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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 the PtdIns(4)P-binding domain of SidM. A, SidM fragments fused to GST were affinity-purified and used in protein-lipid overlay assay to test binding to 100 pmol (B) or serial 2-fold dilutions of PtdIns(4)P and PtdIns(4,5)P2 spotted onto nitrocellulose membranes (C).
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fig3: Identification of the PtdIns(4)P-binding domain of SidM. A, SidM fragments fused to GST were affinity-purified and used in protein-lipid overlay assay to test binding to 100 pmol (B) or serial 2-fold dilutions of PtdIns(4)P and PtdIns(4,5)P2 spotted onto nitrocellulose membranes (C).

Mentions: Identification of the PtdIns(4)P-binding Domain of SidM—To map the PtdIns(4)P-binding domain of SidM, we constructed N-terminal fusions of GST with fragments of SidM of different lengths and visualized binding of the fusion proteins to PtdIns(4)P by protein-lipid overlay assays (Fig. 3A). Full-length SidM (73 kDa) and the C-terminal fragments M7 (49 kDa, SidM-(214–647)), M9 (23 kDa, SidM-(444–647)), and M13 (12 kDa, SidM-(544–647)) bound to PtdIns(4)P but not to PtdIns(4,5)P2, which was used as a negative control (Fig. 3B). M13 was the smallest PtdIns(4)P-binding fragment identified, and upon further cleavage into the N- and C-terminal fragments M17 and M19, PtdIns(4)P binding activity was completely lost. The M13 PtdIns(4)P-binding domain does not show any homology to the PtdIns(4)P-binding pleckstrin homology (PH) domain of the eukaryotic adaptor protein FAPP1 (40), the P4C domain of L. pneumophila SidC (33), or any other prokaryotic or eukaryotic PI-binding protein. Thus, we termed this novel module the “P4M” (PtdIns4P binding of SidM/DrrA) domain.


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 the PtdIns(4)P-binding domain of SidM. A, SidM fragments fused to GST were affinity-purified and used in protein-lipid overlay assay to test binding to 100 pmol (B) or serial 2-fold dilutions of PtdIns(4)P and PtdIns(4,5)P2 spotted onto nitrocellulose membranes (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Identification of the PtdIns(4)P-binding domain of SidM. A, SidM fragments fused to GST were affinity-purified and used in protein-lipid overlay assay to test binding to 100 pmol (B) or serial 2-fold dilutions of PtdIns(4)P and PtdIns(4,5)P2 spotted onto nitrocellulose membranes (C).
Mentions: Identification of the PtdIns(4)P-binding Domain of SidM—To map the PtdIns(4)P-binding domain of SidM, we constructed N-terminal fusions of GST with fragments of SidM of different lengths and visualized binding of the fusion proteins to PtdIns(4)P by protein-lipid overlay assays (Fig. 3A). Full-length SidM (73 kDa) and the C-terminal fragments M7 (49 kDa, SidM-(214–647)), M9 (23 kDa, SidM-(444–647)), and M13 (12 kDa, SidM-(544–647)) bound to PtdIns(4)P but not to PtdIns(4,5)P2, which was used as a negative control (Fig. 3B). M13 was the smallest PtdIns(4)P-binding fragment identified, and upon further cleavage into the N- and C-terminal fragments M17 and M19, PtdIns(4)P binding activity was completely lost. The M13 PtdIns(4)P-binding domain does not show any homology to the PtdIns(4)P-binding pleckstrin homology (PH) domain of the eukaryotic adaptor protein FAPP1 (40), the P4C domain of L. pneumophila SidC (33), or any other prokaryotic or eukaryotic PI-binding protein. Thus, we termed this novel module the “P4M” (PtdIns4P binding of SidM/DrrA) domain.

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.

Show MeSH
Related in: MedlinePlus