Pertussis toxin (PT) is an AB-type protein toxin that consists of a catalytic A subunit (PT S1) and an oligomeric, cell-binding B subunit. It belongs to a subset of AB toxins that move from the cell surface to the endoplasmic reticulum (ER) before A chain passage into the cytosol. Toxin translocation is thought to involve A chain unfolding in the ER and the quality control mechanism of ER-associated degradation (ERAD). The absence of lysine residues in PT S1 may allow the translocated toxin to avoid ubiquitin-dependent degradation by the 26S proteasome, which is the usual fate of exported ERAD substrates. As the conformation of PT S1 appears to play an important role in toxin translocation, we used biophysical and biochemical methods to examine the structural properties of PT S1. Our in vitro studies found that the isolated PT S1 subunit is a thermally unstable protein that can be degraded in a ubiquitin-independent fashion by the core 20S proteasome. The thermal denaturation of PT S1 was inhibited by its interaction with NAD, a donor molecule used by PT S1 for the ADP-ribosylation of target G proteins. These observations support a model of intoxication in which toxin translocation, degradation, and activity are all influenced by the heat-labile nature of the isolated toxin A chain.Pertussis toxin (PT) is an AB-type protein toxin that consists of an enzymatic A moiety and a cell-binding B moiety (reviewed in (1,2)). PT A (the S1 subunit) activates certain Gα proteins by an ADP-ribosylation reaction that utilizes NAD as a donor molecule. PT B is composed of an S2 subunit, an S3 subunit, two S4 subunits, and an S5 subunit. The oligomeric PT B complex forms a ring-like structure that is stable for temperatures up to 60ºC-70ºC (3). Non-covalent interactions position the catalytic S1 subunit within and on top of the B ring to form the PT holotoxin.PT B binds to glycoproteins or glycolipids on the plasma membrane of a target cell (4-6). The surface-bound toxin then travels by vesicular transport to the Golgi apparatus and most likely to the endoplasmic reticulum (ER) as well (7-12).