Transmembrane Insertion of theToxoplasma gondiiGRA5 Protein Occurs after Soluble Secretion into the Host Cell

Abstract

The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV.Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite. INTRODUCTIONAmong the varied lifestyles adopted by intracellular parasites is the residence in host cell vacuoles that do not fuse with lysosomes, a strategy exhibited by the protozoan parasite Toxoplasma and the bacterial pathogens Legionella, Chlamydia, and Mycobacteria (Garcia delPortillo and Finlay, 1995). The Toxoplasma-containing vacuole, called the parasitophorous vacuole (PV) 1 , forms by active penetration of the parasite causing invagination of the plasma membrane bilayer (Suss-Toby et al., 1996). This compartment subsequently resists fusion with endosomes and lysosomes by a mechanism that is poorly understood; however, it is clear that the fate of the vacuole is determined at the time of formation (Sibley et al., 1985;Joiner, 1991;Mordue and Sibley, 1997). As the parasite grows and replicates within the cell, it forms a network of tubular membranes that are continuous with the delimiting membrane of the vacuole (Nichols et al., 1983;Sibley et al., 1995).Secretion of proteins stored in apical organelles (micronemes, rhoptries, and dense granules) is a prominent feature of Toxoplasma invasion and presumably contributes both to the nonfusigenic status of the PV and subsequent nutrient uptake by the parasite (Carruthers and Sibley, 1997). Segregated from the endocytic system and cut off from the extracellular fluid, § Corresponding author. E-mail address: mcesbron@infobiogen. fr. † Present address: Laboratory of Molecular Parasitology, Université Libre de Bruxelles, Rhode Saint Genese, Belgium. 1 Abbreviations used: EM, electron microscopy; FBS, fetal bovine serum; F/T, freeze/thaw; HFF, human foreskin fibroblasts; HSP, high-speed pellet; HSS, high-speed supernatant; LSP, lowspeed pellet; LSS, low-speed supernatant; NP-40...

Transmembrane Domain Modulates Sorting of Membrane Proteins in Toxoplasma gondii

“…Furthermore, the extractability of GRA4 may provide an explanation for the previous enigmatic observations that GRA proteins containing transmembrane domains, when liberated from parasites or infected cells by mechanical disruption, fractionate by differential centrifugation as partially soluble and partially membrane-associated molecules (29 -31). In the case of GRA5 these earlier results had been interpreted to suggest that the molecule inserts post-translationally into membranes of the parasitophorous vacuole following secretion as a soluble protein (31). In view of the current results, it is possible instead or in addition that transmembrane-anchored GRA proteins (GRA3-GRA8) are synthesized as membrane proteins that behave anomalously in traditional subcellular fractionation assays.…”

Host cell manipulation by the human pathogen Toxoplasma gondii

“…For this reason, ROP14 is a candidate for the formation of the pore associated with the diffusion of small molecules across the PVM [41]. The dense granule proteins, GRA3, GRA5, GRA7, GRA8 and GRA10, are also localized at the PVM after the invasion of the parasite [56][57][58][59]. Future experiments may reveal their specific roles in host cell interactions via the PVM.…”

GRA Proteins of Toxoplasma gondii: Maintenance of Host-Parasite Interactions across the Parasitophorous Vacuolar Membrane

“…This molecular structure is supported by ultrastructural studies showing the association of the P21 antigen with the PVM. GRA5 is present both in a soluble form and in hydrophobic aggregates [55] in the targeted PVM. GRA5 is secreted as a soluble form into the PV after which becomes stably associated with the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its Cterminus in the vacuole lumen.…”