seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol 10, 1999: pp. 443 454 Article No. scdb.1999.0315, available online at http:www.idealibrary.com on Protein folding in the ER Fred J. Stevens  and Yair Argon† ( ) The endoplasmic reticulum ER is a major protein folding compartment for secreted, plasma membrane and organelle proteins. Each of these newly-synthesized polypeptides folds in a deterministic process, affected by the unique conditions that exist in the ER. An understanding of protein folding in the ER is a fundamental biomolecular challenge at two levels. The first level addresses how the amino acid sequence programs that polypeptide to efficiently arrive at a particular fold out of a multitude of alternatives, and how different sequences obtain similar folds. At the second level are the issues introduced by folding not in the cytosol, but in the ER, including the risk of aggregation in a molecularly crowded environment, accommodation of post-translational modifications and the compatibility with subsequent intracellular trafficking. This review discusses both the physicochemical and cell biological constraints of folding, which are the challenges that the ER molecular chaperones help overcome. Key words: chaperones  ER quality control  cooperativ- ity  thermodynamics 1999 Academic Press The ongoing international Human Genome Project will soon make known sequences of the approxi- mately 100 000 proteins encoded by the genome. For these proteins to perform their functions, each must fold into a distinct three-dimensional arrangement, and remain folded for a biologically appropriate time. The characteristic fold is determined by each amino acid sequence, a concept known as Anfinsen’s dogma. 1 Each fold has to be compatible with the life history of the protein, the environment where it acts and the partner proteins with which it must interact. Thousands of proteins reside either in the plasma From the  Biosciences Division, Argonne National Lab, Ar - gonne, IL 60439, and the †Department of Pathology, The Univer - sity of Chicago, Chicago, IL 60637, USA 1999 Academic Press 1084-9521  99  050443 12 $30.00  0 membrane of human cells, within membranous or- ganelles or in the extracellular spaces. All of these proteins share a common origin in the endoplasmic Ž . reticulum ER , the compartment where they fold. Here, we describe the environmental constraints that the ER imposes on the folding of these proteins and discuss how these conditions necessitate careful coor- dination and regulation. It is against this background that the actions of the individual molecular chaper- ones described elsewhere in this issue should be gauged. Levinthal’s Paradox and Anfinsen’s Dogma Two mutually exclusive world views can be applied to the issue of protein folding. According to the pes- simistic view, it should be almost impossible for these linear polymers of 20 different amino acids to en- twine themselves reproducibly into stable three-di- mensional structures. After all, for a given linear array of N amino acids, the three tetrahedral posi- Ž tions for the phi angle  , rotation about the N C  . bond, Figure 1 and three corresponding positions Ž . for psi , about the C  C bond result in nine relative arrangements for the backbone atoms of any two successive amino acids. Therefore, a polypeptide of N amino acids has 9 N1 possible arrangements of backbone atoms. This number vastly underestimates the total number of conformations available for the protein sequence, since the side chains of amino acids other than glycine and alanine are all capable of alternative arrangements themselves. If a protein sampled all the possible arrangements, its folding would be very slow. This problem is known as Levinthal’s paradox. 2,3 Steric interference by the side chains reduces the number of possible arrangements. Polyalanine, with its single methyl group side chain, should come closest to achieving the statistical esti- mate. However, polyalanine has a very predictable structure and is widely used as a model for the -helix. If polyalanine has a predictable structure, then a 443