Transport-dependence: sIgM degradation was non-lysosomal and occurred prior to the medial- Golgi (Amitay et al., 1991), yet required vesicular export from the ER (Amitay et al., 1992). In pre-B cells that do not express any Ig light chains, the free ms was also rapidly degraded but this degradation was transport-independent (Rabinovich et al., 1993). We reconstituted the compartmentalized sIgM degradation along the secretory pathway in vitro, in permeabilized cells (Winitz et al., 1996) and pioneered the concept of transport-dependent ERAD-L. We demonstrated in pre-B cells that differentiated into light chain-expressing B-cells shifted ms degradation from vesicular transport-independent process into a mechanism that required budding of vesicles from the ER (Elkabetz et al., 2003).

IgG assembly: Moving from endogenous sIgM in B-cells to non-lymphoid mammalian cells allowed us to address quality control processes operating in Ig production. The g2b heavy chain of mouse IgG2b is retained but not degraded. When expressed in non-lymphoid COS-7 cells, we found intriguing interrelations between Cys residues involved in g2b folding and IgG2b assembly (Elkabetz et al., 2005). We identified unique active antibody composed of two light chains associated non-covalently with the heavy chains homodimer, which formed between them an unusual S-S bond (Elkabetz et al., 2008). Interrelations between assembly and secretion and a role played by Cys were also discovered in our collaborative study on human acetylcholine esterase (Kerem et al., 1993).

sIgM is an ERAD substrate: We found that ALLN, now known as a proteasome inhibitor, blocked sIgM degradation (Amitay et al., 1992). Subsequently, we demonstrated that several specific proteasome inhibitors blocked sIgM degradation and in their presence, poly-ubiquitin was conjugated to the accumulating sIgM, hence sIgM is a bona fide luminal ERAD substrate (Elkabetz et al., 2003). 

ERAD signals – the mstp: We established the mstp as an ERAD signal that conferred ER retention and targeted otherwise stable proteins to the ubiquitin-proteasome system. The mstp was fused to two reporter secretory proteins, a truncated secreted version of thyroid peroxidase (TPO) and yellow fluorescent protein led to the secretory pathway by a signal sequence (ssYFP). When expressed in several non-lymphoid cells, mstpCys fusion proteins were retained in the ER, conjugated to poly-ubiquitin and degraded by the proteasome, whereas those fused to mstpSer were stable proteins that were efficiently secreted (Shapira et al., 2007). These data are consistent with the contribution of the penultimate Cys in the mstp to the intracellular retention of ms and with the importance of the oxidizing thiol milieu within the ER lumen (Shachar et al., 1994).

 

The mstp is highly conserved in evolution, including its penultimate Cys and its N-glycan. To address the role of N-glycan in regulating protein folding and degradation, we generated unglycosylated mstp. We found accelerated degradation of the unglycosylated mutants and ssYFP-mstpSer turned from secreted protein into an ERAD substrate (Shapira et al., 2007). The effect of the N-glycan could not be attributed to limiting amounts of processing enzymes or lectins acting as chaperones or cargo receptors. A refined characterization of the mstp revealed that its  last 15 residues were sufficient to confer ER retention and rapid degradation. This dual function was hampered upon linking N-glycan or if a predicted b-strand was shortened or broken. A b-strand structure, rather than the actual sequence of the 15 residues, was essential for tight retention and rapid degradation, as both functions were conferred by a b-strand constructed from different residues and hampered if this b-strand was broken. The initial distinction between glycosylated and unglycosylated ssYFP was by the ER chaperone BiP, which strongly preferred the unglycosylated species and could account, in part, for their tighter retention and accelerated degradation. However, BiP could not act single-handedly, as all unglycosylated species were associated equally well with BiP, irrespective of their targeting to secretion or degradation. It appeared that a b-strand structure was responsible for executing the tight retention and rapid degradation (Shapira et al., in preparation).

Glucosidases: The importance of the N-glycan and of chaperones/lectins and enzymes that recognize and process the N-glycan led to a theoretical study. Based on in vitro studies published by our collaborators, we constructed a mathematical model that tested key steps in the N-glycan-dependent quality control mechanisms. Our model predicted that glucosidase II was inhibited by its end product, the unglucosylated N-glycan and that the inhibition was more effective if the end product was also trimmed by mannosidase. This physiologically significant prediction was confirmed experimentally by our collaborators. In addition, our model showed that the N-glycan dissociation from the lectin/chaperone calreticulin was spontaneous and independent of glucosidase II, while this enzyme prevented the re-association of the unglucosylated N-glycan with calreticulin (Bosis et al., 2008). 

Competition for ERAD substrates between dislocation and secretion:  Using yeast mutants and invertase fused to µstp, we revealed in the ER lumen competition between secretion and ERAD. The μstpCys, unlike μstpSer, conferred retention onto invertase. The penultimate Cys contribution was corroborated by improved secretion of invertase-μstpCys upon incubation with β-mercaptoethanol or deletion of Eug1, identifying this ER-resident thiol oxidoreductase as a key player in the μstpCys-dependent retention. The retained invertase-μstpCys dislocated to the cytosol in a process involving the E3 ubiquitin ligase Doa10 and the AAA-ATPase Cdc48 and Rpt4 and was degraded by the ubiquitin-proteasome system involving the E2 Ubc7 and the proteasomal Rpt2. Hence, the μstpCys was recognized as an ERAD signal also in yeast. Coupling between retention and dislocation of invertase-μstpCys was deduced from improved secretion under conditions of blocked dislocation. However, while the proximal dislocation step competed with secretion, the distal ERAD steps of ubiquitination or proteasomal degradation were dispensable for either retention or dislocation of invertase-µstpCys (Nadel et al., submitted).