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 mitochondriaassociated 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 GlcNAc-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.

Colocalization of SCD1 and DGAT2: implying preference for endogenous monounsaturated fatty acids in triglyceride synthesis

“…As expected, no activity was detected in mitochondria. The specific enzyme activity was z2-fold higher in ER than in MAM, which is in accordance with the characteristic 2-fold difference reported by other groups (16,17,22). Figure 1E shows the specific activity of the mitochondrion marker cytochrome C oxidase in the different subcellular fractions.…”

“…The cells were incubated at 37jC in a humidified atmosphere with 5% CO 2 . The transfection of the different constructs into the HeLa cells was performed as described (17). Briefly, HeLa cells were cultured to 80-100% confluence.…”

N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase from Entamoeba histolytica

“…Human PIG-L is a 252-amino-acid-long protein with a large cytoplasmic tail (7) showing 77% homology with rat PIG-L (8). Unlike GPI12, it has two ER localization signals and is uniformly localized on the ER as well as in the mitochondria-associated ER membrane (9). Among PIG-L proteins, there appears to be species-specific differences not only in terms of location and conformation but also in terms of specificity of the active sites.…”

Presenilins Are Enriched in Endoplasmic Reticulum Membranes Associated with Mitochondria

“…16 More than two dozen proteins are concentrated in MAM (see Supplemental Table S1 at http://ajp.amjpathol.org), 15,[17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] including proteins involved in calcium homeostasis (eg, inositol triphosphate receptor isoform 3), in lipid metabolism (eg, fatty acid co-A ligase 4 [FACL4]), in intermediate metabolism (eg, glucose-6-phosphatase), in cholesterol metabolism (eg, acyl-coenzyme A:cholesterol acyltransferase 1), and in the transfer of lipids between the ER and mitochondria. A few nonenzymatic proteins are also concentrated in MAM (see Supplemental Table S1 at http://ajp.amjpathol.org), 15,[17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] suggesting that it is a domain of the ER with specialized functions. Contacts between the two organelles are maintained by MAMassociated proteins, such as phosphofurin acidic cluster sorting protein 2, which controls the apposition of mitochondria with the ER and which appears to stabilize and regulate the interaction of ER and mitochondria.…”