François Letourneur

Phg1p Is a Nine-transmembrane Protein Superfamily Member Involved in Dictyostelium Adhesion and Phagocytosis

To identify the molecular mechanisms involved in phagocytosis, we generated random insertion mutants ofDictyostelium discoideum and selected two mutants defective for phagocytosis. Both represented insertions in the same gene, namedPHG1. This gene encodes a polytopic membrane protein with an N-terminal lumenal domain and nine potential transmembrane segments. Homologous genes can be identified in many species; however, their function is yet to be elucidated. Disruption of PHG1 caused a selective defect in phagocytosis of latex beads and Escherichia coli, but not Klebsiella aerogenes bacteria. This defect in phagocytosis was caused by a decrease in the adhesion of mutant cells to phagocytosed particles. These results indicate that the Phg1 protein is involved in the adhesion of Dictyosteliumto various substrates, a crucial event of phagocytosis and demonstrate the usefulness of a genetic approach to dissect the molecular events involved in the phagocytic process.

Delta- and zeta-COP, two coatomer subunits homologous to clathrin-associated proteins, are involved in ER retrieval.

Two new thermosensitive yeast mutants defective in retrieval of dilysine‐tagged proteins from the Golgi back to the endoplasmic reticulum (ER) were characterized. While both ret2–1 and ret3–1 were defective for ER retrieval, only ret2–1 exhibited a defect in forward ER‐to‐Golgi transport at the non‐permissive temperature. Coatomer (COPI) from both mutants could efficiently bind dilysine motifs in vitro. The corresponding RET2 and RET3 genes were cloned by complementation and found of encode the delta and zeta subunits of coatomer respectively. Both proteins show significant homology to clathrin adaptor subunits. These results emphasize the role of coatomer in retrieval of dilysine‐tagged proteins back to the ER, and the similarity between clathrin and coatomer coats.

Coatomer (COPI)-coated vesicles: role in intracellular transport and protein sorting

Coatomer-coated vesicles have been proposed to play a role in many distinct steps of intracellular transport. Coatomer potentially plays a role in forward transport from the endoplasmic reticulum to the Golgi apparatus and through the Golgi apparatus. It may also function in retrograde transport and in the endocytic pathway. There are limitations to the various approaches used to study the role of coatomer, and looking at these helps us to better define the questions that remain to be answered.

Ent3p Is a PtdIns(3,5)P2 Effector Required for Protein Sorting to the Multivesicular Body

PtdIns(3,5)P(2) is required for cargo-selective sorting to the vacuolar lumen via the multivesicular body (MVB). Here we show that Ent3p, a yeast epsin N-terminal homology (ENTH) domain-containing protein, is a specific PtdIns(3,5)P(2) effector localized to endosomes. The ENTH domain of Ent3p is essential for its PtdIns(3,5)P(2) binding activity and for its membrane interaction in vitro and in vivo. Ent3p is required for protein sorting into the MVB but not for the internalization step of endocytosis. Ent3p is associated with clathrin and is necessary for normal actin cytoskeleton organization. Our results show that Ent3p is required for protein sorting into intralumenal vesicles of the MVB through PtdIns(3,5)P(2) binding via its ENTH domain.

New COP1‐binding motifs involved in ER retrieval

Coatomer‐mediated sorting of proteins is based on the physical interaction between coatomer (COP1) and targeting motifs found in the cytoplasmic domains of membrane proteins. For example, binding of COP1 to dilysine (KKXX) motifs induces specific retrieval of tagged proteins from the Golgi back to the endoplasmic reticulum (ER). Making use of the two‐hybrid system, we characterized a new sequence (δL) which interacts specifically with the δ‐COP subunit of the COP1 complex. Transfer of δL to the cytoplasmic domain of a reporter membrane protein resulted in its localization in the ER, in yeast and mammalian cells. This was due to continuous retrieval of tagged proteins from the Golgi back to the ER, in a manner similar to the ER retrieval of KKXX‐tagged proteins. Extensive mutagenesis of δL identified an aromatic residue as a critical determinant of the interaction with COP1. Similar COP1‐binding motifs containing an essential aromatic residue were identified in the cytoplasmic domain of an ER‐resident protein, Sec71p, and in an ER retention motif previously characterized in the CD3ϵ chain of the T‐cell receptor. These results emphasize the role of the COP1 complex in retrograde Golgi‐to‐ER transport and highlight its functional similarity with clathrin–adaptor complexes.

Membrane sorting in the endocytic and phagocytic pathway of Dictyostelium discoideum.

To study sorting in the endocytic pathway of a phagocytic and macropinocytic cell, monoclonal antibodies to membrane proteins of Dictyostelium discoideum were generated. Whereas the p25 protein was localized to the cell surface, p80 was mostly present in intracellular endocytic compartments as observed by immunofluorescence as well as immunoelectron microscopy analysis. The p80 gene was identified and encodes a membrane protein presumably involved in copper transport. Expression of chimeric proteins revealed that the cytoplasmic domain of p80 was sufficient to cause constitutive endocytosis and localization of the protein to endocytic compartments. Dileucine- and tyrosine-based endocytic signals described previously in mammalian systems were also capable of targeting chimera to endocytic compartments. In phagocytosing cells no membrane sorting was observed during formation of the phagosome. Both p25 and p80 were incorporated non-selectively in nascent phagosomes, and then retrieved shortly after phagosome closure. Our results emphasize the fact that very active membrane traffic takes place in phagocytic and macropinocytic cells. This is coupled with precise membrane sorting to maintain the specific composition of endocytic compartments.

An adhesion molecule in free-living Dictyostelium amoebae with integrin beta features

The study of free‐living amoebae has proven valuable to explain the molecular mechanisms controlling phagocytosis, cell adhesion and motility. In this study, we identified a new adhesion molecule in Dictyostelium amoebae. The SibA (Similar to Integrin Beta) protein is a type I transmembrane protein, and its cytosolic, transmembrane and extracellular domains contain features also found in integrin β chains. In addition, the conserved cytosolic domain of SibA interacts with talin, a well‐characterized partner of mammalian integrins. Finally, genetic inactivation of SIBA affects adhesion to phagocytic particles, as well as cell adhesion and spreading on its substrate. It does not visibly alter the organization of the actin cytoskeleton, cellular migration or multicellular development. Our results indicate that the SibA protein is a Dictyostelium cell adhesion molecule presenting structural and functional similarities to metazoan integrin β chains. This study sheds light on the molecular mechanisms controlling cell adhesion and their establishment during evolution.

Selective membrane exclusion in phagocytic and macropinocytic cups

Specialized eukaryotic cells can ingest large particles and sequester them within membrane-delimited phagosomes. Many studies have described the delivery of lysosomal proteins to the phagosome, but little is known about membrane sorting during the early stages of phagosome formation. Here we used Dictyostelium discoideum amoebae to analyze the membrane composition of newly formed phagosomes. The membrane delimiting the closing phagocytic cup was essentially derived from the plasma membrane, but a subgroup of proteins was specifically excluded. Interestingly the same phenomenon was observed during the formation of macropinosomes, suggesting that the same sorting mechanisms are at play during phagocytosis and macropinocytosis. Analysis of mutant strains revealed that clathrin-associated adaptor complexes AP-1, -2 and -3 were not necessary for this selective exclusion and, accordingly, ultrastructural analysis revealed no evidence for vesicular transport around phagocytic cups. Our results suggest the existence of a new, as yet uncharacterized, sorting mechanism in phagocytic and macropinocytic cups.

The ADP-ribosylation factor GTPase-activating protein GIo3p is involved in ER retrieval

Retrograde transport of proteins from the Golgi to the endoplasmic reticulum (ER) has been the subject of some interest in the recent past. Here a new thermosensitive yeast mutant defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum was characterized. The ret4-1 mutant also exhibited a selective defect in forward ER-to-Golgi transport of some secreted proteins at the non-permissive temperature. The corresponding RET4 gene was found to encode Glo3p, a GTPase-activating protein (GAP) specific for ADP-ribosylation factor (ARF). In vitro, the Glo3 thermosensitive mutant showed a reduced ARF1-GAP activity. The Glo3 protein belongs to a family of zinc finger proteins that may include additional ARF-GAPs. Gene deletion experiments of other family members showed that only GLO3 deletion resulted in impaired retrieval of dilysine-tagged proteins back to the ER. These results demonstrate that Glo3p is the main ARF-GAP specifically involved in ER retrieval.

Tyrosine dephosphorylation of the syndecan-1 PDZ binding domain regulates syntenin-1 recruitment.

Heparan sulfate proteoglycan receptor syndecan-1 interacts with the carboxyl-terminal LG4/5 domain in laminin 332 (α3LG4/5) and participates in cell adhesion and spreading. To dissect the function of syndecan-1 in these processes, we made use of a cell adhesion model in which syndecan-1 exclusively interacts with a recombinantly expressed α3LG4/5 fragment. Plating HT1080 cells on this fragment induces the formation of actin-containing protrusive structures in an integrin-independent manner. Here we show that syndecan-1-mediated formation of membrane protrusions requires dephosphorylation of tyrosine residues in syndecan-1. Accordingly, inhibition of phosphatases with orthovanadate decreases cell adhesion to the α3LG4/5 fragment. We demonstrate that the PDZ-containing protein syntenin-1, known to connect cytoskeletal proteins, binds to syndecan-1 in cells plated on the α3LG4/5 fragment and participates in the formation of membrane protrusions. We further show that syntenin-1 recruitment depends on the dephosphorylation of Tyr-309 located within syndecan-1 PDZ binding domain EFYA. We propose that tyrosine dephosphorylation of syndecan-1 may regulate its association with cytoskeleton components.