The intracellular parasite Toxoplasma gondii resides within a membrane-bound parasitophorous vacuole (PV) and secretes an array of proteins to establish this replicative niche. It has been shown previously that Toxoplasma secretes kinases and that numerous proteins are phosphorylated after secretion. Here, we assess the role of the phosphorylation of strand-forming protein 1 (SFP1) and the related protein GRA29, two secreted proteins with unknown function. We show that both proteins form stranded structures in the PV that are independent of the previously described intravacuolar network or actin. SFP1 and GRA29 can each form these structures independently of other Toxoplasma secreted proteins, although GRA29 appears to regulate SFP1 strands. We show that an unstructured region at the C termini of SFP1 and GRA29 is required for the formation of strands and that mimicking the phosphorylation of this domain of SFP1 negatively regulates strand development. When tachyzoites convert to chronic-stage bradyzoites, both proteins show a dispersed localization throughout the cyst matrix. Many secreted proteins are reported to dynamically redistribute as the cyst forms, and secreted kinases are known to play a role in cyst formation. Using quantitative phosphoproteome and proteome analyses comparing tachyzoite and early bradyzoite stages, we reveal widespread differential phosphorylation of secreted proteins. While we found no direct evidence for phosphorylation playing a dominant role for SFP1/GRA29 redistribution in the cyst, these data support a model in which secreted kinases and phosphatases contribute to the regulation of secreted proteins during stage conversion. IMPORTANCE Toxoplasma gondii is a common parasite that infects up to one-third of the human population. Initially, the parasite grows rapidly, infecting and destroying cells of the host, but subsequently switches to a slow-growing form and establishes chronic infection. In both stages, the parasite lives within a membrane-bound vacuole within the host cell, but in the chronic stage, a durable cyst wall is synthesized, which provides protection to the parasite during transmission to a new host. Toxoplasma secretes proteins into the vacuole to build its replicative niche, and previous studies identified many of these proteins as phosphorylated. We investigate two secreted proteins and show that a phosphorylated region plays an important role in their regulation in acute stages. We also observed widespread phosphorylation of secreted proteins when parasites convert from acute to chronic stages, providing new insight into how the cyst wall may be dynamically regulated.

The ability of cells to mount an interferon response to virus infections depends on intracellular nucleic acid sensing pattern recognition receptors (PRRs). RIG-I is an intracellular PRR that binds short double stranded viral RNAs to trigger MAVS-dependent signalling. The RIG-I/MAVS signalling complex requires the coordinated activity of multiple kinases and E3 ubiquitin ligases to activate the transcription factors that drive type I and type III interferon production from infected cells. The linear ubiquitin chain assembly complex (LUBAC) regulates the activity of multiple receptor signalling pathways in both ligase-dependent and -independent ways. Here, we show that the three proteins that constitute LUBAC have separate functions in regulating RIG-I signalling. Both HOIP, the E3 ligase capable of generating M1-ubiquitin chains, and LUBAC accessory protein HOIL-1 are required for viral RNA sensing by RIG-I. The third LUBAC component, SHARPIN, is not required for RIG-I signalling. These data cement the role of LUBAC as a positive regulator of RIG-I signalling and as an important component of antiviral innate immune responses.

cyst wall is synthesized that provides protection to the parasite during transmission to a new 46 host. Toxoplasma secretes proteins into the vacuole to build its replicative niche and previous 47 studies identified many of these proteins as phosphorylated. We investigate two secreted 48 proteins and show that phosphorylation plays an important role in their regulation. We also 49 observed widespread phosphorylation of secreted proteins when parasites convert from acute 50 to chronic stages, providing new insight into how the cyst wall may be dynamically regulated. 51During infection Toxoplasma secretes proteins that subvert host cell signalling and develops its 67 replicative niche within the PV (6, 7). The repertoire of secreted proteins is thought to include 68 up to 200 proteins (ToxoDB, LOPIT dataset) including 50 kinases/pseudokinases (8), although 69 the functions of many of these remain unknown. These proteins are secreted from the 70 rhoptries or dense granules and are called ROPs or GRAs, respectively. Secreted proteins 71 extensively modify the PV allowing selective passage of molecules and proteins across the PV 72 membrane. For example, GRA17 and GRA23 form pores in the PV membrane allowing the 73 passage of small molecules into the PV (9), while the MYR complex mediates the transport of 74 secreted proteins to the host cell (10, 11). Additionally, the parasite develops a complex set of 75 membrane structures within the PV including GRA7-lined invaginations (12) and a network of 76 tubules called the intravacuolar network (IVN) (13). The formation of this network is dependent 77 on GRA2 and the accessory protein GRA6, and is required for full virulence in mice (14,15). 78Both structures have been reported to play a role in scavenging nutrients from the host cell 79 with the uptake of host cell proteins (16), lipid droplets (17), and endolysosomal compartments 80 (18). 81Previous phosphoproteome analysis revealed that a large number of Toxoplasma secreted 82 proteins are phosphorylated after their release (19), raising the intriguing possibility that their 83 function is dynamically regulated. Indeed, the PV localised kinase WNG1 was shown to 84 contribute to formation of a functional IVN (20). Furthermore, it was recently shown that IVN 85 associated GRAs show dynamic localisation patterns as the parasite remodels the PV to form 86 the chronic stage cyst (21). Here we analyse two secreted, strand forming proteins that are 87 phosphorylated after secretion and show that phosphorylation regulates their localisation, 88 disrupting normal strand formation. Both proteins disperse in chronic stage cysts, so we 89

Protein pseudokinases comprise a considerable fraction of the human kinome, as well as other forms of life. Recent studies have demonstrated that their lack of key catalytic residues compared to their kinase counterparts does not negate their ability to intersect with molecular signal transduction.