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The immunity-related GTPases (IRGs) constitute an interferon-induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion-driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild-type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

Toxoplasma gondii is a widespread protozoan parasite infecting all warm-blooded animals and causing disease in immunocompromised individuals and in utero. The pathogen depends on the intracellular life style residing in a specialized organelle, termed parasitophorous vacuole (PV), in order to survive and replicate. Cell-autonomous immunity, regulated by IFNg, is essential to restrict growth of the parasite in mouse and man. In mouse cells, resistance to T. gondii is mediated by the family of IFN-inducible IRG proteins (p47 GTPases). Upon infection several IRG proteins associate with the PV and participate in vesiculation of the PV membrane leading to demise of the parasite and necrotic death of the cell. Until now, despite intrinsic interest, the phenomenon of IRG protein loading onto PV has not been well studied. In human cells, depletion of cellular tryptophan by IDO has been reported as the major mechanism of restriction of T. gondii proliferation exerted by IFNg. IDO-independent restriction of T. gondii growth has been reported but not followed up. The process of IRG protein association with T. gondii vacuoles emerged as a rapid, organized and diffusion-driven event where multiple resistance proteins sequentially bind to the vacuolar membrane forming homomeric and heteromeric complexes. The efficient loading process requires the autophagy factor Atg5 regulating correct localisation of IRG proteins prior to infection. Virulent strains of T. gondii inhibit IRG protein association with PVs independently of individual virulence determinants ROP5, ROP16 and ROP18. Impaired loading of IRG proteins onto T. gondii vacuoles leads to reduced elimination of the parasite in IFNg-stimulated cells, underlining the importance of the phenomenon in cell-autonomous immunity to T. gondii. This study shows that density of cultured cells is the key factor in determining the mode of T. gondii control in primary human cells. IFNg-induced, proliferating cells control parasite replication independently of IDO. Consistent with absence of the IRG system in humans, the vacuolar membrane and enclosed parasite remain intact in IFNg-induced human cells. However, similar to mouse cells, human cells die by necrosis, when infected with T. gondii and stimulated with IFNg. This may not only suppress parasite growth but also amplify an antimicrobial response due to release of the proinflammatory �danger� signal HMGB1. Programmed necrosis could be efficiently suppressed at high densities of primary cells and in HeLa cell line, and tryptophan depletion becomes the main source of T. gondii control.

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication(1). Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process(2-8). However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans(9) are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.