and soluble forms in neurosecretory vesicles. The features of DBH req uired for glycosylation and incorporation into membranes were studied in a cell-free system. Translation of full-length DBH with microsomal membranes generated two glycosylated products (GH and GL) depending on the magnesium concentration. Carboxyl-terminal, in contrast to aminot erminal, truncations gave translation products that were glycosylated by microsomal membranes. Site-directed mutants were generated with the second AUG codon and the region of a putative signal sequence cleavag e site modified. Translation without membranes indicated that the seco nd AUG is not used to initiate translation. The mutant with Glu(41) -- > Leu(41) and Ser(43) --> Thr(43) yielded only the GH form with membra nes, whereas mutation of Ser(43) --> Ala(43) generated both GH and GL forms. Both glycosylated forms comigrated with the microsomal membrane s on sucrose gradients. Endoglycosidase H digestion indicated that the differences between the GH and GL forms are not due to the sugar moie ty. The results suggest a role for cleavage of a signal sequence in th e formation of different forms of DBH. The possibility that these muta tions change the secondary structure near the signal cleavage site, af fecting processing, is discussed.