Short transmembrane domains target type II proteins to the Golgi apparatus and type I to the endoplasmic reticulum.

Transmembrane domains (TMDs) contain information targeting membrane proteins to various compartments of the secretory pathway. In previous studies, short or hydrophilic TMDs have been shown to target membrane proteins either to the endoplasmic reticulum (ER), or to the Golgi apparatus. The basis for differential sorting to the ER and to the Golgi apparatus remained however unclear. To clarify this point, we analyzed quantitatively the intracellular targeting of a collection of proteins exhibiting a single TMD. Our results reveal that membrane topology is a major targeting element in the early secretory pathway: type I proteins with a short transmembrane domain are targeted to the ER, and type II proteins to the Golgi apparatus. A combination of three features accounts for the sorting of simple membrane proteins in the secretory pathway: membrane topology, length and hydrophilicity of the TMD, and size of the cytosolic domain. By clarifying the rules governing sorting to the ER and to the Golgi apparatus, our study may revive the search for sorting mechanisms in the early secretory pathway.

Transcriptional Responses of Dictyostelium discoideum Exposed to Different Classes of Bacteria.

Dictyostelium discoideum amoebae feed by ingesting bacteria, then killing them in phagosomes. Ingestion and killing of different bacteria have been shown to rely on largely different molecular mechanisms. One would thus expect that D. discoideum adapts its ingestion and killing machinery when encountering different bacteria. In this study, we investigated by RNA sequencing if and how D. discoideum amoebae respond to the presence of different bacteria by modifying their gene expression patterns. Each bacterial species analyzed induced a specific modification of the transcriptome. Bacteria such as Bacillus subtilis, Klebsiella pneumoniae, or Mycobacterium marinum induced a specific and different transcriptional response, while Micrococcus luteus did not trigger a significant gene regulation. Although folate has been proposed to be one of the key molecules secreted by bacteria and recognized by hunting amoebae, it elicited a very specific and restricted transcriptional signature, distinct from that triggered by any bacteria analyzed here. Our results indicate that D. discoideum amoebae respond in a highly specific, almost non-overlapping manner to different species of bacteria. We additionally identify specific sets of genes that can be used as reporters of the response of D. discoideum to different bacteria.

Weevil pgrp-lb prevents endosymbiont TCT dissemination and chronic host systemic immune activation.

Long-term intracellular symbiosis (or endosymbiosis) is widely distributed across invertebrates and is recognized as a major driving force in evolution. However, the maintenance of immune homeostasis in organisms chronically infected with mutualistic bacteria is a challenging task, and little is known about the molecular processes that limit endosymbiont immunogenicity and host inflammation. Here, we investigated peptidoglycan recognition protein (PGRP)-encoding genes in the cereal weevil Sitophilus zeamais's association with Sodalis pierantonius endosymbiont. We discovered that weevil pgrp-lb generates three transcripts via alternative splicing and differential regulation. A secreted isoform is expressed in insect tissues under pathogenic conditions through activation of the PGRP-LC receptor of the immune deficiency pathway. In addition, cytosolic and transmembrane isoforms are permanently produced within endosymbiont-bearing organ, the bacteriome, in a PGRP-LC-independent manner. Bacteriome isoforms specifically cleave the tracheal cytotoxin (TCT), a peptidoglycan monomer released by endosymbionts. pgrp-lb silencing by RNAi results in TCT escape from the bacteriome to other insect tissues, where it chronically activates the host systemic immunity through PGRP-LC. While such immune deregulations did not impact endosymbiont load, they did negatively affect host physiology, as attested by a diminished sexual maturation of adult weevils. Whereas pgrp-lb was first described in pathogenic interactions, this work shows that, in an endosymbiosis context, specific bacteriome isoforms have evolved, allowing endosymbiont TCT scavenging and preventing chronic endosymbiont-induced immune responses, thus promoting host homeostasis.

TM9SF4 levels determine sorting of transmembrane domains in the early secretory pathway.

Previous studies have shown that TM9SF4 interacts with glycine-rich transmembrane domains (TMDs) and promotes their surface localization, presumably by escorting them along the secretory pathway. Here, we delineated the role of TM9 proteins in the sorting of TMDs. Our results indicate that TM9SF4 interacts with and sorts a variety of TMDs. In human embryonic kidney (HEK) cells, a TMD carrying a positively charged residue (T-R1) or a negatively charged residue (T-D1) was localized to the endoplasmic reticulum (ER), but partially relocated to the Golgi complex upon overexpression of TM9SF4. These results show that TM9SF4 controls the sorting of TMDs at the ER-Golgi interface. Remarkably, sorting of T-R1 in HCT116 cells was different from that in HEK cells: in HCT116 cells, a substantial fraction of T-R1 was localized to the Golgi complex, and it was relocated to the ER by genetic ablation of TM9SF4. This observation indicates that TM9SF4 sorting activity differs in HEK and HCT116 cells, resulting in different sorting of TMDs in these two cell types. Although TM9SF1 associated with several TMDs, it did not visibly alter their intracellular transport in the secretory pathway and may function in other intracellular transport pathways.

Role of the HIV-1 envelope transmembrane domain in intracellular sorting.

BACKGROUND: The envelope protein of lentiviruses are type I transmembrane proteins, and their transmembrane domain contains conserved potentially charged residues. This highly unusual feature would be expected to cause endoplasmic reticulum (ER) localization. The aim of this study was to determine by which means the HIV-1 Env protein is transported to the cell surface although its transmembrane domain contains a conserved arginine residue. RESULTS: We expressed various chimeric proteins and analyzed the influence of their transmembrane domain on their intracellular localization. The transmembrane domain of the HIV-1 Env protein does not cause ER retention. This is not due to the presence of conserved glycine residues, or to the position of the arginine residue, but to the length of the transmembrane domain. A shortened version of the Env transmembrane domain causes arginine-dependent ER targeting. Remarkably, the transmembrane domain of the HIV-1 Env protein, although it does not confer ER retention, interacts efficiently with negatively charged residues in the membrane. CONCLUSION: These results suggest that the intrinsic properties of the HIV-1 Env transmembrane domain allow the protein to escape ER-retention mechanisms, while maintaining its ability to interact with cellular proteins and to influence cellular physiology.

Vps13F links bacterial recognition and intracellular killing in Dictyostelium.

Bacterial sensing, ingestion, and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. The cellular mechanisms involved in intracellular killing, their relative importance, and their specificity towards different bacteria are however poorly defined. In this study, we used Dictyostelium discoideum, a phagocytic cell model amenable to genetic analysis, to identify new gene products involved in intracellular killing. A random genetic screen led us to identify the role of Vps13F in intracellular killing of Klebsiella pneumoniae. Vps13F knock-out (KO) cells exhibited a delayed intracellular killing of K. pneumoniae, although the general organization of the phagocytic and endocytic pathway appeared largely unaffected. Transcriptomic analysis revealed that vps13F KO cells may be functionally similar to previously characterized fspA KO cells, shown to be defective in folate sensing. Indeed, vps13F KO cells showed a decreased chemokinetic response to various stimulants, suggesting a direct or indirect role of Vps13F in intracellular signaling. Overstimulation with excess folate restored efficient killing in vps13F KO cells. Finally, genetic inactivation of Far1, the folate receptor, resulted in inefficient intracellular killing of K. pneumoniae. Together, these observations show that stimulation of Dictyostelium by bacterial folate is necessary for rapid intracellular killing of K. pneumoniae.

TM9 family proteins control surface targeting of glycine-rich transmembrane domains.

TM9 family proteins (also named Phg1 proteins) have been previously shown to control cell adhesion by determining the cell surface localization of adhesion proteins such as the Dictyostelium SibA protein. Here, we show that the glycine-rich transmembrane domain (TMD) of SibA is sufficient to confer Phg1A-dependent surface targeting to a reporter protein. Accordingly, in Dictyostelium phg1A-knockout (KO) cells, proteins with glycine-rich TMDs were less efficiently transported out of the endoplasmic reticulum (ER) and to the cell surface. Phg1A, as well as its human ortholog TM9SF4 specifically associated with glycine-rich TMDs. In human cells, genetic inactivation of TM9SF4 resulted in an increased retention of glycine-rich TMDs in the endoplasmic reticulum, whereas TM9SF4 overexpression enhanced their surface localization. The bulk of the TM9SF4 protein was localized in the Golgi complex and a proximity-ligation assay suggested that it might interact with glycine-rich TMDs. Taken together, these results suggest that one of the main roles of TM9 proteins is to serve as intramembrane cargo receptors controlling exocytosis and surface localization of a subset of membrane proteins.

The nonaspanins TM9SF2 and TM9SF4 regulate the plasma membrane localization and signalling activity of the peptidoglycan recognition protein PGRP-LC in Drosophila.

Transmembrane 9 (TM9) proteins, or nonaspanins, are a family of proteins conserved throughout evolution and characterized by 9 transmembrane domains. In Drosophila, TM9 superfamily protein member 4 (TM9SF4) and its closest paralogue, TM9SF2, contribute to phagocytosis of various types of particles, while TM9SF4 displays non-redundant requirement in Gram-negative bacteria engulfment. In addition, the two TM9 proteins control the actin cytoskeleton in larval haemocytes and in Drosophila S2 cells. Here, we show that TM9SF4 and TM9SF2 co-immunoprecipitate with the peptidoglycan recognition protein (PGRP)-LC, which triggers the Drosophila immune response to bacterial infection. Furthermore, both TM9 proteins co-localize with this receptor in intracellular vesicles and at the plasma membrane in Drosophila S2 cells in culture and in the fly fat body. Silencing TM9SF4 prevents plasma membrane localization of PGRP-LC, whereas silencing TM9SF2 does not, which may account for the non-redundant role of TM9SF4 in phagocytosis of Gram-negative bacteria. Finally, we provide a set of data suggesting that TM9 proteins can prevent inappropriate signalling from the unstimulated receptor.

Anchors aweigh: protein localization and transport mediated by transmembrane domains.

The transmembrane domains (TMDs) of integral membrane proteins have emerged as major determinants of intracellular localization and transport in the secretory and endocytic pathways. Unlike sorting signals in cytosolic domains, TMD sorting determinants are not conserved amino acid sequences but physical properties such as the length and hydrophilicity of the transmembrane span. The underlying sorting machinery is still poorly characterized, but several mechanisms have been proposed, including TMD recognition by transmembrane sorting receptors and partitioning into membrane lipid domains. Here we review the nature of TMD sorting determinants and how they may dictate transmembrane protein localization and transport.

TM9/Phg1 and SadA proteins control surface expression and stability of SibA adhesion molecules in Dictyostelium.

TM9 proteins form a family of conserved proteins with nine transmembrane domains essential for cellular adhesion in many biological systems, but their exact role in this process remains unknown. In this study, we found that genetic inactivation of the TM9 protein Phg1A dramatically decreases the surface levels of the SibA adhesion molecule in Dictyostelium amoebae. This is due to a decrease in sibA mRNA levels, in SibA protein stability, and in SibA targeting to the cell surface. A similar phenotype was observed in cells devoid of SadA, a protein that does not belong to the TM9 family but also exhibits nine transmembrane domains and is essential for cellular adhesion. A contact site A (csA)-SibA chimeric protein comprising only the transmembrane and cytosolic domains of SibA and the extracellular domain of the Dictyostelium surface protein csA also showed reduced stability and relocalization to endocytic compartments in phg1A knockout cells. These results indicate that TM9 proteins participate in cell adhesion by controlling the levels of adhesion proteins present at the cell surface.

TM9SF4 is required for Drosophila cellular immunity via cell adhesion and phagocytosis.

Nonaspanins are characterised by a large N-terminal extracellular domain and nine putative transmembrane domains. This evolutionarily conserved family comprises three members in Dictyostelium discoideum (Phg1A, Phg1B and Phg1C) and Drosophila melanogaster, and four in mammals (TM9SF1-TM9SF4), the function of which is essentially unknown. Genetic studies in Dictyostelium demonstrated that Phg1A is required for cell adhesion and phagocytosis. We created Phg1A/TM9SF4-null mutant flies and showed that they were sensitive to pathogenic Gram-negative, but not Gram-positive, bacteria. This increased sensitivity was not due to impaired Toll or Imd signalling, but rather to a defective cellular immune response. TM9SF4-null larval macrophages phagocytosed Gram-negative E. coli inefficiently, although Gram-positive S. aureus were phagocytosed normally. Mutant larvae also had a decreased wasp egg encapsulation rate, a process requiring haemocyte-dependent adhesion to parasitoids. Defective cellular immunity was coupled to morphological and adhesion defects in mutant larval haemocytes, which had an abnormal actin cytoskeleton. TM9SF4, and its closest paralogue TM9SF2, were both required for bacterial internalisation in S2 cells, where they displayed partial redundancy. Our study highlights the contribution of phagocytes to host defence in an organism possessing a complex innate immune response and suggests an evolutionarily conserved function of TM9SF4 in eukaryotic phagocytes.

Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model.

The human pathogen Pseudomonas aeruginosa has been shown previously to use similar virulence factors when infecting mammalian hosts or Dictyostelium amoebae. Here we randomly mutagenized a clinical isolate of P. aeruginosa, and identified mutants with attenuated virulence towards Dictyostelium. These mutant strains also exhibited a strong decrease in virulence when infecting Drosophila and mice, confirming that P. aeruginosa makes use of similar virulence traits to confront these very different hosts. Further characterization of these bacterial mutants showed that TrpD is important for the induction of the quorum-sensing circuit, while PchH and PchI are involved in the induction of the type III secretion system. These results demonstrate the usefulness and the relevance of the Dictyostelium host model to identify and analyse new virulence genes in P. aeruginosa.

Rac2 is a major actor of Drosophila resistance to Pseudomonas aeruginosa acting in phagocytic cells.

Pathogen recognition and engulfment by phagocytic cells of the blood cell lineage constitute the first line of defense against invading pathogens. This cellular immune response is conserved throughout evolution and depends strictly on cytoskeletal changes regulated by the RhoGTPases family. Many pathogens have developed toxins modifying RhoGTPases activity to their own benefit. In particular, the Exoenzyme S (ExoS) toxin of the Gram-negative bacteria Pseudomonas aeruginosa is directly injected into the host cell cytoplasm and contains a GAP domain (ExoSGAP) targeting RhoGTPases. Searching for the contribution of each RhoGTPases, Rho1, Rac1, Rac2, Mtl (Mig2-like) and Cdc42 to fly resistance to P. aeruginosa infections, we found that Rac2 is required to resist to P. aeruginosa and to other Gram-negative or Gram-positive bacteria. The Rac2 immune-deficient phenotype is attributable to defective engulfment of pathogens since Rac2-mutant macrophages exhibited strong reduction in the phagocytosis level of both Gram-negative and Gram-positive bacterial particles whereas systemic immune signaling pathways, including Toll, Immune deficiency and Jun kinases, were not affected. Co-expression of Rac2 and ExoSGAP rescued the increased sensitivity to P. aeruginosa observed in ExoSGAP-expressing flies suggesting that Rac2 is the main host factor whose function is inhibited by the GAP domain of the ExoS toxin.