Salmonella enterica serovar Typhimurium effector SigD/SopB is membrane-associated and ubiquitinated inside host cells.
SigD/SopB is an effector protein translocated into host cells by one of the type III secretion systems of Salmonella enterica serovar Typhimurium (serovar Typhimurium). It is an inositol phosphatase that has activity towards several inositol phospholipids in vitro, including phosphatidylinositol 3,4,5- triphosphate. SigD activates Akt in epithelial cells and indirectly activates Cdc42 through one of its products, inositol 1,4,5,6-tetrakisphosphate. As phospholipid targets of SigD activity are localized to host cell membranes, we sought to investigate the intracellular localization of translocated SigD. We show here that SigD is a membrane-associated protein that is ubiquitinated inside host cells. SigD was extracted from host cell membranes with a high pH buffer but not by high salt. Fractionation and deletion analysis using transfected SigD-green fluorescent protein fusions revealed that amino acid residues 117-167 of SigD are essential for membrane association, and that a fragment containing residues 29-116 was ubiquitinated. This is the first direct evidence of a bacterial effector protein being ubiquitinated. Treatment of cells with the proteasome inhibitor MG-132 revealed that, unlike the host cell protein inhibitor of nuclear factor kappa B (IkappaBalpha), SigD does not appear to be rapidly degraded by the proteasome. We speculate that ubiquitination serves to downregulate SigD activity by an alternative mechanism, such as by targeting it for lysosomal degradation.
Marcus
Sandra L
SL
Biotechnology Laboratory, University of British Columbia, 237 Wesbrook Building, 6174 University Boulevard, Vancouver, BC, V6T 1Z3, Canada.
Knodler
Leigh A
LA
Finlay
B Brett
BB
D001426:Bacterial Proteins; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D046988:Proteasome Endopeptidase Complex; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism / Q000382:microbiology
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C060972:SopB protein, Bacteria; D025801:Ubiquitin; D010744:Phosphoric Monoester Hydrolases; C489224:SopB protein, Salmonella; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex