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Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.

Brombacher E, Urwyler S, Ragaz C, Weber SS, Kami K, Overduin M, Hilbi H - J. Biol. Chem. (2008)

Bottom Line: 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.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.Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.

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.

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Identification of SidM in a screen for PI-binding L. pneumophila proteins. Pulldown of lysate from L. pneumophila wild-type (A and B), and ΔsidC or ΔsidM mutant strains (C) using agarose beads coated with different PIs or PtdIns. Bacterial proteins retained by washed beads were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue (A and C) or Silver (B). The dominant protein with an apparent molecular mass of ∼75 kDa eluting from beads coated with PtdIns(4)P or PtdIns(3,4)P2 and to a smaller extent from beads coated with PtdIns(4,5)P2 or PtdIns(3,4,5)P3 was identified by mass spectrometry as the Rab1 GEF SidM/DrrA.
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fig1: Identification of SidM in a screen for PI-binding L. pneumophila proteins. Pulldown of lysate from L. pneumophila wild-type (A and B), and ΔsidC or ΔsidM mutant strains (C) using agarose beads coated with different PIs or PtdIns. Bacterial proteins retained by washed beads were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue (A and C) or Silver (B). The dominant protein with an apparent molecular mass of ∼75 kDa eluting from beads coated with PtdIns(4)P or PtdIns(3,4)P2 and to a smaller extent from beads coated with PtdIns(4,5)P2 or PtdIns(3,4,5)P3 was identified by mass spectrometry as the Rab1 GEF SidM/DrrA.

Mentions: Identification of SidM as a PI-binding Protein—The signaling lipid PtdIns(4)P was recently discovered to be specifically recognized by the Icm/Dot substrate SidC on LCVs (32, 33). To identify additional PI-binding proteins of L. pneumophila, we performed pulldown assays using agarose beads coated with individual PIs. Staining of the proteins eluting from washed beads by Coomassie Brilliant Blue revealed large amounts of a protein with an apparent molecular mass of ∼75 kDa, which predominantly bound to PtdIns(4)P (Fig. 1A). The protein also displayed weaker interactions with PtdIns(3,4)P2. The major PtdIns(4)P interactor was identified by mass spectrometry as the 73-kDa protein SidM, a known Icm/Dot substrate previously characterized as a Rab1 GEF (19, 20).


Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.

Brombacher E, Urwyler S, Ragaz C, Weber SS, Kami K, Overduin M, Hilbi H - J. Biol. Chem. (2008)

Identification of SidM in a screen for PI-binding L. pneumophila proteins. Pulldown of lysate from L. pneumophila wild-type (A and B), and ΔsidC or ΔsidM mutant strains (C) using agarose beads coated with different PIs or PtdIns. Bacterial proteins retained by washed beads were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue (A and C) or Silver (B). The dominant protein with an apparent molecular mass of ∼75 kDa eluting from beads coated with PtdIns(4)P or PtdIns(3,4)P2 and to a smaller extent from beads coated with PtdIns(4,5)P2 or PtdIns(3,4,5)P3 was identified by mass spectrometry as the Rab1 GEF SidM/DrrA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2643517&req=5

fig1: Identification of SidM in a screen for PI-binding L. pneumophila proteins. Pulldown of lysate from L. pneumophila wild-type (A and B), and ΔsidC or ΔsidM mutant strains (C) using agarose beads coated with different PIs or PtdIns. Bacterial proteins retained by washed beads were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue (A and C) or Silver (B). The dominant protein with an apparent molecular mass of ∼75 kDa eluting from beads coated with PtdIns(4)P or PtdIns(3,4)P2 and to a smaller extent from beads coated with PtdIns(4,5)P2 or PtdIns(3,4,5)P3 was identified by mass spectrometry as the Rab1 GEF SidM/DrrA.
Mentions: Identification of SidM as a PI-binding Protein—The signaling lipid PtdIns(4)P was recently discovered to be specifically recognized by the Icm/Dot substrate SidC on LCVs (32, 33). To identify additional PI-binding proteins of L. pneumophila, we performed pulldown assays using agarose beads coated with individual PIs. Staining of the proteins eluting from washed beads by Coomassie Brilliant Blue revealed large amounts of a protein with an apparent molecular mass of ∼75 kDa, which predominantly bound to PtdIns(4)P (Fig. 1A). The protein also displayed weaker interactions with PtdIns(3,4)P2. The major PtdIns(4)P interactor was identified by mass spectrometry as the 73-kDa protein SidM, a known Icm/Dot substrate previously characterized as a Rab1 GEF (19, 20).

Bottom Line: 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.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.Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.

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.

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Related in: MedlinePlus