Jolanta Vidugiriene

A Cell-free Assay for Glycosylphosphatidylinositol Anchoring in African Trypanosomes DEMONSTRATION OF A TRANSAMIDATION REACTION MECHANISM

We established an in vitro assay for the addition of glycosyl-phosphatidylinositol (GPI) anchors to proteins using procyclic trypanosomes engineered to express GPI-anchored variant surface glycoprotein (VSG). The assay is based on the premise that small nucleophiles, such as hydrazine, can substitute for the GPI moiety and effect displacement of the membrane anchor of a GPI-anchored protein or pro-protein causing release of the protein into the aqueous medium. Cell membranes containing pulse-radiolabeled VSG were incubated with hydrazine, and the VSG released from the membranes was measured by carbonate extraction, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis/fluorography. Release of VSG was time- and temperature-dependent, was stimulated by hydrazine, and occurred only for VSG molecules situated in early compartments of the secretory pathway. No nucleophile-induced VSG release was seen in membranes prepared from cells expressing a VSG variant with a conventional transmembrane anchor (i.e. a nonfunctional GPI signal sequence). Pro-VSG was shown to be a substrate in the reaction by assaying membranes prepared from cells treated with mannosamine, a GPI biosynthesis inhibitor. When a biotinylated derivative of hydrazine was used instead of hydrazine, the released VSG could be precipitated with streptavidin-agarose, indicating that the biotin moiety was covalently incorporated into the protein. Hydrazine was shown to block the C terminus of the released VSG hydrazide because the released material, unlike a truncated form of VSG lacking a GPI signal sequence, was not susceptible to proteolysis by carboxypeptidases. These results firmly establish that the released material in our assay is VSG hydrazide and strengthen the proof that GPI anchoring proceeds via a transamidation reaction mechanism. The reaction could be inhibited with sulfhydryl alkylating reagents, suggesting that the transamidase enzyme contains a functionally important sulfhydryl residue.

Segregation of Glycosylphosphatidylinositol Biosynthetic Reactions in a Subcompartment of the Endoplasmic Reticulum

Glycosylphosphatidylinositols (GPIs) are synthesized in the endoplasmic reticulum (ER) via the sequential addition of monosaccharides, fatty acid, and phosphoethanolamine(s) to phosphatidylinositol (PI). While attempting to establish a mammalian cell-free system for GPI biosynthesis, we found that the assembly of mannosylated GPI species was impaired when purified ER preparations were substituted for unfractionated cell lysates as the enzyme source. To explore this problem we analyzed the distribution of the various GPI biosynthetic reactions in subcellular fractions prepared from homogenates of mammalian cells. The results indicate the following: (i) the initial reaction of GPI assembly, i.e. the transfer of GlcNAc to PI to form GlcNAc-PI, is uniformly distributed in the ER; (ii) the second step of the pathway, i.e.de-N-acetylation of GlcNAc-PI to yield GlcN-PI, is largely confined to a subcompartment of the ER that appears to be associated with mitochondria; (iii) the mitochondria-associated ER subcompartment is enriched in enzymatic activities involved in the conversion of GlcN-PI to H5 (a singly mannosylated GPI structure containing one phosphoethanolamine side chain; and (iv) the mitochondria-associated ER subcompartment, unlike bulk ER, is capable of the de novosynthesis of H5 from UDP-GlcNAc and PI. The confinement of these GPI biosynthetic reactions to a domain of the ER provides another example of the compositional and functional heterogeneity of the ER. The implications of these findings for GPI assembly are discussed.

Cell Surface Display and Intracellular Trafficking of Free Glycosylphosphatidylinositols in Mammalian Cells

In addition to serving as membrane anchors for cell surface proteins, glycosylphosphatidylinositols (GPIs) can be found abundantly as free glycolipids in mammalian cells. In this study we analyze the subcellular distribution and intracellular transport of metabolically radiolabeled GPIs in three different cell lines. We use a variety of membrane isolation techniques (subcellular fractionation, plasma membrane vesiculation to isolate pure plasma membrane fractions, and enveloped viruses to sample cellular membranes) to provide direct evidence that free GPIs are not confined to their site of synthesis, the endoplasmic reticulum, but can redistribute to populate other subcellular organelles. Over short labeling periods (2.5 h), radiolabeled GPIs were found at similar concentration in all subcellular fractions with the exception of a mitochondria-enriched fraction where GPI concentration was low. Pulse-chase experiments over extended chase periods showed that although the total amount of cellular radiolabeled GPIs decreased, the plasma membrane complement of labeled GPIs increased. GPIs at the plasma membrane were found to populate primarily the exoplasmic leaflet as detected using periodate oxidation of the cell surface. Transport of GPIs to the cell surface was inhibited by Brefeldin A and blocked at 15 °C, suggesting that GPIs are transported to the plasma membrane via a vesicular mechanism. The rate of transport of radiolabeled GPIs to the cell surface was found to be comparable with the rate of secretion of newly synthesized soluble proteins destined for the extracellular space.

Soluble constituents of the ER lumen are required for GPI anchoring of a model protein.

Transfer of a glycosylphosphatidylinositol (GPI) anchor to proteins carrying a C‐terminal GPI‐directing signal sequence occurs after protein translocation across the endoplasmic reticulum (ER). We describe the translocation and GPI modification of a model protein, preprominiPLAP, in ER microsomes depleted of lumenal content by high pH washing. In untreated microsomes preprominiPLAP was processed to prominiPLAP and GPI‐anchored miniPLAP. Both products were fully translocated, since they resisted proteinase K treatment of the microsomes, and both behaved as membrane proteins by the carbonate extraction criterion. Microsomes depleted of lumenal content were able to translocate and process preprominiPLAP to give protease‐protected prominiPLAP, but were unable to convert prominiPLAP to miniPLAP. Loss of GPI anchoring capacity occurred with a wash of pH > 9.5. If the alkaline wash was performed after formation of prominiPLAP conversion to miniPLAP was relatively unimpaired. The results indicate that constituents of the ER lumen, possibly chaperones interacting with the proprotein and/or the GPI anchor precursor, are required in the initial steps of GPI anchoring.

[38] Biosynthesis of glycosylphosphatidylinositol anchors

Publisher Summary This chapter describes radiolabeling techniques for studying glycosylphosphatidylinositol (GPI) biosynthesis by cell lysates, subcellular fractions, and permeabilized cells. GPIs are synthesized by all eukaryotic cells and are typically found covalently linked to cell surface glycoproteins. GPIs serve as an important alternative mechanism for anchoring proteins to cell membranes and a wide spectrum of functionally diverse proteins rely on a GPI anchor for membrane association. The GPI moiety is synthesized in the endoplasmic reticulum and then transferred to proteins containing a carboxyl-terminal GPI-attachment signal sequence. The methods described in the chapter are used to generate well-characterized radiolabeled GPI biosynthetic intermediates and determine the topological arrangement of the lipids in the membrane bilayer. The completed GPI structure in bloodstream trypanosomes undergoes fatty acid remodeling reactions in which the glycerol-linked fatty acids are replaced by myristic acid.