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-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT), an endoplasmic reticulum enzyme that synthesizes GlcNAc-P-P-dolichol and, thus, catalyzes the committed 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, thermal stability, tunicamycin binding, reticular localization, or consumption of cellular dolichol-P. However, in the absence of residues 398-408, the removal of three additional residues (Phe395-Ser396-Ile397), or their change to Leu395-Met396-Trp397 fully eliminated enzyme expression in vivo. By reattaching residues 398-408 to Leu395-Met396-Trp397, expression was restored. Thus, the carboxyl-terminal region of GPT is essential for stable expression. Either of two sequences (395-397 and 398-408) is sufficient for expression, but neither is necessary. Expression of GPT in the absence of residues 398-408 specifically required the Phe395-Ser396-Ile397 sequence, since most scramble and termination mutations within this sequence were inhibitory. One scramble mutant (Ile395-Ser396-Phe397-Stop398) was enzymatically active, but unusually thermolabile. Thus, the function of Phe395-Ser396-Ile397 may be to stabilize GPT.
Zara
J
J
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Lehrman
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006224:Cricetinae; D015870:Gene Expression; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000235:genetics; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000378:metabolism*
D012333:RNA, Messenger; D017855:Transferases (Other Substituted Phosphate Groups); C023222:UDPacetylglucosamine-dolichyl-phosphate acetylglucosamine-1-phosphate transferase