Subcellular localization and targeting of N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the second enzyme in the glycosylphosphatidylinositol biosynthetic pathway.

Abstract

The second step in glycosylphosphatidylinositol biosynthesis is the de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) catalyzed by N-acetylglucosaminylphosphatidylinositol deacetylase (PIG-L). Previous studies of mouse thymoma cells showed that GlcNAc-PI de-N-acetylase activity is localized to the endoplasmic reticulum (ER) but enriched in a mitochondria-associated ER membrane (MAM) domain. Because PIG-L has no readily identifiable ER sorting determinants, we were interested in learning how PIG-L is localized to the ER and possibly enriched in MAM. We used HeLa cells transiently or stably expressing epitope-tagged PIG-L variants or chimeric constructs composed of elements of PIG-L fused to Tac antigen, a cell surface protein. We first analyzed the subcellular distribution of PIG-L and Glc-NAc-PI-de-N-acetylase activity and then studied the localization of Tac-PIG-L chimeras to identify sequence elements in PIG-L responsible for its subcellular localization. We show that human PIG-L is a type I membrane protein with a large cytoplasmic domain and that, unlike the result with mouse thymoma cells, both PIG-L and GlcNAc-PI-de-N-acetylase activity are uniformly distributed between ER and MAM in HeLa cells. Analyses of a series of Tac-PIG-L chimeras indicated that PIG-L contains two ER localization signals, an independent retention signal located between residues 60 and 88 of its cytoplasmic domain and another weak signal in the luminal and transmembrane domains that functions autonomously in the presence of membrane proximal residues of the cytoplasmic domain that themselves lack any retention information. We conclude that PIG-L, like a number of other ER membrane proteins, is retained in the ER through a multi-component localization signal rather than a discrete sorting motif.

• Authors: Anita Pottekat, Anant K Menon
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 279 Issue 16 Pg. 15743-51 (Apr 16 2004) ISSN: 0021-9258 [Print] United States
• PMID: 14742432 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Epitopes
• Glycosylphosphatidylinositols
• N-acetylglucosaminylphosphatidylinositol
• Phosphatidylinositols
• Amidohydrolases
• N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase
• Acetylglucosamine

Topics

• Acetylglucosamine (analogs & derivatives, metabolism)
• Amidohydrolases (metabolism)
• Animals
• Cricetinae
• Endoplasmic Reticulum (metabolism)
• Epitopes (metabolism)
• Glycosylphosphatidylinositols (metabolism)
• HeLa Cells
• Humans
• Mice
• Phosphatidylinositols (metabolism)
• Protein Transport
• Signal Transduction