ROLE OF THE CARBOXYL-TERMINUS IN STABLE EXPRESSION OF HAMSTER UDP-GLCNAC-DOLICHOL-P GLCNAC-1-P TRANSFERASE

In order to examine the function of the carboxyl terminus of UDP-GlcNA c:dolichol-P GlcNAc-1-P transferase (GPT), an endoplasmic reticulum en zyme that synthesizes GlcNAc-P-P-dolichol and, thus, catalyzes the com mitted step for N-linked glycosylation, a series of carboxyl-terminal truncation mutations was examined. Removal of the last 11 amino acids (398-408) from GPT had no significant effect on catalytic activity, th ermal stability, tunicamycin binding, reticular localization, or consu mption of cellular dolichol-P. However, in the absence of residues 398 -408, the removal of three additional residues (Phe(395)-Ser(396)-Ile( 397)), or their change to Leu(395)-Met(396)-Trp(397) fully eliminated enzyme expression in vivo. By reattaching residues 398-408 to Leu(395) -Met(396)-Trp(397), expression was restored. Thus, the carboxyl-termin al region of GPT is essential for stable expression. Either of two seq uences (395-397 and 398-408) is sufficient for expression, but neither is necessary. Expression of GPT in the absence of residues 398-408 sp ecifically required the Phe(395)-Ser(396)-Ile(397) sequence, since mos t scramble and termination mutations within this sequence were inhibit ory. One scramble mutant (Ile(395)-Ser(396)-Phe(397)-Stop(398)) was en zymatically active, but unusually thermolabile. Thus, the function of Phe(395)-Ser(396)-Ile(397) may be to stabilize GPT.