Sevlever, Daniel

Glycosylphosphatidylinositol (GPI)-anchored proteins are resistant to solubilization with Triton X-100 at 4 °C, and they can be recovered in Triton-insoluble membranes (TIMs) that float to a characteristic buoyant density. Because the GPI structure itself has been shown to target GPI-anchored proteins to TIMs, we investigated the association of GPI-anchor intermediates with TIMs. GPI-anchor biosynthesis involves a pathway of some 10 steps that take place in the endoplasmic reticulum (ER). These intermediates include glucosaminyl-acylphosphatidylinositol [GlcN-(acyl)PI] and later mannosylated GPIs, denoted H6, H7 and H8, that are present not only in the ER but also in other cell compartments, including the plasma membrane. At least two-thirds of the GlcN-(acyl)PI in HeLa D cells and mannosylated GPIs in K562 cells were found in TIMs. Although previous reports have considered TIMs to be derived primarily from the plasma membrane, we recovered TIMs from subcellular fractions enriched in ER membranes. The ER marker calnexin and GPI-anchored proteins as well as N-acetylglucosaminyl-phosphatidylinositol and mannosylated GPIs were present in ER-TIMs. Interestingly, GlcN-PI and H7 were less enriched in ER-TIM than the other GPIs, suggesting that ER-TIMs might reflect a compartmentalization of the GPI-anchor biosynthetic pathway in the ER.

GPI-PLD [glycosylphosphatidylinositol (GPI)-specific phospholipase D (PLD)] is a secreted mammalian enzyme that specifically cleaves GPI-anchored proteins. In addition, the enzyme has been shown to cleave GPI anchor intermediates in cell lysates. The biosynthesis of the GPI anchor is well characterized; however, the mechanisms by which the levels of GPI anchor intermediates are regulated are still unknown. To investigate whether GPI-PLD plays a role in this regulation, we isolated stable HeLa cells overexpressing the enzyme. GPI-PLD-HeLa (GPI-PLD-transfected HeLa) cells showed a 3-fold increase in intracellular GPI-PLD activity and drastically decreased the levels of GPI-anchored proteins when compared with untransfected HeLa controls. Intracellular cleavage of GPI-anchored proteins has been suggested to occur early in the secretory pathway and, in agreement with this proposal, GPI-PLD activity in GPI-PLD-HeLa cells was detected not only in the endoplasmic reticulum and Golgi apparatus, but also in the plasma membrane. The enzyme was also active in lipid rafts, membrane microdomains in which GPI-anchored proteins and GPI anchor intermediates are concentrated, indicating that intracellular GPI-PLD cleavage may also occur in this compartment. Pulse–chase paradigms revealed the turnover rate of the last intermediate of the GPI anchor pathway in GPI-PLD-HeLa cells to be accelerated compared with the controls. Furthermore, 1,10-phenanthroline, a GPI-PLD inhibitor, reversed this effect. Our studies demonstrated that GPI-PLD can cleave not only GPI-anchored proteins, but also GPI anchor intermediates intracellularly. This observation opens the possibility that GPI-PLD can influence the steady-state levels of GPI-anchored proteins by hydrolysing the anchor before and after its attachment to proteins.