Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway

Terminally misfolded proteins are selectively recognized and cleared by the endoplasmic reticulum-associated degradation (ERAD) pathway. SEL1L, a component of the ERAD machinery, plays an important role in selecting and transporting ERAD substrates for degradation. We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1Lcent). Strikingly, SEL1Lcent forms a homodimer with two-fold symmetry in a head-to-tail manner. Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. We identified that the full-length SEL1L forms a self-oligomer through the SEL1Lcent domain in mammalian cells. Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery.ope

Ras GTPase-like protein MglA, a controller of bacterial social-motility in Myxobacteria, has evolved to control bacterial predation by Bdellovibrio

Bdellovibrio bacteriovorus invade Gram-negative bacteria in a predatory process requiring Type IV pili (T4P) at a single invasive pole, and also glide on surfaces to locate prey. Ras-like G-protein MglA, working with MglB and RomR in the deltaproteobacterium Myxococcus xanthus, regulates adventurous gliding and T4P-mediated social motility at both M. xanthus cell poles. Our bioinformatic analyses suggested that the GTPase activating protein (GAP)-encoding gene mglB was lost in Bdellovibrio, but critical residues for MglABd GTP-binding are conserved. Deletion of mglABd abolished prey-invasion, but not gliding, and reduced T4P formation. MglABd interacted with a previously uncharacterised tetratricopeptide repeat (TPR) domain protein Bd2492, which we show localises at the single invasive pole and is required for predation. Bd2492 and RomR also interacted with cyclic-di-GMP-binding receptor CdgA, required for rapid prey-invasion. Bd2492, RomRBd and CdgA localize to the invasive pole and may facilitate MglA-docking. Bd2492 was encoded from an operon encoding a TamAB-like secretion system. The TamA protein and RomR were found, by gene deletion tests, to be essential for viability in both predatory and non-predatory modes. Control proteins, which regulate bipolar T4P-mediated social motility in swarming groups of deltaproteobacteria, have adapted in evolution to regulate the anti-social process of unipolar prey-invasion in the “lone-hunter” Bdellovibrio. Thus GTP-binding proteins and cyclic-di-GMP inputs combine at a regulatory hub, turning on prey-invasion and allowing invasion and killing of bacterial pathogens and consequent predatory growth of Bdellovibrio

Modulation of the surface proteome through multiple ubiquitylation pathways in African Trypanosomes

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes

Multi-genome identification and characterization of chlamydiae-specific type III secretion substrates: the Inc proteins

<p>Abstract</p> <p>Background</p> <p><it>Chlamydiae </it>are obligate intracellular bacteria that multiply in a vacuolar compartment, the inclusion. Several chlamydial proteins containing a bilobal hydrophobic domain are translocated by a type III secretion (TTS) mechanism into the inclusion membrane. They form the family of Inc proteins, which is specific to this phylum. Based on their localization, Inc proteins likely play important roles in the interactions between the microbe and the host. In this paper we sought to identify and analyze, using bioinformatics tools, all putative Inc proteins in published chlamydial genomes, including an environmental species.</p> <p>Results</p> <p>Inc proteins contain at least one bilobal hydrophobic domain made of two transmembrane helices separated by a loop of less than 30 amino acids. Using bioinformatics tools we identified 537 putative Inc proteins across seven chlamydial proteomes. The amino-terminal segment of the putative Inc proteins was recognized as a functional TTS signal in 90% of the <it>C. trachomatis </it>and <it>C. pneumoniae </it>sequences tested, validating the data obtained <it>in silico</it>. We identified a <it>macro </it>domain in several putative Inc proteins, and observed that Inc proteins are enriched in segments predicted to form coiled coils. A surprisingly large proportion of the putative Inc proteins are not constitutively translocated to the inclusion membrane in culture conditions.</p> <p>Conclusions</p> <p>The Inc proteins represent 7 to 10% of each proteome and show a great degree of sequence diversity between species. The abundance of segments with a high probability for coiled coil conformation in Inc proteins support the hypothesis that they interact with host proteins. While the large majority of Inc proteins possess a functional TTS signal, less than half may be constitutively translocated to the inclusion surface in some species. This suggests the novel finding that translocation of Inc proteins may be regulated by as-yet undetermined mechanisms.</p

The complex interactions of Chs5p, the ChAPs and the cargo Chs3p

The exomer complex is a putative vesicle coat required for the direct transport of a subset of cargoes from the trans-Golgi network (TGN) to the plasma membrane. Exomer comprises Chs5p as well as the ChAPs family of proteins (Chs6p, Bud7p, Bch1p and Bch2p), which are thought to act as cargo receptors, and in particular Chs6p is required for the transport of the chitin synthase Chs3p to the bud neck. However, how the ChAPs associate with Chs5p and recognize cargo is not well understood. Using domain-switch chimeras of Chs6p and Bch2p we show that four tetratricopeptide repeats (TPRs) are involved in interaction with Chs5p. Since these roles are conserved between the ChAPs, the TPRs are interchangeable between different ChAP proteins. In contrast, the N-terminal and the central parts of the ChAPs contribute to cargo specificity. While the entire N-terminal domain of Chs6p is required for Chs3p export at all cell cycle stages, the central part seems to predominantly favor Chs3p export in small-budded cells. The cargo Chs3p probably also uses a complex motif for the interaction with Chs6, as the C-terminus of Chs3p interacts with Chs6p and is necessary, but not sufficient, for TGN export

A Trypanosomal Pentatricopeptide Repeat Protein Stabilizes the Mitochondrial mRNAs of Cytochrome Oxidase Subunits 1 and 2

The pentatricopeptide repeat (PPR) protein family consists of organellar proteins predicted to bind to specific RNA sequences. Plants have hundreds of distinct PPR proteins, whereas other eukaryotes generally have many fewer. The genome of the parasitic protozoon Trypanosoma brucei is predicted to encode more than 30 different PPR proteins, which is an extraordinarily high number for a nonplant organism. Here we report the characterization T. brucei PPR9 (TbPPR9). Epitope tagging shows that the protein is exclusively mitochondrially localized. Interestingly, while in induced RNA interference cell lines TbPPR9 is efficiently downregulated, the level of its mRNA is not affected. Ablation of TbPPR9 selectively abolishes oxidative but not mitochondrial substrate-level phosphorylation. The molecular basis of this phenotype is the fact that TbPPR9 is required for the stability of the cytochrome oxidase subunit 1 (COX1) and COX2 mRNAs. This is supported by the observation that ablation of TbPPR9 destabilizes the COX complex but not the cytochrome bc1 or the ATP synthase complex. Moreover, it was shown by blue native gel electrophoresis that TbPPR9 is present in a large complex of unknown composition