Role of the carboxyl terminus in stable expression of hamster UDP- GlcNAc:Dolichol-P GlcNAc-1-P transferase

Jane Zara, Mark A. Lehrman

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)19108-19115
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number29
StatePublished - Jan 1 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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