title: Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.
abstract: The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the alpha-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIbeta. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.
authors.affiliation: Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.
authors.forename: Eva
authors.initials: E
authors.lastname: Brombacher
authors.forename: Simon
authors.initials: S
authors.lastname: Urwyler
authors.forename: Curdin
authors.initials: C
authors.lastname: Ragaz
authors.forename: Stefan S
authors.initials: SS
authors.lastname: Weber
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Kami
authors.forename: Michael
authors.initials: M
authors.lastname: Overduin
authors.forename: Hubert
authors.initials: H
authors.lastname: Hilbi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004330:Drosophila; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016952:Legionella pneumophila / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D007877:Legionnaires' Disease / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010449:Peptide Mapping; D010586:Phagocytes / Q000378:metabolism / Q000382:microbiology; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D014617:Vacuoles / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D020761:rab1 GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D018129:Phosphatidylinositol Phosphates; C497516:RalF protein, Legionella pneumophila; C539733:SidM protein, Legionella pneumophila; C037178:phosphatidylinositol 4-phosphate; D020761:rab1 GTP-Binding Proteins