Transmembrane substrates of type three secretion system injectisomes.

The type three secretion system injectisome of Gram-negative bacterial pathogens injects virulence proteins, called effectors, into host cells. Effectors of mammalian pathogens carry out a range of functions enabling bacterial invasion, replication, immune suppression and transmission. The injectisome secretes two translocon proteins that insert into host cell membranes to form a translocon pore, through which effectors are delivered. A subset of effectors also integrate into infected cell membranes, enabling a unique range of biochemical functions. Both translocon proteins and transmembrane effectors avoid cytoplasmic aggregation and integration into the bacterial inner membrane. Translocated transmembrane effectors locate and integrate into the appropriate host membrane. In this review, we focus on transmembrane translocon proteins and effectors of bacterial pathogens of mammals. We discuss what is known about the mechanisms underlying their membrane integration, as well as the functions conferred by the position of injectisome effectors within membranes.

The Salmonella transmembrane effector SteD hijacks AP1-mediated vesicular trafficking for delivery to antigen-loading MHCII compartments.

SteD is a transmembrane effector of the Salmonella SPI-2 type III secretion system that inhibits T cell activation by reducing the amounts of at least three proteins -major histocompatibility complex II (MHCII), CD86 and CD97 -from the surface of antigen-presenting cells. SteD specifically localises at the trans-Golgi network (TGN) and MHCII compartments; however, the targeting, membrane integration and trafficking of SteD are not understood. Using systematic mutagenesis, we identify distinct regions of SteD that are required for these processes. We show that SteD integrates into membranes of the ER/Golgi through a two-step mechanism of membrane recruitment from the cytoplasm followed by integration. SteD then migrates to and accumulates within the TGN. From here it hijacks the host adaptor protein (AP)1-mediated trafficking pathway from the TGN to MHCII compartments. AP1 binding and post-TGN trafficking require a short sequence in the N-terminal cytoplasmic tail of SteD that resembles the AP1-interacting dileucine sorting signal, but in inverted orientation, suggesting convergent evolution.

CD97 stabilises the immunological synapse between dendritic cells and T cells and is targeted for degradation by the Salmonella effector SteD.

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII ß chain. This requires the Nedd4 family HECT E3 ubiquitin ligase Wwp2 and a tumor-suppressing transmembrane protein adaptor Tmem127. Here, through a proteomic screen of dendritic cells, we found that SteD targets the plasma membrane protein CD97 for degradation by a similar mechanism. SteD enhanced ubiquitination of CD97 on K555 and mutation of this residue eliminated the effect of SteD on CD97 surface levels. We showed that CD97 localises to and stabilises the immunological synapse between dendritic cells and T cells. Removal of CD97 by SteD inhibited dendritic cell-T cell interactions and reduced T cell activation, independently of its effect on MHCII. Therefore, SteD suppresses T cell immunity by two distinct processes.

The Tumour Suppressor TMEM127 Is a Nedd4-Family E3 Ligase Adaptor Required by Salmonella SteD to Ubiquitinate and Degrade MHC Class II Molecules.

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII ß chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4+ T cell activation. We found that TMEM127 contains a canonical PPxY motif, which was required for binding to WWP2. SteD bound to TMEM127 and enabled TMEM127 to interact with and induce ubiquitination of mature MHCII. Furthermore, SteD also underwent TMEM127- and WWP2-dependent ubiquitination, which both contributed to its degradation and augmented its activity on mMHCII.

SrcA is a chaperone for the Salmonella SPI-2 type three secretion system effector SteD.

Effector proteins of type three secretion systems (T3SS) often require cytosolic chaperones for their stabilization, to interact with the secretion machinery and to enable effector delivery into host cells. We found that deletion of srcA, previously shown to encode a chaperone for the Salmonella pathogenicity island 2 (SPI-2) T3SS effectors SseL and PipB2, prevented the reduction of mature Major Histocompatibility Complex class II (mMHCII) from the surface of antigen-presenting cells during Salmonella infection. This activity was shown previously to be caused by the SPI-2 T3SS effector SteD. Since srcA and steD are located in the same operon on the Salmonella chromosome, this suggested that the srcA phenotype might be due to an indirect effect on SteD. We found that SrcA is not translocated by the SPI-2 T3SS but interacts directly and forms a stable complex with SteD in bacteria with a 2 : 1 stoichiometry. We found that SrcA was not required for SPI-2 T3SS-dependent, neutral pH-induced secretion of either SseL or PipB2 but was essential for secretion of SteD. SrcA therefore functions as a chaperone for SteD, explaining its requirement for the reduction in surface levels of mMHCII.

Quantification of cytosolic interactions identifies Ede1 oligomers as key organizers of endocytosis.

Clathrin-mediated endocytosis is a highly conserved intracellular trafficking pathway that depends on dynamic protein-protein interactions between up to 60 different proteins. However, little is known about the spatio-temporal regulation of these interactions. Using fluorescence (cross)-correlation spectroscopy in yeast, we tested 41 previously reported interactions in vivo and found 16 to exist in the cytoplasm. These detected cytoplasmic interactions included the self-interaction of Ede1, homolog of mammalian Eps15. Ede1 is the crucial scaffold for the organization of the early stages of endocytosis. We show that oligomerization of Ede1 through its central coiled coil domain is necessary for its localization to the endocytic site and we link the oligomerization of Ede1 to its function in locally concentrating endocytic adaptors and organizing the endocytic machinery. Our study sheds light on the importance of the regulation of protein-protein interactions in the cytoplasm for the assembly of the endocytic machinery in vivo.

The initiation of clathrin-mediated endocytosis is mechanistically highly flexible.

Clathrin-mediated endocytosis is driven by a complex machinery of proteins, which assemble in a regular order at the plasma membrane. The assembly of the endocytic machinery is conventionally thought to be a continuous process of mechanistically dependent steps, starting from a defined initiation step. Indeed, several initiation mechanisms involving single proteins have been proposed in mammalian cells. Here, we demonstrate that the initiation mechanism of endocytosis is highly flexible. We disrupted the long early phase of endocytosis in yeast by deleting seven genes encoding early endocytic proteins. Surprisingly, membrane uptake and vesicle budding dynamics were largely normal in these mutant cells. Regulated cargo recruitment was, however, defective. In addition, different early endocytic proteins were able to initiate vesicle budding when anchored to a plasma membrane domain where endocytosis does not normally take place. Our results suggest that the cargo-recruiting early phase is not mechanistically required for vesicle budding, but early-arriving proteins can recruit the budding machinery into position at the plasma membrane. Separable early and late phases allow for a robust process of vesicle budding to follow from variable initiation mechanisms. Such a modular design could easily adapt and evolve to respond to different cellular requirements.

Review series: From uncertain beginnings: initiation mechanisms of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is a central and well-studied trafficking process in eukaryotic cells. How this process is initiated is likely to be a critical point in regulating endocytic activity spatially and temporally, but the underlying mechanisms are poorly understood. During the early stages of endocytosis three components-adaptor and accessory proteins, cargo, and lipids-come together at the plasma membrane to begin the formation of clathrin-coated vesicles. Although different models have been proposed, there is still no clear picture of how these three components cooperate to initiate endocytosis, which may indicate that there is some flexibility underlying this important event.

ATP hydrolysis is required for relocating cohesin from sites occupied by its Scc2/4 loading complex.

BACKGROUND: The Cohesin complex that holds sister chromatins together until anaphase is comprised of three core subunits: Smc1 and Smc3, two long-rod-shaped proteins with an ABC-like ATPase head (nucleotide-binding domain [NBD]) and a dimerization domain linked by a 50 nm long intramolecular antiparallel coiled-coil, and Scc1, an α-kleisin subunit interconnecting the NBD domains of Smc1 and Smc3. Cohesin's stable association with chromosomes is thought to involve entrapment of chromatin fibers by its tripartite Smc1-Smc3-Scc1 ring via a poorly understood mechanism dependent on a separate Scc2/4 loading complex. A key issue concerns where entrapment initially takes place: at sites where cohesin is found stably associated or at distinct "loading" sites from which it translocates. RESULTS: In this study, we find transition state mutant versions (Smc1E1158Q and SmcE1155Q) defective in disengagement of their nucleotide binding domains (NBDs), unlike functional cohesin, colocalize with Scc2/4 at core centromeres, sites that catalyze wild-type cohesin's recruitment to sequences 20 kb or more away. In addition to Scc2/4, the unstable association of transition state complexes with core centromeres requires Scc1's association with Smc1 and Smc3 NBDs, ATP-driven NBD engagement, cohesin's Scc3 subunit, and its hinge domain. CONCLUSION: We propose that cohesin's association with chromosomes is driven by two key events. NBD engagement driven by ATP binding produces an unstable association with specific loading sites like core centromeres, whereas subsequent ATP hydrolysis triggers DNA entrapment, which permits translocation along chromatin fibers.