Abstract
The active subunit (S1) of pertussis toxin (PT), a major virulence factor of Bordetella pertussis, ADP-ribosylates Gi proteins in the mammalian cell cytosol to inhibit GPCR signaling. The intracellular pathway of PT includes endocytosis and retrograde transport to the trans-Golgi network (TGN) and endoplasmic reticulum (ER). Subsequent translocation of S1 to the cytosol is presumably preceded by dissociation from the holotoxin. In vitro, such dissociation is stimulated by interaction of PT with ATP. To investigate the role of this interaction in cellular events, we engineered a form of PT (PTDM) with changes to two amino acids involved in the interaction with ATP. PTDM was reduced in (1) binding to ATP, (2) dissociability by interaction with ATP, (3) in vitro enzymatic activity and (4) cellular ADP-ribosylation activity. In cells treated with PTDM carrying target sequences for organelle-specific modifications, normal transport to the TGN and ER occurred, but N-glycosylation patterns of the S1 and S4 subunits were consistent with an inability of PTDM to dissociate in the ER. These results indicate a requirement for interaction with ATP for PT dissociation in the ER and cellular activity. They also indicate that the retrograde transport route is the cellular intoxication pathway for PT.