In yeast, the bulk of ER-imported SLTxA1 is O
-mannosylated in the ER, which may be a consequence of its relatively long ER residence (T1/2
~60 min, estimated from ) compared to RTA (T1/2
~20 min 
). A number of models have been presented for the role of Pmt2p-dependent O
-mannosylation in ERAD. For mutant α-factor precursor, O
-mannosylation slightly stabilizes this protein, suggesting a minor role in protecting substrates from ERAD by interfering with dislocation 
. However for the misfolded substrate HA-Gas1*p, Pmt2p-dependent O
-mannosylation is important for proteasomal degradation, and in the absence of this modification the substrate is primarily degraded in vacuoles 
) shows considerable Pmt2p-stimulated O
-mannosylation that is enhanced by the absence of the Hrd1p, Hrd3p, Usa1p and Der1p members of the Hrd1 ubiquitin ligase dislocation complex, but we see no evidence for a switching of the degradative compartment between the proteasome and the vacuole. Instead O
-mannosylation may maintain solubility of SLTxA1 in the ER, as proposed for the ERAD substrates mutant prepro-α-factor and a pro-region-deleted derivative of aspartic proteinase-I 
. The toxic fraction of SLTxA1 appears to be derived from the non-O
-mannosylated forms, but increasing the proportion of these (in a PMT2 null strain) does not increase toxicity, suggesting that there are limiting factors in the yeast ER for dislocation and subsequent recovery of activity. In mammalian cells, ER trafficking of STx/SLTx appears to be efficient, since HRP-conjugated STx can be visualized in the ER by electronmicroscopy 
and Cy-2 labelled SLTx can be visualized in the ER by fluorescence microscopy 
. However, the dislocation frequency of the activated A1 chain is very low 
, suggesting that in mammalian cells also, there is an ER bottleneck for pre-dislocation events or dislocation itself.
There are two broad populations of ER-imported SLTxA1(N−); a major non-toxic fraction and a critical fraction that recovers activity in the cytosol and is responsible for toxicity and a resulting growth defect. We therefore used pulse-chase analysis to define the behavior of the bulk population of SLTxA1(N−) and used growth studies (drop tests) to describe the fate of the fraction of toxin that is destined to recover activity. Accurate quantitation of smeared bands after pulse-chase analysis is challenging, so stabilization in the ER was also judged visually by noting increased O-mannosylation of the toxin subunit. For the growth studies, a number of strains were unsuitable because growth would need to be performed at the restrictive temperature for growth (e.g. the cold-sensitive CDC48 mutant). Nevertheless, taking the data as a whole, we find that the bulk behavior of SLTxA1 is consistent with that of an authentic ERAD substrate that is extracted via the Hrd1/Hrd3/Der1 complex by Npl4p-adapted Cdc48p and which is terminally dispatched by the proteasome.
The fraction of the SLTxA1(N−
) population that is destined to recover activity is also dislocated via a Hrd1p-dependent mechanism. A hrd1
mutant previously characterized as specific for ERAD-M substrates, with no measurable effects on ERAD-L substrates 
, is partly defective in dislocation of the toxic sub-population of RTA, a protein that alters conformation in the presence of negatively charged lipids 
, and which embeds its hydrophobic C terminus into microsomal membranes 
. The C-terminal region of SLTxA1 also contains a relatively hydrophobic stretch of amino acids that is important for cytotoxicity 
, and peptides based on this region interact with lipid membranes at low pH, possibly inserting at neutral pH 
, which may allow the toxin subunit to be perceived as ‘misfolded’ by ER quality control surveillance. However, effects of ERAD-M specific hrd1
mutants on SLTxA1(N−
) dislocation are only apparent following their overexpression, so our results suggest only a minor physiological role for this potential membrane piercing. It is clear that the two toxins negotiate dislocation idiosyncratically: although Hrd1p residues E78 and W123 play a minor role for dislocation of both toxin subunits, Hrd1p L74 is strongly required for RTA dislocation but has no obvious role for SLTxA1.
Cdc48 acts as a nexus for converging ERAD pathways that then targets substrates for proteasomal destruction 
. However, RTA can uncouple from ERAD by dislocating in a ubiquitin-independent manner, thereby avoiding interactions with Cdc48p and subsequent proteasomal degradation 
. Similarly, the K28 viral killer toxin dislocates without being ubiquitylated and without assistance from Cdc48p and its Npl4p and Ufd1p co-factors, and is not degraded by the proteasomal core 
. Curiously though, the catalytic cysteine of Hrd1p is
apparently required for the dislocation of the toxic fraction of SLTxA1, suggesting that this population is also extracted as a ubiquitylated protein. Presumably, such a modified polypeptide would need to be stripped of polyubiquitin chains to allow refolding to a functional conformation, which would suggest the intervention of a deubiquitylase. Since recovery of toxin activity did not depend on a battery of Cdc48p co-factors (the substrate recruiting Npl4p, the E4 ubiquitin chain extending factor Ufd2 and its competing Ufd3p antagonist, the de-ubiquitylase Otu1p and the substrate release factor Vms1p) such de-ubiquitylation would have to occur upstream of Cdc48 interactions. In mammalian cells, SLTx dislocation and toxicity are not influenced by chemical perturbation of the ubiquitin-proteasome system by eeyarestatin 1 (an inhibitor of p97 associated activities) or clasto-Lactacystin ß-lactone (an inhibitor of the proteasomal proteolytic activities) 
, again suggesting that the fraction of ER-delivered toxin that recovers activity in the cytosol does not interact with p97/VCP, the mammalian equivalent of Cdc48p. It appears that avoidance of Cdc48p interactions may be a common strategy for proteins that dislocate and avoid proteasomal proteolysis, but it remains unclear how a small fraction of SLTxA1 can perform this and whether an early de-ubiquitylation reaction of a type not normally sanctioned for ERAD substrates is involved. Furthermore, it is also not obvious why SLTxA1 requires the catalytic cysteine residue of Hrd1p for dislocation, but has no obvious requirements for Ubc6 and the Ubc7p/Cue1p co-factors that normally provide E2 ubiquitin-conjugating activities. This contrasts quite strikingly with a partial requirement for Ubc7/Cue1p and the total lack of requirement for an active Hrd1p for RTA 
. Although we have not identified any E2 involved (directly or indirectly) for the toxic fraction of SLTxA1, the combined observations point to mechanistic differences between the way these two toxins reach the cytosol.
Since mammalian HRD1 is required for the dislocation of substrates that are not necessarily ubiquitylated 
the requirement of SLTxA1 for the E3 ligase activity of Hrd1p may be an indirect one, perhaps reflecting auto-ubiquitylation of HRD1/Hrd1p 
or the turnover of an unknown ER factor that is utilized by the small population of SLTxA1 that can recover activity in the cytosol. Such questions will be addressed in future studies.