Abstract
The in vivo conformational substrates of the GroE chaperonins have been difficult to identify, in part because of limited information on in vivo polypeptide chain folding pathways. Temperature-sensitive folding (tsf) mutants have been characterized for the coat protein and tailspike protein of phage P22. These mutations block intracellular folding at restrictive temperature by increasing the lability of folding intermediates without impairing the stability or function of the native state. Overexpression of GroEL/ES suppressed the defects of tsf mutants at 17 sites in the coat protein, by improving folding efficiency rather than assembly efficiency or protein stability. Immunoprecipitation experiments demonstrated that GroEL interacted transiently with newly synthesized wild-type coat protein and that this interaction was prolonged by the tsf mutations. Folding defects of the tailspike polypeptide chains were not suppressed. A fraction of the tsf mutant tailspike chains bound to GroEL but were inefficiently discharged. The results suggest that 1) thermolabile folding intermediates are natural substrates of GroEL/ES; 2) although GroEL may bind such intermediates for many proteins, the chaperoning function is limited to a subset of substrate proteins; and 3) a key reason for the heat-shock response may be to stabilize thermolabile folding intermediates at elevated temperatures.
Highlights
Heptameric rings, while HsplO is a single ring of 7 subunits chaperonins have been difficult to identify,in part be- (Hendrix, 1979;Zwickl et al, 1990; Hartman et al, 1992)
Strate proteins; and3) a key reason for the heat-shock Hsp6O has been shown to reduce the extent of denaturation response may be to stabilize thermolabile folding inter-of native proteins (Martin et al, 1992)
On the pLS cellsp, lating eficiency increased to near permissive levels. These results demonstrated that overexpression of both GroEL and GroES was required for rescue of the ts coat protein strains
Summary
The P22coat protein, encoded by gene 5, is composed of 430 28 "C. At the indicated times, aliquots were removed and lysed by amino acidsand hasa molecular mass of 47 kDa (Eppler et al, 1991). 420 icosahedrally arranged coat protein molecules are the major proteinconstitutents of phage heads, withinwhich is located P22 DNA (King and Casjens, 1974).A set of 18temperature-sensitive mutants, at 17 different sites in the coat protein, have been characterized. Radiolabeling Experiment-Overnight cultures of DB7136/pBR322 and DB7136/pOF39 growing inMinimaUAmp were diluted1:50in fresh media and grown toa concentration of 1x 108/mlat 32 "C. Cells were pelleted, resuspended in fresh media to a concentration of 4 x 108/ml and placed on ice. Cells were infected with phageat a multiplicity of 7 phage particles (procapsids)carrying these mutationws ere not at 29 "C (time 0). To lyse the infected cells, samples were frozen at -20 "C, genic complementation, and cannot be rescued by wild-type thawed, frozen again ina dry-ice/ethanol bath, and thawed. The tsf mutants allow one togenerateintracellularpartially disordered folding intermediates a t temperatures optimal for cell were incubated with 1.5 ml phage, a t a multiplicity of infection of 7 phagehell, at 37 "C with aeration. 50 min after infection, cells were labeled with 20 pci/ml I4C-amino-acids (to 3 pci/ml). Dilution fluid, used for serial above DnaK, where no protein bands were visible
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