THERMOLABILE FOLDING INTERMEDIATES - INCLUSION-BODY PRECURSORS AND CHAPERONIN SUBSTRATES

An unexpected aspect of the expression of cloned genes is the frequent failure of newly synthesized polypeptide chains to reach their native state, accumulating instead as insoluble inclusion bodies, Amyloid de posits represent a related state associated with a variety of human di seases, The critical folding intermediates at the juncture of producti ve folding and the off-pathway aggregation reaction have been identifi ed for the phage P22 tailspike and coat proteins, Though the parallel beta coil tailspike is thermostable, an early intracellular folding in termediate is thermolabile. As the temperature of intracellular foldin g is increased, this species partitions to inclusion bodies, a kinetic trap within the cell, The earliest intermediates along the in vitro a ggregation pathway, sequential multimers of the thermolabile folding i ntermediates, have been directly identified by native gel electrophore sis, Temperature-sensitive folding (tsf) mutations identify sites in t he beta coil domain, which direct the junctional intermediate down the productive pathway. Global suppressors of tsf mutants inhibit the pat hway to inclusion bodies, rescuing the mutant chains, These mutants id entify sites important for avoiding aggregation. Coat folding intermed iates also partition to inclusion bodies as temperature is increased, Coat tsf mutants are suppressed by overexpression of the GroE chaperon in, indicating that the thermolabile intermediate is a physiological s ubstrate for GroE. We suggest that many proteins are likely to have th ermolabile intermediates in their intracellular folding pathways, whic h will be precursors to inclusion body formation at elevated temperatu res and therefore substrates for heat shock chaperonins.