FUNCTION OF GROEL IN THE BACTERIAL CYTOPLASM

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The proposed studies are aimed at defining the function of the homooligomeric double ring 14mer complex, groEL, in folding and assembly of newly-translated proteins in the bacterial cytoplasm. During the current project period we have identified an apparently global role for a homologue in the mitochondrial matrix, hsp60 complex, in mediating folding and assembly of proteins newly-imported into the organelles. We now wish to assess whether groEL could have a similar general role for assisting newly-translated proteins to reach their native forms. such a possibility is suggested by observations: that groEL is essential for cell viability at all temperatures; that bacteriophage assembly is impaired in several nonlethal groEL mutants; and that groEL and its cooperating component groES can mediate refolding in vitro of several polypeptides diluted from denaturant. We will assess the role of groEL in vivo by producing temperature-sensitive or cold-sensitive lethal mutants. Our approach will involve direct homologous insertion of in vitro-mutagenized groEL sequences into the E. coli chromosome. Mutagenesis approaches will include directed codon changes, doped oligonucleotide mutagenesis, and alanine scanning mutagenesis. After shift to nonpermissive temperature, we will compare wild-type and conditional lethal mutant cells for: fractionation behavior of groEL complexes; rat of protein translation and pattern of translation products; half-life and solubility of the newly-translated proteins; translation and acquisition of enzyme activity of several proteins specifically induced at nonpermissive temperature, including beta- galactosidase and galactokinase. groEL complexes from both conditional lethal mutants and slow-growing nonlethal mutants will be overproduced and purified, to allow functional studies in vitro to distinguish specific defects, enabling ultimately, once a groEL crystal structure is determined, structure-function understandings. We will also examine where polypeptides are bound at groEL, whether at its outer surface or within the cavity of the rings, by examination i the scanning transmission electron microscope of undecagold-conjugated dihydrofolate reductase bound to groEL.