Intracellular membrane localization of pseudomonas ExoS and Yersinia YopE in mammalian cells.
ExoS (453 amino acids) is a bi-functional type-III cytotoxin of Pseudomonas aeruginosa. Residues 96-233 comprise the Rho GTPase-activating protein (Rho GAP) domain, while residues 234-453 comprise the 14-3-3-dependent ADP-ribosyltransferase domain. Residues 51-72 represent a membrane localization domain (MLD), which targets ExoS to perinuclear vesicles within mammalian cells. YopE (219 amino acids) is a type-III cytotoxin of Yersinia that is also a Rho GAP. Residues 96-219 comprise the YopE Rho GAP domain. While the Rho GAP domains of ExoS and YopE share structural homology, unlike ExoS, the intracellular localization of YopE within mammalian cells has not been resolved and is the subject of this investigation. Deletion mapping showed that the N terminus of YopE was required for intracellular membrane localization of YopE in CHO cells. A fusion protein containing the N-terminal 84 amino acids of YopE localized to a punctate-perinuclear region in mammalian cells and co-localized with a fusion protein containing the MLD of ExoS. Residues 54-75 of YopE (termed YopE-MLD) were necessary and sufficient for intracellular localization in mammalian cells. The YopE-MLD localized ExoS to intracellular membranes and targeted ExoS to ADP-ribosylate small molecular weight membrane proteins as observed for native type-III delivered ExoS. These data indicate that the YopE MLD functionally complements the ExoS MLD for intracellular targeting in mammalian cells.
Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Rebecca
R
Krall
Yue
Y
Zhang
Joseph T
JT
Barbieri
D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism* / Q000382:microbiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011552:Pseudomonas Infections / Q000378:metabolism / Q000382:microbiology; D017384:Sequence Deletion; D015009:Yersinia Infections / Q000378:metabolism / Q000382:microbiology
D001425:Bacterial Outer Membrane Proteins; D001427:Bacterial Toxins; C063239:yopE protein, Yersinia; D036002:ADP Ribose Transferases; C444820:exoenzyme S