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Throughout the 50 year history of the protein folding field, the tracking of disulfide bonds and their formation has played a central role. In pioneering days when folding studies were limited to refolding of denatured proteins in vitro, the making, breaking and rearrangement of disulfide bonds provided the only reliable way to monitor kinetics and conformations during the refolding process. Later, when it became possible to study folding in live cells, the stepwise generation of intramolecular disulfides in nascent and newly synthesized proteins was what we and others followed. The assembly of oligomeric proteins could also be easily detected if they acquired interchain disulfides. Misfolding could often be identified by the formation of disulfide-bonded aggregates. For larger proteins, such as influenza virus hemagglutinin, it was possible to characterize both co-translational and post-translational folding events simply by following mobility differences of folding intermediates in nascent and fully synthesized polypeptide chains using non-reducing SDS-PAGE.

By combining such “disulfide tracking” approaches with radioactive pulse and chase, sufficient time resolution was achieved to follow the entire folding process, especially of larger multi-domain proteins. When combined with conformation-specific antibodies, inhibitors, mutations, limited proteolysis, expression of recombinant proteins and other perturbations, this approach revealed insights into the rules that govern the folding program of many proteins. The role of chaperones and folding enzymes and the influence of covalent modifications such as glycosylation could be analyzed.

The reviews in this volume show that great progress has been made in understanding protein maturation, quality control, secretion and endoplasmic reticulum (ER)-associated degradation. A deeper understanding of the compartments where disulfide bonds are formed is emerging. We have, for example, learned that the ER lumen provides an exquisitely complex, adaptable environment for the oxidative folding of protein products produced by a cell. It contains a powerful mixture of folding factors that together support a continuous process of folding, unfolding, misfolding and refolding. For most newly synthesized proteins, this is accompanied by oxidation, reduction and rearrangement of disulfides. The mixture of chaperones, thiol oxidoreductases, folding sensors, ER-associated degradation components, modifying enzymes, Ca2+ buffers, membrane transporters and other critical factors are fine tuned according to cell function and physiological condition.

It is important to realize that our understanding of in vivo folding is still superficial. The cell biology of oxidative protein folding still presents us with formidable challenges. Folding factors work together via complicated synergies in a highly complex, dynamic milieu that we cannot successfully mimic in vitro. In the ER, we do not know the concentrations of chaperones or their distribution within the luminal space. We do not comprehend their mutual interactions and influences and we cannot predict the choice and combination of factors needed for specific protein substrates. We hardly have a clue about the physical state of the interior of the ER. Is it a fluid or gel like? We do not know how selective transport of cargo from the ER is maintained and how quality control really works. A connection between defects in protein folding and ER function is evident in various disease states, but we cannot address such problems effectively. Hence there is still a lot to accomplish in this important field of research.

Ari Helenius

Institute of Biochemistry

ETH Zürich

Zürich, Switzerland

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